science experiments plant growth

Top 17 Plant Science Experiments: Exploring Plant Growth

Join us as we embark on a journey of scientific exploration, unveiling the wonders of plant life one experiment at a time.

We have selected the best plant-related science experiments for this collection. These hands-on, educational activities are suitable for students of all age groups and not only satiate our curiosity about the natural world but also anchor our understanding of ecology and biology.

Let’s get started, and hopefully, this botanical journey will inspire a lifelong appreciation for the marvels of mother nature.

1. Grow Your Own Plants

This experiment offers an immersive learning experience, allowing students to witness firsthand the stages of plant growth, understand the requirements for healthy development, and observe the effects of various environmental factors.

2. Chlorophyll Paintings

“Chlorophyll Paintings” offers an innovative and artistic approach to plant science experimentation that both students and teachers should explore.

This unique experiment combines the worlds of biology and art, allowing participants to create captivating masterpieces while exploring the wonders of chlorophyll, the pigment responsible for a plant’s green color.

Learn more: Chlorophyll Paintings

3. Color Changing Flowers

This experiment provides an excellent opportunity to explore the process of water uptake in plants and how it affects the distribution of pigments within the flowers.

Learn more: Color Changing Flowers

4. Low-Prep Flower Dissection

“Low-Prep Flower Dissection” presents an accessible and engaging plant science experiment that is ideal for both students and teachers seeking hands-on learning experiences with minimal preparation.

This experiment offers a fascinating glimpse into the intricate anatomy of flowers and the functions of their various parts.

Learn more: Low-Prep Flower Dissection

5. Acid Rain Science

“Acid Rain Science” presents an impactful and relevant plant science experiment that offers valuable insights into the environmental effects of acid rain.

Students and teachers should engage in this experiment to understand the detrimental consequences of pollution on plant life and ecosystems.

6. Reveal a Plant’s Vascular System

“Reveal a Plant’s Vascular System” offers an exciting and enlightening plant science experiment that allows students and teachers to explore the hidden wonders of a plant’s circulatory system.

Learn more: Reveal a Plant’s Vascular System

7. Make Oxygen at Home

Through the process of photosynthesis, plants convert carbon dioxide into oxygen, a vital component for supporting life on Earth.

This experiment offers a unique opportunity to understand the connection between plants, photosynthesis, and the oxygen we breathe.

8. How Water Travels Through Leaves

Students and teachers should engage in this experiment to gain a deeper understanding of how plants absorb and distribute water, while also exploring the concepts of transpiration and the importance of water in plant survival.

Learn more: How Water Travels Through Leaves

9. Growing a Bean Plant

By following simple steps, participants can cultivate their own bean plants and observe the stages of germination, root development, and leaf growth.

This experiment offers an excellent opportunity to explore plant anatomy, photosynthesis, and the importance of environmental factors for healthy plant growth.

10. Easy Seed Sprouting

“Easy Seed Sprouting” offers a simple yet rewarding plant science experiment that students and teachers should embrace to witness the wonder of seed germination and plant growth.

Learn more: Easy Seed Sprouting

11. Leaf Color Chromatography

By conducting this experiment, participants can explore the fascinating world of pigments and chromatography, gaining a deeper understanding of the diverse hues present in plant leaves.

12. How to Revive Any Dying Plant

This experiment offers a hands-on opportunity to understand the factors influencing plant health and to develop skills in plant care and problem-solving.

By exploring various techniques such as adjusting watering schedules, providing appropriate light exposure, and optimizing soil conditions, participants can revive and rejuvenate struggling plants.

13. Make Your Own Fun Light Maze for Plants

By constructing a maze using various light sources, participants can investigate how plants respond to different light conditions and orientations.

14. How Plants Breathe

By engaging in this experiment, participants can gain a deeper understanding of how plants exchange gases and respire, just like humans and animals.

Through this experiment, students will discover the importance of oxygen and carbon dioxide in plant metabolism and growth.

15. The Color-Changing Celery Experiment

This experiment provides a unique opportunity to witness the movement of water and the transportation of pigments through the xylem vessels of celery stalks.

16. Growing Seeds in Eggshells

This experiment not only promotes sustainable practices by repurposing waste materials but also provides an opportunity to explore the principles of seed germination, root development, and plant nutrition.

Learn more: Growing Seeds in Eggshells

17. Make a 3D Flower Model with Parts

By constructing a three-dimensional model using various materials, participants can explore the different parts of a flower and their functions.

Learn more: Make a 3D Flower Model with Parts

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23 Ideas for Science Experiments Using Plants

ThoughtCo / Hilary Allison

• Cell Biology
• Weather & Climate
• B.A., Biology, Emory University
• A.S., Nursing, Chattahoochee Technical College

Plants are tremendously crucial to life on earth. They are the foundation of food chains in almost every ecosystem. Plants also play a significant role in the environment by influencing climate and producing life-giving oxygen. Plant project studies allow us to learn about plant biology and potential usage for plants in other fields such as medicine, agriculture, and biotechnology. The following plant project ideas provide suggestions for topics that can be explored through experimentation.

Plant Project Ideas

• Do magnetic fields affect plant growth?
• Do different colors of light affect the direction of plant growth?
• Do sounds (music, noise, etc.) affect plant growth?
• Do different colors of light affect the rate of photosynthesis ?
• What are the effects of acid rain on plant growth?
• Do household detergents affect plant growth?
• Can plants conduct electricity?
• Does cigarette smoke affect plant growth?
• Does soil temperature affect root growth?
• Does caffeine affect plant growth?
• Does water salinity affect plant growth?
• Does artificial gravity affect seed germination?
• Does freezing affect seed germination?
• Does burned soil affect seed germination?
• Does seed size affect plant height?
• Does fruit size affect the number of seeds in the fruit?
• Do vitamins or fertilizers promote plant growth?
• Do fertilizers extend plant life during a drought?
• Does leaf size affect plant transpiration rates?
• Can plant spices inhibit bacterial growth ?
• Do different types of artificial light affect plant growth?
• Does soil pH affect plant growth?
• Do carnivorous plants prefer certain insects?
• 8th Grade Science Fair Project Ideas
• Plant and Soil Chemistry Science Projects
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• Chemistry Science Fair Project Ideas
• 11th Grade Science Fair Projects
• Magnetism Science Fair Projects
• 9th Grade Science Fair Projects
• Science Fair Project Ideas
• 4th Grade Science Fair Projects
• Caffeine Science Fair Projects
• Science Fair Experiment Ideas: Food and Cooking Chemistry

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• Science & Plants for Schools (SAPS): Primary Resources/

Primary science investigations with plants

A collection of investigations around the topic of plants, looking at life cycles, factors affecting growth, parts of a plant, composting and plants that we eat. Investigations provided by Science & Plants for Schools (SAPS) are:  

Holly leaves : investigate questions about holly leaves.

Tree rings : investigate cut tree trunks to determine the age of the tree, how fast it grew and climatic conditions during its growth.

Investigating if plants grow better with fertiliser : plan and carry out a fair test looking at the effect of fertiliser on growing radishes

The life cycle of a flowering plant : a sequencing activity looking at the lifecycle of Brassica, a fast cycling flowering plant.

How does light affect growing plants? : Plan and carry out a fair test looking at the effect of light on plant growth.

Having fun growing plants: provide the conditions for germination and observe a variety of seeds over time.

Finding out about the number of flower parts : three ways to dissect a flower in order to identify and name the different parts.

Designing a seed:  design and make a seed from a newly discovered plant using junk materials.

Are you a plant eater?: Develop an awareness of the parts of a plant that are eaten.

Composting, recycling and the curriculum : investigate what happens during the composting process.

• Cross curricular
• Activity sheet
• Presentation
• Teacher guidance
• Include Physical Resources

Holly Leaves - Themes and Variations

This resource, aimed at primary level and linked to the curriculum area of plants, investigates holly leaves. Designed to be used in an outdoor area where holly plants grow, it provides questions about holly leaves which children may investigate, for example, how many...

This teaching package, aimed at Key Stages Two and Three, investigates the science of tree rings (dendrochronology). Linked to the topics of plants and living things and their habitats, it looks at cut tree trunks to determine the age of the tree, how fast it grew and...

Investigating if Plants Grow Better with Fertiliser

This resource, aimed at primary level, helps to develop an understanding of what plants need to grow well. It provides an opportunity to plan and carry out a fair test looking at the effect of fertilizer on growing radishes. It includes detailed notes on setting up and...

The Life Cycle of a Flowering Plant

Aimed at primary level, this resource provides a sequencing activity looking at the lifecycle of Brassica, a fast cycling flowering plant. It is designed to help children demonstrate their understanding of the progression through the lifecycle and reinforce their...

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Plants Science Experiments & Teaching How Plants Grow

I love doing plant experiments and sprouting seeds with young children in the spring. Not only do they get excited to see how plants grow but planting seeds also teaches them patience and how to wait for gratification which is very important in this fast-paced, instant gratification world in which we now live.  In this post I’m sharing some of my favorites from over the years.

What Liquid is Best for Growing Seeds? Experiment

This experiment tests what type of liquid is best for growing seeds and can be done using a wide variety of liquids. Since we already discussed that plants need water to grow, we first tested different types of water to see if it made a difference. We decided to test tap water, bottled water, sugar water (1 cup of water with 1 Tbsp sugar), and salt water (1 cup water with 1 Tbsp salt). I used grass seed for this experiment because it sprouts fairly quickly but you can use bean seeds (lima beans soaked overnight in water work well) or any other type of seed you wish.

We added the same amount of soil and seed to each cup and labeled them.  We then measured out the same amount of water for each cup and watered the seeds with the different types of water and set them by the window. Students make predictions as to which one they feel will work best.

We observed the seeds for 5 days and were a little surprised that the bottled water didn’t grow as well as the tap water. The tap water grew the best, followed by the bottled water, the sugar water had a few blades come up, and the salt water did not have any.

When looking at the label of the bottled water we found that additional ingredients are added (calcium chloride, sodium bicarbonate, and magnesium sulfate) which most likely lead to mineral imbalances in the soil that slowed growth. Liquids with very high sugar or salt levels can actually pull water away from the plant or seed rather than allowing the water to be absorbed. In conclusion, simple pure tap water worked best.

We then do the experiment with liquids other than water to see if another type of liquid could be used if water isn’t available. You can use any liquids you have on hand, just make sure that one of them is water to use as the comparison. We have tried vinegar, oil, rubbing alcohol, lemon juice. As expected, water always works best.  Last year students had the idea to test liquids that we drink to see if plants would drink them too. I thought this was very creative! We tested vitamin water, pop (soda), and juice.

We added the same amount of soil and seed to each cup and labeled them.  We then measured out the same amount of liquid for each cup and watered the seeds with the different types of liquids and set them by the window. Students made predictions as to which liquid they feel would work best.

We observed them for a week. Our results were that water was best, followed by the vitamin water. Neither the juice or pop had any sprouts.

Using liquids that are very acidic or very alkaline lead to mineral imbalances in the soil that will kill plants or slow growth.  Liquids with very high sugar or salt levels can actually pull water away from the plant or seed rather than allowing the water to be absorbed.

I have students record their results.

How Plants Drink Science Experiment

This experiment has been around for years and is a great way to  demonstrate to students how plants get water from their roots all the way up to their leaves.

It is very simple to set up. Celery stalks that have leaves at the top work best. The stalks on the inside of the bundle of celery usually have the most leaves.

Cut about an inch or so off the bottom of the celery stalks.

Fill each container about halfway with water and drop 10-15 drops of food coloring in each glass.  Place the celery stalks in the water.

I also like to do a split stalk one. Cut one stalk in half part way up and place one half in one color and the other in a different color.

Observe the celery at the end of the school day.  You may see a little color in the stalk or the leaves. Observe them again the next day and you should see color in the leaves.  After 48 hours you will really notice changes and color in the stalks and leaves showing that the water traveled up through the stalk to the leaves.

The split celery stalk should show the separate colors on each side and then a mix of the colors in the leaves in the middle. In the pictures below the blue is on the left, red on the right, and some purple leaves in the center.

You can cut open the stalks to allow students to see the small tubes inside the stalks that carried up the colored water to the leaves.

After cutting open the celery we discuss the results. I introduce some bigger vocabulary to them when we talk about the science behind the experiment, but I basically just want them to understand that the water travels up the stem through tiny tubes to the leaves.  Here is a simple explanation:

The Science Behind It:

This experiment demonstrates how plants use capillary action to draw water up their stems. Capillary action is the process in which a liquid, like water, moves up something solid, like the tubes (xylem) in the stem.  The leaves help pull the water up the xylem through transpiration. The leaves have little holes that let out the water that the plant is done using. This makes room for more water to come rushing up through the stem.

I have students record their observations by coloring the celery on their recording page (I created pages with the celery already drawn to make it easier for my young students).  Then they write what they learned along the bottom.

Do Plants Need Light? Experiment

This experiment tests whether plants need light to grow.  You can choose to plant 2 containers of seeds and set one in direct sunlight near a window and one in complete darkness OR plant 3 containers and set one in complete sunlight near a window, one in partial light, and one in complete darkness (it is important that there is NO light).

Plant the same number of seeds in each container with the same amount of soil and label each container.

Have students help you decide the best places in the room to place each container (by a sunny window, in a closet that gets NO light, in a file cabinet drawer, on a shelf in partial light, inside a closed box, etc.)

Observe the containers for about 2 weeks (or however long it takes to see growth) watering as needed.  At the end of the experiment, put the containers side by side and discuss the results.

We do 3 containers – one by the window in full sunlight, one on a shelf that gets partial light, and one in the back of the closet behind a box.

The one near the window shows the most growth, the one in the partial light has growth on the side of the container that received partial light and grows towards the light, the container in total darkness has no growth.

Plants need light to grow because it is an important part of photosynthesis, the process plants use to convert carbon dioxide and water into food. Without light, photosynthesis does not work properly and therefore the plant does not get enough food.  However, not all plants need the same amount of sunlight. There are types of plants that need a lot of bright sunshine and some that can survive with only a little light, but in the absence of ALL light plants will not survive. If you had a seed sprout in the dark, it may have used energy stored up in the seed to begin growing but it will not continue to grow without light.

I have students record their results on recording pages.

Growing Grass Science Activity

Growing grass is a great activity to do with young children because it is easy to plant and grows fairly quickly. It also teaches them about the needs of plants and develops patience because they have to wait for the results and observe changes over time.

A fun option that I like to do is put faces on the cups or containers and have the grass be the face’s hair.  You can glue on actual photos of the students’ faces or have them draw faces on the cup or use accessories such as wiggle eyes. You can also do this activity around St. Patrick’s Day and put leprechaun faces on the containers and grow green leprechaun “hair”.

I have students use plastic spoons to fill their cups about ¾ full with dirt/soil. Then have them sprinkle grass seed on top of the dirt. There is no need to measure out the seed, however I usually tell students to cover the dirt with seed (the more seed, the more grass that will grow).  Then have them cover the seeds with a small amount of dirt.

Lastly, I have students water their seeds with a spray bottle. I like using a spray bottle because it prevents over watering (and then once the grass “hair” starts to grow, students pretend the water is hairspray lol).

I have students help determine the best location in the room for their grass seed (next to a sunny window) and guess how many days they think it will take for their grass to grow.

We usually see some type of growth by day 3 or so.

Once it sprouts the grass grows fairly quickly.

I’ve done several different activities with students. One is having them predict how long they think it will take their grass to grow and then recording the actual results.

We practice measurement skills by measuring how tall the grass has grown.

After students’ grass hair grows, I let cut their hair with scissors and then estimate how long they think it will be until it grows back.

Growing Bean Sprouts

This is another experiment that has been around for years but is a wonderful way for students to observe beans sprouting and see what happens underground when a seed is planted.

I have done this experiment 3 different ways.

Growing Beans in a Jar

This is a good method to use if you want to do a class experiment and you do NOT want each student to grow their own seeds.

Stuff a large jar with paper towels.  Students can help.

Slowly pour some water in the jar to wet the paper towels but do not flood it.  If you have any excess water at the bottom pour it out. You want the paper towels to be damp not soaking wet.

Push your seeds down in between the jar and paper towels and make sure they are firmly in place (a snug fit between the jar and towels).

Place several seeds around each side of the jar.  Place the jar near a sunny window.

Check on the jar daily.  You should see a root come out of the seed first within 3 days.  If you used bean seeds you should be able to observe the plant until it grows to the top of the jar.

I like having students keep plant journals because they improve their observation and recording skills and give them a record of the seed’s growth. Students do a recording page for each observation.

Sprouting Beans in Baggies on a Sunny Window

This method requires a bright sunny window on which you can hang baggies that contain the seeds.  You are making a plastic baggie “greenhouse” for the seeds.  You can choose to have each student plant their own beans in their own baggie or plant a few baggies as a class.  If you choose to have students do their own seeds and baggies, it’s a good idea to plant extra seeds in case some students’ seeds do not grow.  If this happens, switch out the seeds when students are not there to ensure that each child has at least one bean that sprouts.

If doing individual bags for each student, have students write their name on their baggie with a marker. Optional: you can also have them write the date. If doing a class experiment, you can write the date on the baggies.

For each baggie, place a dampened, folded paper towel along the bottom. It should have a fair amount of water but not be soaking or dripping wet.

Place one or several bean seeds between the paper towel and the baggie.

Tape or Sticky Tac them on a bright, sunny window.

Check them daily.  You should see a root come out of the seed first within 3 days.

I have students keep plant journals similar to the one shown above but the recording pages are slightly different. I have the baggie already drawn for them to make it easier. Students can also upload real photos to Pic Collage and complete their journals using the app.

Growing Seeds in a Greenhouse on a Window

This method is the same as the baggie method shown above except students make a greenhouse from construction paper and place their baggie in the opening.

Hang them on a sunny window and make daily observations.

The journal pages I use for this method have the greenhouse already drawn to make it easier for students to record results.

We take the bean plants that have grown to the top of the jar or baggies and carefully put them in soil. I explain to students that the plant needs the support and nutrients from soil to continue to grow larger.

What Do Plants Need to Grow? Pages

I like using these pages to check individual student understanding of what plants need to grow.  On the first page they have to circle the correct pictures. On the second page they unscramble the words and write the correct words on the lines.

If you would like to use the printables, activities, word wall cards, label cards, play dough recipe, and more with your students they are available in my  Plants & Flowers Science Activities resource .  It also includes experiments for plants & seeds, step by step directions with photos for easy set-up, plant journal pages, and more.  Click here  to see complete details and photos of each activity.

Have engaging science experiments and STEM activities throughout the entire school year with this money-saving Science & STEM Bundle !

You may also like:

Flower science experiments & parts of a flower activities, water cycle, rain cycle science experiments and craftivity.

Hi! Thanks for stopping by!

I’m Tina and I’ve taught preK and K for 20+ years. I share fun and creative ideas that spark your students’ love for learning. 

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Plant Growth and Root Development

Water is a critical element for plant growth, but certain soils or sediments can actually block water from getting to the deeper soil layers. For example, caliche is a hard, shallow layer of soil often found in the southwest and other arid and semiarid parts of the world. Caliche inhibits water from reaching the deeper soils, making it harder for plants to grow. It also prevents roots from reaching the deeper soils, depriving the plant of nutrients. Consequently, this “barrier” could stunt a plant’s growth, or cause the plant to wilt and die. (See Appendix A for more information)

About the Experiment

For this experiment, we’re going to test the effect a hard soil layer has on plant growth and root development.

 What You'll Need

• 3 clear plastic cups (e.g. Solo cups)
• 3 non-clear plastic cups
• Potting soil (Small bag, 6-8 qt.)
• Wheatgrass or cat grass seed (100 seeds, available online or at local pet store)
• Piece of cardboard
• Baking soda
• Measuring cups
• Drill & small bit

Let's Do This!

Step 1 . Drill 3 small holes in the bottom of 3 clear plastic cups. (Have an adult help with this step for safety).

Step 2 . Cut 2 pieces of cardboard so it fits tightly about 1.5” from the top of one clear cup (cup with holes). Trace the opening of the cup and cut 1/4” inside the line.

Make sure the cardboard fits tightly. Mark where the cardboard is on the cup, remove cardboard and fill up to the line with soil. Place cardboard on top of the soil.

Step 3 . Using duct tape, tear skinny strips and place around the edge of the cardboard to seal to the cup. Make sure to only cover edges of the cardboard with tape.

Step 4 . Fill the cup with soil ½” from the top (label this “Cup 1”). Repeat the process, but place cardboard about 3” (halfway) from the top of the second cup.

For a tight fit, trace the top of the cup and cut cardboard 1/2” inside line. (label this “Cup 2”).

Fill 3rd clear cup ½” from the top with soil (No cardboard) (Label this “Cup 3”).

Place the cups inside 3 non-clear cups.

Step 5 . Pour ½ cup water in each cup. Wait 1 minute and pour another ½ cup of water in each cup. OBSERVE: Which cup has standing water after a minute? Record answer on data sheet .

Step 6 . Place 30 grass seeds in each cup and cover with 1/8” soil. Gently add a little more water to wet topsoil. 

Let seeds germinate and grow for 1 week.

Let’s Look At The Results!

After 1 week count the number of plants in each cup and measure the tallest blades of grass in each cup.

Remove clear cups from non-clear cups and observe the amount of water in the non-clear cup. Which cup has the most?

Observe the amount of roots in the clear cups. Are the roots below the “caliche” layer? Which cup has the most roots? Record your answers and observations on the data sheet.

After 1 week, add ¼ cup water to each cup. Do Not add any more water .

Observe how many days till plants in each cup wilt (may take a week or more) and record answers on data sheet.

Record any other observations you have during the experiment on the data sheet.

Finally, summarize what you think occurred in your experiment and why based on the observations you made and the data you collected.

Botany Science Projects - Learn about plant growth by Science Made Simple

Botany and plant growth science projects.

Use these experiments as described, or expand and modify them based on your own interests and imagination.

1. What affect does the brightness of light have on the growth rate of a plant?

How do light and dark conditions affect the germination and growth of seedlings?

Materials: Greenhouse or sunny window sill, 10 bean seeds, 10 small pots, water, ruler, potting soil, pencil.

• Fill the 10 small pots with equal amounts of dampened potting soil.
• With a pencil, make holes about 2 centimeters deep in each pot.
• Place the 10 bean seeds, one per pot, and cover the seeds with some of the soil.
• Place 5 of the pots in the greenhouse or on a window sill on the sunny side of the house.
• Place the other 5 on a window sill that does not receive bright sunlight.
• Be sure to water the plants as needed.
• Seeds will germinate within 7 days, and you can begin making stem measurements. Take stem measurements for 14 days. Note the difference in stem length for each set of plants, and write down your observations.

Results: What differences did you observe between seedlings that grew in the bright sunlight compared to less bright light? (color of leaves, length of stems, etc.) What caused those differences?

2. How do different types of fertilizers affect plant growth?

Fertilizers differ in their amounts of the nutrients nitrogen, phosphorus and potassium. Get different fertilizers from a garden shop or nursery and apply them to groups of the same plant. Do the different fertilizers change how the plants grow? You could measure height, width, number of leaves, how fast the plants grow, number of flowers or yield.

3. Which way is up? - Tropism and Auxin

Many seeds and bulbs have a definite top and bottom. What happens if you plant them upside down or sideways? Will the seeds still grow; will it take longer for leaves to start showing up?

What happens if you change a seed's direction once it starts to sprout? You'll learn about the chemical auxin, which affects where roots and stems grow.

• Divide 10 bean seeds into 2 groups of 5. - a control group and the experimental group.
• Spread the seeds out on moist paper towels then wrap them a pieces of folded aluminum foil.
• Label one side of control group packet "Up". Label the sides of the experimental group "A" and "B". Place the sprouts where they will not be observed.
• Allow the beans to sprout for 3 days.
• Carefully open the foil and towels and observe the seedlings. Moisten the towels if necessary, then refold the foil. Turn the experimental set of the seeds upside down. Make sure to keep the control seeds right-side up.
• Open and observe the sprouts every 2 days, making sure to keep the control sprouts right-side up and turning the experimental group over.

If you have access to an old record player turntable, you can take it a step further by using it to simulate changing gravity's pull on seeds. Tape the experimental packet onto the turntable and set it for 78 RPM. Allow the machine to rotate continuously for 5 days. After the 5 days are up, turn off the record player and without changing the position of the foil, open them up and observe the beans. The rotating turntable creates a gravity with an outward force instead of the normal down.

4. What happens when you grow sweet potatoes next to other plants? - Allelopathy

Compare how fast other plants grow at different distances from sweet potatoes. Remember to grow some control plants nowhere near the sweet potato.

Background Info: Allelopathy is a chemical process that a plant uses to keep other plants from growing too close to it. Some plants that use allelopathy are black walnut trees, sunflowers, wormwoods, sagebrushes, and trees of heaven.

There are several ways in which an allelopathic plant can release its protective chemicals:

• Volatilization - Allelopathic trees release a chemical in the form of a gas through small openings in their leaves. Other plants absorb the toxic chemical and die.
• Leaching - Some plants store protective chemicals in the leaves they drop. When the leaves fall to the ground, they decompose, giving off chemicals that protect the plant.
• Exudation - Some plants release defensive chemicals into the soil through their roots. Those chemicals are absorbed by the roots of other nearby plants, which are damaged.

Fruit Ripening

5. how do different conditions affect the speed at which fruit and vegetables ripen.

Temperature, light, placement in sealed bags, exposure to other ripe fruit--all have different effects on different fruits and vegetables. Design an experiment to test two or more of these variables.

Background Info: Ethylene gas is the ripening agent that many fruits and vegetables produce naturally. Ethylene causes them to ripen--and then overripen. While refrigeration and humidity slow the effects of ripening, they don't stop the production of ethylene gas.

The more the fruit ripens, the more ethylene gas it makes. This has a big effect on how--and when--farmers harvest their fruits and vegetables for market. Most commercial tomatoes are picked before ripening is completed, so the fruit won't spoil before it gets to your market. But picking early also means the tomato spends less time on the vine, where ethylene would help build more of the sugars and acids that create tip-top tomato flavor.

6. The effects of light on seedlings germination

How do light and dark conditions affect the germination and growth of seedlings....

Get the rest of this experiment and many more on the next page.

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Easy Plant Experiments For Kids

Plants come in all shapes, sizes, and colors and are essential for life on Earth. Learn more about how incredible plants are with these hands-on plant experiments and printable plant worksheets. You will find easy plant activities and ideas for elementary to middle school. We love do-able science experiments for kids!

Plant Science

Plant science or botany is a fun topic to teach to kids of all ages. We have a range of plant experiments and projects that would work well in a wide range of settings, from home to the classroom.

Our science activities are designed with you, the parent or teacher, in mind. Easy to set up and quick to do, most activities take only 15 to 30 minutes and are fun. Plus, you don’t need a ton of expensive materials!

Learn about…

• How living things form part of systems.
• How energy flows through simple systems.
• Develop observational skills and practice making predictions.
• Understand the role of variables in measuring changes.

These plant experiments below are great for elementary to middle school students. For our younger kiddos, check out our list of plant activities for preschoolers .

Plant Facts For Kids

• Most plants need water, soil, and sunlight to grow.
• We need plants because they produce oxygen, clean the air we breathe, provide food, they are homes and food for many other living things, and more.
• Some plants are carnivorous. That means they eat animals (like spiders and insects)!
• 80% of flowering plants have adaptions so that they can be pollinated by bees and other insects or birds.
• Some plants do not have flowers or seeds, moss, and ferns. They reproduce by making spores.
• There are over 390,000 different types of plants in the world. Over 90% of which are flowering plants.
• Some plants live underwater and are called aquatic plants.
• About half of all plants are edible. Yet we only eat about 200 plants, and three plants, rice, wheat, and corn, make up over 50% of the plants we eat.

Tips For A Plant Science Project

Science projects are an excellent tool for kids to show what they know about science! Plus, they can be used in various environments, including classrooms, homeschool, and groups.

Kids can take what they have learned about using the scientific method , stating a hypothesis, choosing variables , and analyzing and presenting data.

Our seed germination experiment and plastic bottle greenhouse are both great plant growth experiments to consider for a science project.

Want to turn one of these experiments into an awesome science fair project? Check out these helpful resources.

• Science Project Tips From A Teacher
• Science Fair Board Ideas
• Easy Science Fair Projects

Get your quick and easy spring STEM challenges!  

Science Experiments With Plants

More than plant growth experiments, we have lots of fun ways for you to explore plant science for kids. Learn about capillary action, osmosis, respiration, and more.

Acid Rain Experiment

What happens to plants when rain is acidic? Set up an easy acid rain project with this flowers in vinegar experiment. Explore what causes acid rain and what can be done about it.

Celery Experiment

This celery food coloring experiment is a great way to show how water travels through a plant.

Color Changing Flowers

Turn white flowers into all sorts of fun colors! Observe capillary action at work as water moves up the stem to the flower.

ALSO CHECK OUT: Color Changing Carnations

Flower Dissection

Grab some flowers, and do a simple flower dissection to identify and name the parts of a flower. Pair it with our printable parts of a flower diagram.

Glowing Spinach

Transform ordinary spinach that you eat into a glowing green mixture under ultraviolet light! Learn about the pigments present in plants, particularly chlorophyll and how certain pigments can absorb light at one wavelength and emit light at another, resulting in the observed glow.

How Do Plants Breath

This fun plant science experiment is a great way to teach kids about plant respiration. All you need are some green leaves and water to observe how plants breathe.

Leaf Chromatography

Have you ever wondered how leaves get their color? Discover the hidden pigments that are in leaves with this fun chromatography experiment. Chromatography is a technique used in chemistry that separates the components of a mixture into its individual parts.

ALSO CHECK OUT: Marker Chromatography Experiment

Learn about how water travels through leaves with this simple science experiment. Watch what happens when you put leaves into a jar of colored water!

Mini Greenhouse

Enjoy the wonder of growing plants by making an easy mini greenhouse from plastic bottles. Includes suggestions for turning it into a plant growth experiment.

Potato Osmosis Lab

Plant roots absorb water from the soil through osmosis. Learn about osmosis with this fun potato osmosis experiment. Investigate what happens to potatoes when you put them in a concentration of salt water and then pure water.

Regrow Lettuce

Did you know that you can regrow certain vegetables from their stalks right on the kitchen counter? Give it a try!

Seed Germination Experiment

Investigate what factors affect the germination of seeds with a simple germination jar. Kids love being able to watch the growth of the seeds!

Bonus Plant Activities & Worksheets

Ever noticed that different plants live in different parts of the world? Learn about what a biome is and examples of biomes around the world with this fun biomes lapbook project. 

Carbon Cycle

Plants have an important role in the carbon cycle which sustains life on earth. Find out what the carbon cycle is , and how plants are involved.

Explore the important role plants have as producers in the food chain . Includes printable food chain worksheets.

Honey Bee Life Cycle

Bees are important pollinators for flowering plants. Find out some fun facts about honey bees with this printable bee life cycle lapbook activity.

Life Cycle Of A Bean Plant

Learn about green bean plants with these fun and free printable life cycle of a bean plant worksheets! Find out more about how beans grow and learn about the stages of bean growth.

Life Cycle Of A Pine Tree

Learn about pine trees, and how they are different to flowering plants with these printable pine tree life cycle worksheets.

Parts of a Flower

Learn about the parts of a flower and what they do with this fun printable parts of a flower diagram.

Parts of a Leaf

A fun and easy way to learn the parts of a leaf . Grab this printable leaf coloring page!

Photosynthesis

How do plants get their food? Green plants make their own food and food for us through the process of photosynthesis . Use these printable worksheets to introduce the steps of photosynthesis to kids.

Plant Cells

Color in and label the parts of a plant cell as you explore what makes plant cells different to animal cells.

Pollinators

Explore the important role of pollinators in the reproduction of flowering plants with our printable pollinator activity guide.

Printable Spring Pack

If you’re looking to grab all of the printables in one convenient place plus exclusives with a spring theme, our 300+ page Spring STEM Project Pack is what you need!

Weather, geology, plants, life cycles, and more!

Subscribe to receive a free 5-Day STEM Challenge Guide

~ projects to try now ~.

How Wee Learn

Out of the box learning ideas, playful art, exploring nature, and simple living - that is How We Learn!

Plant Science Experiments

May 28, 2024 by Sarah Leave a Comment

In this blog post, I’ve gathered my favorite plant science experiments, from sprouting seeds to discovering how light, water, and soil influence plant growth, to flower and leaf experiments, and beyond.

These hands-on activities will cultivate a deeper appreciation for the environment and inspire your child to embrace curiosity and get their hands dirty—literally!

Free Printable Seed Growth Tracker

Before we get into all of the plant science experiments, you’ll want to grab your FREE Seed Growth Tracker. This page is perfect for recording observations as you watch your seeds grow!

Plant Science Experiments with Seeds

Sprout Seeds in a Mason Jar – The “mason jar and paper towel method” of seed germinating is perfect for comparing and tracking how seeds grow!

Do Seeds Need Their Seed Coat to Grow? by Gift of Curiosity – What is the purpose of a seed’s coat? Is it really needed? Find out with a seed germination experiment!

Light and Plant Growth Experiments

How Plant Growth is Affected by Light by Life with Moore Learning – Discover how light affects a plant’s growth with this simple set up.

Maze Potato Plant Experiment by 123 Homeschool 4 Me – Now that you know plants need light to grow, how can we have some fun with that knowledge?! Make a maze! This is such a neat one to watch as the plant makes it’s way through the maze to reach the light.

Water and Plant Growth Experiments

What Liquid is Best for Growing Seeds? Experiment by Lessons for Little Ones – Discover what type of water is best for growing seeds with this plant experiment! Little ones can make predictions and track each seed’s growth. 

What Solution Keeps Flowers Fresh Longest? by We Have Kids – With the last experiment, you found out what kind of water helps plants grow, but what helps cut flowers stay fresh?

Soil Experiments

Soil Erosion Experiment by Life is a Garden – This soil experiment is so cool! Kids will learn how plants and their roots help to protect soil from eroding.

What Soil Type is Best for Growing Seeds? by STEAMsational – Which type of soil is best for growing your plant? It may not be the one you think!

Learning About Leaves

Why Do Leaves Change Color? – Have you ever wondered why leaves change color? With just a few simple supplies, you can learn about chlorophyll and how leaves change color in the fall.

How Do Leaves Breathe? by KC Edventures – Did you know that leaves breathe!? For this experiment, all you will need is a bowl of water and a leaf. Easy peasy!

Flower Science Experiments

Reveal a Plant’s Vascular System by Tamara Horne – You’ve likely seen experiments where you plop cut flowers into vases filled with water dyed different colors. Well, if you happen to have a highlighter and a black light, you can take that experiment to a whole new level!

Dissecting Daffodils to Explore Pollination by Sloely – Explore all of the different parts of a plant by dissecting one!

What Happens When You Submerge a Dandelion? by Mud and Bloom – Have you ever tried dipping a dandelion in water? You would think those fragile little seeds would fall right off, considering how easily they blow away in the wind, but… well, you’ll just have to try it!

Which of these plant science experiments are you excited to try!? My kids are super excited to try out the maze experiment!

If you’re ready to dive into learning all about seeds and plants through fun, hands-on activities, I encourage you to check out my Seeds and Plants Family Unit Study. You’ll learn about the different types of plants, seed anatomy, photosynthesis, pollination, plant adaptations, and so much more!

Seeds and Plants Family Unit Study

https://shop.howweelearn.com/products/family-unit-study-seeds-and-plants

I hope your week is off to a wonderful start, my friend. Take care, and don’t forget to water your plants!

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• Science Fair Project Ideas for Kids, Middle & High School Students

Ideas for a Science Fair Project on how Different Liquids Affect Plant Growth

Science Fair Project for Testing Different Soils With Plant Growth

So you're planning an experiment on how different liquids affect plant growth for this year's science fair. Even though you already have the general idea, you'll need to think through all of the details in order to give your science fair project the best chance. Make sure you focus on the different conditions of the experiment, as well as how you will display the information about your experiment, in order to make sure that the judges will take notice of your thought-out project.

Types of Liquids to Use

You can use several different groups of liquids in this type of experiment. The most basic (but least scientifically interesting) experiment would have you using random liquids from around the house, from orange juice and apple juice to liquid cleaner or even urine. Most would guess, however, that plain water would work better than any of these variations. Instead, consider trying out different types of water--distilled water, tap water, mineral water, and water from a nearby stream or swamp. You could also try out different liquid fertilizers to see which does the best job.

Ways to Measure Plant Growth

You may think that it is easy to measure plant growth--until you find that one of your plants has grown 3 inches fewer than the others but has dozens of flowers all over it. In order to take accurate data, you'll need to define your methods of measuring the flowers before you begin the experiment. You may want to rely solely on height, especially if you're trying to simplify the experiment. Alternately, you can create a chart with a column for each factor that measures plant growth: height, number of leaves, number of flowers, thickness of stem, or any other factors that may affect the specific plants you choose.

Type of Plants

Make sure to choose fast-growing plants for your science experiment--unless you plan on spending months taking data. Examples of fast-growing plants are marigolds, zinnias, sunflowers, radishes, beans, cucumbers and cress. Make sure to use seeds from the same package in your experiment so that the type of liquid is your only variable.

Display Ideas

If possible, time your experiment so that you will be able to put your plants on display while they are still alive and thriving. You can place them in front of your display board, or off to the side of your display, if room allows. Make sure to take photographs of your plants at each stage of development, and attach one from each stage (clearly labeled) to the bottom center of your tri-fold board to show the plants' development. For an artistic touch, you can paint vines crawling up your display board in extremely light background colors before attaching your information to the board. Make sure that the background vines do not overwhelm the important information.

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• Redwood Barn Nursery: Seeds That Are Easy to Grow!

About the Author

Keren (Carrie) Perles is a freelance writer with professional experience in publishing since 2004. Perles has written, edited and developed curriculum for educational publishers. She writes online articles about various topics, mostly about education or parenting, and has been a mother, teacher and tutor for various ages. Perles holds a Bachelor of Arts in English communications from the University of Maryland, Baltimore County.

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9 Plant Activities & Garden STEM Projects

By: Author Jacquie Fisher

Posted on Published: August 10, 2020

Categories Nature & Outdoor Activities

Explore some science & math in the garden with these FUN plant STEM activities for kids !

Gardening is the perfect way to inspire kids to explore botany (the study of flowers & plants) , habitats and pollination. It’s also a wonderful family activity since everyone can get involved in planting, growing and harvesting from your own backyard.

Even if you don’t have the time or space for a full-blown garden, your kids would enjoy starting some flowers and veggies in a few posts on your porch.  And these STEM activities are a wonderful way to learn while you grow!

9 Plant Activities & Garden STEM Projects

We are joining the Storybook Science series again this week with the focus of science in the garden!  So we are, of course, including some great book recommendations along with STEM activities and affiliate links to some of our favorite plant & garden items for kids too.

One of the first things many kids will notice as they explore plants is that they grow 🙂 

This is very obvious to us but for kids, it’s a small revelation.  You’ll hear lots of questions:

“How does it grow?”

“How tall will it be?”

“Why is it growing?”

So before we head straight to the dirt, let’s talk about a few books that will inspire your kids to learn more about plants and gardening:

Garden & Plant Books for Kids

One of my favorites if you plan to grow flowers is Zinnia’s Flower Garden!  The story shows the reader each of the steps you take to set up and cultivate a garden.  I love the playful illustrations along with the details about seeds, weather and the garden environment.  And your kids will LOVE reading the mini journal entries on each page too.  This is a great preschool garden book !

The next two books we chose based on the extensive details and gorgeous illustrations in each one! 

Jack’s Garden is the story of how a boy plants & grows a garden in his backyard.  The illustrations are SO detailed you can explore the garden habitat and talk about the tools and science involved in gardening.  Readers will see what insects visit the garden, what animals & organisms live in the dirt to help the garden grow and tips for starting your own garden!

How Does My Garden Grow? is a wonderful story of Sophie, a city girl who visits her grandparents in the country one summer and learns about all the plants you can grow in a garden.  This is a longer picture book with TONS of details about the different types of vegetable families, garden tools and animals & insects that visit her garden.

And if you’re looking for more great garden reads, visit our 30 Gardening Books & Activities for Kids!

Once you have an idea of which plants you’ll grow, explore STEM (science, technology, engineering & math) with these fun ideas!

Garden Science Activities

The books we’ve recommended above are all perfect when it comes to dicussing the various science concepts of plants & gardening!

• HABITAT:  As you plant your garden or pot, talk about the habitat you are working with — what insects live in the soil?  What animals visit your garden for food (such as bees) or might live there all the time (for example, snails or ladybugs)?  Grab this FREE printable Garden Scavenger Hunt to help with this!
• GROWTH & ENVIRONMENT:  Plants need four things to grow — water, sunlight (for most), air and good soil (dirt with nutrients).  Mention these items to your kids as you plant your garden or pots. 
• PLANT BIOLOGY:  Discuss the various parts of a plant (roots, stem, leaves, buds, flower, fruit). The detail of the discussion will vary with the age of your child — here’s a post to get you started: Exploring the Parts of a Flower

Math Project: Measuring Plant Growth Worksheet (FREE printable)

There are many factors involved in plant growth — a few key items that will help kids to understand the idea of botany. 

You can use the FREE plant growth measurement worksheet below to record your plant’s height and growing conditions.

Be sure your kids understand that different plants will grow at different rates — some will grow quickly and others are slower to grow.  Some plants (such as sunflowers) will also be much taller than others when they reach maturity.  And the amount of water, sunlight, type of soil and air temperature can all impact plant growth.

Here are a few suggestions for plants that will grow quickly & quite long (so they’re fun to measure):

Sunflower Seeds “Crazy Mixture” (10+ Varieties)

Cucumber Seeds – perfect for salads

Large Leaf Italian Sweet Basil

Sugar Sweetie Cherry Tomatoes

When you first start your seeds or plant your seedling, check the seed packet or plant tab.  These will tell you approximately how tall or long your plant will grow and will also give you the approximate number of days to maturity.

Engineering & Technology Activities in the Garden

It’s actually easier than you think to integrate these two STEM areas with plants!

• For engineering, you’ll probably need to create something to ‘hold up’ your plant as it grows.  This idea works best with plants such as green beans, cucumbers, tomatoes and sunflowers (or other tall plants). Older kids might even ‘engineer’ a watering system — something that catches rainwater for use in the garden.
• And for technology, use your phone or camera to snap a photo of your plant each week!  It’s a great way to track plant growth and if you stand in the same spot to take the picture every week, you can line up the photos to see how quickly your plant is growing.
• It can also be lots of fun to take some pictures of your kids standing next to their plant every few weeks especially if you are growing sunflowers or vine plants such as beans, pumpkins or cucumbers. 

You might also be inspired by some of the STEM activities we used in our fairy garden last year too!

Art in the Garden

And if you’d like to include some art for a set of STEAM activities, try having your kids make this beautiful & easy rainbow garden pots !

Use this free printable to have the kids measure your plant each week during spring & summer .  

More Plant & Garden Activities:

30+ Gardening Activities for Kids

Great Books about Gardens

And be sure to see all the garden & science activities at the Storybook Science series this month too!

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Plant Growth in Soils – Science Experiment

Updated:  12 Oct 2023

Explore how plants grow in different types of soil with this science experiment perfect for elementary school science lessons.

Editable:  Google Slides

Non-Editable:  PDF

Pages:  10 Pages

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Looking for a Plant Growth Experiment?

If you’ve been studying soil types with your students, they’ll likely already know that different soil types possess different properties (color, texture, particle size, ability to retain water). If you’re looking for a way to demonstrate the real-world effects of these properties, then look no further than this hands-on experiment exploring how plant growth is affected by soil type.

This soil science experiment has been designed by our experienced team of teachers to transform your learners from students into scientists! The students will compare and contrast the growth of plants in a variety of soil types.

Throughout the investigation, the students will:

• Prepare and germinate seeds.
• Transfer the germinated seeds into a pot containing a certain soil type.
• Measure and record the growth of the sprouted seeds over a period of time.
• Compare results with their classmates.
• Draw conclusions about how soil type affects plant growth.

You’ll find everything you need to implement this soil science experiment in the comprehensive student workbook. The workbook contains the following:

• Teacher instructions
• Student instructions
• Experiment planning template
• Procedure text template
• Observations templates
• Growth chart template
• Final observations template
• Results and conclusions template

This plant growth science experiment downloads as a print-and-go PDF or an editable Google Slides file. 

This student-centered science experiment would make the perfect activity to accompany any unit of work on soil types and properties!

Make Sure It’s a Fair Test!

One of the cornerstones of any scientific inquiry is fair testing. A test (experiment) is fair when only one thing causes a change (in this case, the soil type). A fair test can be created by:

• Changing only one thing.
• Measuring something.
• Keeping everything else the same.

This plant growth experiment provides an ideal opportunity to reinforce the principles of fair testing with your students. Our What Is a Fair Test PowerPoint is a great resource to support these teachings!

Download This Plant Growth Experiment

Use the dropdown menu on the Download button above to access the PDF or editable Google Slides file. (Note: You will be prompted to make a copy of the Google Slides template before accessing it).

For sustainability purposes, please consider printing this workbook double-sided.

This resource was created by Caitlyn Phillips, a Teach Starter collaborator.

More Teacher-Made Science Experiments

Looking for more science experiments to excite your learners? Explore this great selection of teacher-created resources!

teaching resource

Making a mini water cycle - experiment.

Investigate the water cycle with this hands-on experiment.

Does the Sun Transfer Heat? – Science Experiment

Discover how the sun transfers thermal energy and which objects absorb more heat with this science experiment for kids.

Water Retention in Soils – Science Experiment

Explore the water retention capabilities of sand, silt, loam and clay with this science experiment for kids.

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How does the amount of water affect plant growth?

Introduction: (initial observation).

Plants are living things. They live in places all around the world. Plants can grow in deserts, rain forests, and even in your own backyard. But no matter where plants grow they all need soil, water, air, and sunshine. A plant’s needs change as it grows. Plants need a lot of water during early growth, flowering and fruit set.

In this project we will try to see how does the amount of water affect the plant growth.

This project guide contains information that you need in order to start your project. If you have any questions or need more support about this project, click on the “Ask Question” button on the top of this page to send me a message.

If you are new in doing science project, click on “How to Start” in the main page. There you will find helpful links that describe different types of science projects, scientific method, variables, hypothesis, graph, abstract and all other general basics that you need to know.

Project advisor

Information Gathering:

Find out about nutrients for plants. Read books, magazines or ask professionals who might know in order to learn about the effect or area of study. Keep track of where you got your information from.

This is a sample of the information you may find:

Indoor Plants – Watering

The main cause of death of potted plants is over-watering. Roots need both water and oxygen, and when surrounded by water, they cannot take up oxygen. These roots may rot and eventually the whole plant may die. The symptoms of over-watering and underwatering are similar. Both lead to poor root health, root decline and possibly death of the plant.

A common question from gardeners is “How often should I water my plants?” There is no pat answer to this question. The amount and frequency of watering depends on many factors, such as the plant species, its growth stage, its location, the type and size of its pot, soil mix characteristics and variable weather conditions.

There is a wide range of watering requirements for different species of plants. Plants with large or very thin leaves and those with fine surface roots usually require more frequent watering than succulent plants with fleshy leaves and stems that are able to store water. Some plants thrive under moist conditions while other plants grow well when kept drier.

Plants may slow in growth after a flush of new growth or a heavy flowering. During these periods and while it is dormant, a plant will need less water.

Water evaporates rapidly from the sides of a porous clay pot, which requires more frequent watering than nonporous, glazed or plastic pots. A large plant in a small pot needs water more often than a small plant in a large pot.

Different soil mixes require different watering schedules. Heavy, fine-textured potting media and those that contain a lot of peat moss hold more moisture than loose, porous mixtures of bark, sand and perlite.

A plant in a warm, dry, sunny location needs more frequent watering than one in a cool, low-light environment.

The rule-of-thumb is to water when necessary. The following methods may be used to determine when to water:

• Touch the soil – The most accurate gauge is to water when the potting mixture feels dry to the touch. Stick your finger into the mix up to the first joint; if it is dry at your fingertip it needs water.
• Tap the pot – When the potting mix in a clay pot begins to dry, it shrinks away from the sides of the pot. Rap the side of the pot with the knuckles or a stick. If the sound is dull, the soil is moist; if the sound is hollow, water is needed.
• Estimate weight – As potting mixtures become dry, a definite loss in weight can be observed.
• Judge soil color – Potting mixtures will change from a dark to lighter color as they dry.

There are a number of watering meters available to measure moisture in the soil, indicating whether water is needed. These products vary widely in accuracy. The readings can be influenced by factors other than soil moisture content. Fertilizer and soil type can affect the reading.

When watering is required, water thoroughly. Apply water until it runs out of the bottom of the pot. This washes out the excess salts, and it guarantees that the bottom two-thirds of the pot, which contains most of the roots, receives sufficient water. Don’t let the pot sit in the water that runs out. Empty the saucer.

Do not allow the soil to become excessively dry. If the salt level in the container is high, root damage may occur. If soil does become very dry and hard to rewet, use the double watering method. Water once and then again half an hour later; or place the pot in a sink or a bucket of water. Remove the pot when the soil surface is moist. Allow the pot to drain completely. If peat is allowed to dry completely, not only is it difficult to rewet, it also will not hold as much water as it could hold before it dried.

Do not water with hot or cold water. The water temperature should be between 62 and 72 °F.

Do not water plants with softened water because sodium and chloride will also be added to the soil mix, possibly causing plant damage.

Although wilting is often an indication of the need to water, it is not always so. Any injury to the root system decreases a plant’s ability to take up water, including root rot, which is caused by too much water. This inability to take up water will cause wilting, and under these conditions, watering may make the problem worse.

Why Do We Fertilize Plants?

For centuries plants grew without any help from human beings, and many are doing so today. Thus, it is obvious that they can do so by themselves, especially in environments to which they are adapted. However, as humans cultivated plants it was learned that the addition of certain materials to the soil sometimes caused plants to respond with characteristics which were considered to be desirable (e.g., more fruit, faster growth, better color, more attractive flowers). Early in recorded history we find accounts of applications of animal manures, wood ashes, and lime to enhance plant performance. Thus was born the practice of fertilization and soil amendment.

We should note here that the plant responses we get from applying fertilizer and other soil amendments are not inherently “good” or “bad.” These terms are subjective and reflect personal judgment as to what is “desirable.” For example, a greater quantity of fruit which is too small for market is not a characteristic desired by a peach farmer. Faster growth is usually not a desirable effect for someone growing bonsai plants. Rank vegetative growth is not desirable in an already-lush lawn nor are profusely-blooming squash plants that are not setting fruit. Thus, a “good” response to fertilization under one set of circumstances may be a “bad” response under another set. It depends on what response the person wants from the plant.

So, why do we apply fertilizer to the soil? Because we want to obtain some desired plant response. We want our plants to “do better.” As we set out to fertilize our plants we should keep in mind how we want them to do better (grow faster, produce better flowers or fruit, etc.) – and we should also know if fertilization will contribute to that improvement.

When Should I Apply Fertilizer?

Stated simply, you need to fertilize whenever you expect to get a desired plant response. However, the difficulty is in predicting. You usually want to know in advance if there will be a response to added fertilizer so that you can avoid growing plants under nutrient-deficient conditions. Since predicting plant responses is difficult, many people apply fertilizer as insurance against nutrient deficiencies. The result: over fertilization in the United States is now as prevalent a problem as under fertilization.

A suggested approach to fertilization involves the following steps:

• Recognize what plant response you are seeking;
• Determine from observation or consultation if fertilizer application is likely to give you the response you want;
• Apply fertilizer only if your desired response is likely;
• Apply only the amount of fertilizer necessary to give the desired response.

What Nutrients Do My Plants Need?

The best way of knowing what your plants need is by observing plant performance and understanding the multiple factors affecting such performance (e.g., light, water, temperature, pests, nutrition).

There is no magical way of knowing which nutrient may be in limited supply in the soil. Soil testing helps predict the need for some of the nutrients, but testing is only one of the tools in plant nutrient management. If you recycle organic matter such as grass clippings (don’t use a bag on your mower) and leaves, you will be returning to the soil the nutrients those plants had absorbed. It is the easiest, least expensive, and most environmentally sound way to fertilize. You may still have to supplement, but you will then apply fewer nutrients–and a lot less fertilizer. Plants need 18 elements for proper growth and reproduction. Under many conditions, plants obtain enough of these elements from the soil, water, and air. It is only in certain environments and growing conditions that one or more of the nutrients are deficient.

The most-commonly applied nutrients are nitrogen (N), phosphorus (P), and potassium (K). Responses to all three elements were fairly widespread in the past, and it became customary to apply the three together. As a result of habit, all three are still applied even though there are now many situations, especially in gardens and landscapes, where plants do not respond to one or more of these fertilizer nutrients.

Other plant-essential nutrients used in fairly large quantities are calcium (Ca), magnesium (Mg), and sulfur (S). However, fertilization with these nutrients is not usually necessary because the Ca and Mg contents of soil are generally sufficient for most plant species. Also, large quantities of Ca and Mg are supplied when acidic soil is limed with dolomite. Sulfur is usually present in sufficient quantities from the slow decomposition of soil organic matter, an important reason for not throwing out grass clippings and leaves.

Micronutrients are those elements essential for plant growth which are needed in only very small (micro) quantities . These elements are sometimes called minor elements or trace elements, but use of the term micronutrient is encouraged by the American Society of Agronomy and the Soil Science Society of America. The micronutrients are iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), boron (B), molybdenum (Mo), Cobalt (Co), Nickel (Ni), and chlorine (Cl). If one of your plant species has a micronutrient deficiency, apply the recommended rate of the deficient nutrient. Recycling organic matter such as grass clippings and tree leaves is an excellent means of providing micronutrients (as well as macronutrients) to growing plants.

What About Organic Matter as a Source of Nutrients?

Organic matter (such as grass clippings, tree leaves, shrubbery and tree trimmings) is an excellent source of plant nutrients. The plants which produced that organic material accumulated all the essential nutrients for their own growth needs. Upon decomposition, those nutrients in the organic material become available for reuse. When you recycle “homegrown” organic matter such as grass clippings, leaves, and shrubbery trimmings you are practicing an excellent method of fertilizing your landscape. You are keeping valuable materials on site and are also greatly reducing the municipal solid wastes placed at curb side. Other organic materials, such as animal manures, biosolids (processed sewage sludge), or various composted materials, are also alternative sources of plant nutrients.

Question/ Purpose:

The purpose of this project is to determine how does the amount of water affect the plant growth (i.e. plant height, stem volume).

Identify Variables:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

Independent variable (also known as the manipulated variable is the amount of water used to grow the plant.

Dependent variable (also known as responding variable) is the plant growth (plant height).

Controlled variables are light and temperature. We grow all plants under the same temperature and light conditions.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis. This is a sample hypothesis.

My hypothesis is that more water will result more growth.

Experiment Design:

In order to test how does the amount of water affect the plant growth, we plant some seeds and use them to test the effect of water.

Experiment 1:

Materials: radish seeds (one packages will be enough for multiple experiments), water, paper towels, plastic beverage cups, aluminum foil, liquid fertilizer (plant food purchased from the grocery or home supply store)

• Soak the radish seeds in water for about an hour.
• Fold a paper towel lengthwise and float it in a shallow pan of water. Remove it and gently wring out the excess water.
• Get 6 soaked radish seeds
• Lay the soaked seeds along the folded edge of the moist paper towel. Roll the paper with the seeds into a cylinder, as in the diagram.
• Repeat with the above steps ten times until you have ten rolled paper towel with 6 seeds in each of them.
• Place the rolled paper cylinders in separate plastic beverage cups
• Label one of the cups as a control and label the others with numbers from 1 to 9 . One represents the least amount of water and 9 represents the highest amount of water.
• Add the necessary nutrients to one gallon of water that will be used for our experiment.
• Each day add 10 drops of water to cup number 1, 20 drops of water to cup number 2, 30 drops of water to cup number 3 and continue with the same increment so the cup number 9 will get 90 drops of water each day. DON’T WATER THE CONTROL CUP.
• Place a piece of aluminum foil loosely over all four cups and allow the cups to remain undisturbed until the seeds germinate (2 to 4 days)
• Once the seeds have germinated, remove the foil and place the cups in a location that provides them with light.
• Measure the roots and the shoots of the growing plants and chart the growth of their seedlings every day or two.
• Describe the effect of water amount on the growth of the seedlings.

Above description is for testing 9 different amounts of water. If you want to test more or less samples, you can modify the experiment as needed.

The reason that we place 6 seeds in each paper towel is to see the average result, not the result of an accidentally large seed.

Experiment 2:

1) Plant 6 bean seeds in 6 small pots filled with potting soil. Place the seeds at the same level in soil for all pots. Place the pots by a sunny window.

2) Label each pot with numbers from 1 to 6

3) Water the pots every day. Each time the pot number 1 will get the least amount of water and the pot number 6 will get the most.

4) Make daily observations and record the height of you plant every day for two to three weeks.

If you need a control, get an additional small pot in which you plant a bean seed, but you don’t water it at all.

Experiment 3:

How does excess amount of water affect plants?

3 plants*, 3 dishes or pans, water, crayons, graph paper

* make sure you choose plants that are the same kind of plant, as close to the same size as possible and healthy

1. Label the plants A, B, and C. Make holes in the bottom of the containers of plants A and B. If your containers already have holes, plug up the holes in the bottom of container C.

2. Place each plant in a dish and put all three plants in the same location where they will receive the same light.

3. Water plant A every other day, keeping the soil moist but not wet. Do not water plant B. Water plant C every day, keeping the soil saturated, very wet.

4. Make a bar graph showing the color of each plants leaves every day for a week.

Think About This:

1. What happened to the color of plant A? plant B? plant C?

2. Does it matter how much water a plant gets?

3. Is drainage important?

Materials and Equipment:

• Radish seeds (one packages will be enough for multiple experiments)
• Paper towels
• Plastic beverage cups
• Aluminum foil
• Samples of liquid fertilizers (plant food purchased from the grocery or home supply store)
• Pots for experiment 2
• Bean seeds for experiment 2

Results of Experiment (Observation):

Record the results of your experiment 1 in tables like this:

This table shows the average height of seedlings in each cup in different days starting day 4.

Questions :

1) Why did you need 10 cups to perform this experiment? What conclusion could you draw if you had performed the experiment with only one cup?

2) What effect did the amount of water have on the plant growth?

If you are performing experiment 2, make a similar table for the results of experiment 2.

Calculations:

You may need to calculate the average heights of seedlings in each paper towel.

Summery of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

Find and review some books about plants, nutrients and fertilizers.

http://collaboratory.nunet.net/timber/scifair/kindto4/8.htm

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AI shows how field crops develop: Software can simulate future growth based on a single initial image

by Johannes Seiler, University of Bonn

Researchers at the University of Bonn have developed software that can simulate the growth of field crops. To do this, they fed thousands of photos from field experiments into a learning algorithm. This enabled the algorithm to learn how to visualize the future development of cultivated plants based on a single initial image. Using the images created during this process, parameters such as leaf area or yield can be estimated accurately.

Which plants should I combine in what ratio to achieve the greatest possible yield? And how will my crop develop if I use manure instead of artificial fertilizers? In the future, farmers should increasingly be able to count on computer support when answering such questions.

The researchers have now taken a crucial step forward on the path towards this goal. "We have developed software that uses drone photos to visualize the future development of the plants shown," explains Lukas Drees from the Institute of Geodesy and Geoinformation at the University of Bonn. The early career researcher is an employee in the PhenoRob Cluster of Excellence.

The large-scale project based at the University of Bonn intends to drive forward the intelligent digitalization of agriculture to help farming become more environmentally friendly, without causing harvest yields to suffer. The findings are published in the journal Plant Methods .

A virtual glimpse into the future to aid decision-making

The computer program now presented by Drees and his colleagues is an important building block. It should eventually make it possible to simulate certain decisions virtually—for instance, to assess how the use of pesticides or fertilizers will affect crop yield.

For this to work, the program must be fed with drone photos from field experiments. "We took thousands of images over one growth period," explains the doctoral researcher. "In this way, for example, we documented the development of cauliflower crops under certain conditions."

The researchers then trained a learning algorithm using these images. Afterwards, based on a single aerial image of an early stage of growth, this algorithm was able to generate images showing the future development of the crop in a new, artificially created image.

The whole process is very accurate as long as the crop conditions are similar to those present when the training photos were taken. Consequently, the software does not take into account the effect of a sudden cold snap or steady rain lasting several days. However, it should learn in the future how growth is affected by influences such as these—as well as an increased use of fertilizers, for example. This should enable it to predict the outcome of certain interventions by the farmer.

"In addition, we used a second AI software that can estimate various parameters from plant photos, such as crop yield," says Drees. "This also works with the generated images. It is thus possible to estimate quite precisely the subsequent size of the cauliflower heads at a very early stage in the growth period."

Focus on polycultures

One area the researchers are focusing on is the use of polycultures. This refers to the sowing of different species in one field—such as beans and wheat. As plants have different requirements, they compete less with each other in a polyculture of this kind compared to a monoculture, where just one species is grown. This boosts yield. In addition, some species—beans are a good example of this—can bind nitrogen from the air and use it as a natural fertilizer. The other species, in this case wheat, also benefits from this.

"Polycultures are also less susceptible to pests and other environmental influences ," explains Drees. "However, how well the whole thing works very much depends on the combined species and their mixing ratio."

When results from many different mixing experiments are fed into learning algorithms, it is possible to derive recommendations as to which plants are particularly compatible and in what ratio.

Plant growth simulations on the basis of learning algorithms are a relatively new development. Process-based models have mostly been used for this purpose up to now. These—metaphorically speaking—have a fundamental understanding of what nutrients and environmental conditions certain plants need during their growth in order to thrive.

"Our software, however, makes its statements solely based on the experience they have collected using the training images," stresses Drees.

Both approaches complement each other. If they were to be combined in an appropriate manner, it could significantly improve the quality of the forecasts. "This is also a point that we are investigating in our study," says the doctoral researcher. "How can we use process- and image-based methods so they benefit from each other in the best possible way?"

Provided by University of Bonn

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• Open access
• Published: 31 May 2024

A comprehensive review of in planta stable transformation strategies

• Jérôme Gélinas Bélanger 1 , 2 ,
• Tanya Rose Copley 1 ,
• Valerio Hoyos-Villegas 2 ,
• Jean-Benoit Charron 2 &
• Louise O’Donoughue 1  

Plant Methods volume  20 , Article number:  79 ( 2024 ) Cite this article

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Metrics details

Plant transformation remains a major bottleneck to the improvement of plant science, both on fundamental and practical levels. The recalcitrant nature of most commercial and minor crops to genetic transformation slows scientific progress for a large range of crops that are essential for food security on a global scale. Over the years, novel stable transformation strategies loosely grouped under the term “in planta” have been proposed and validated in a large number of model (e.g. Arabidopsis and rice), major (e.g. wheat and soybean) and minor (e.g. chickpea and lablab bean) species. The in planta approach is revolutionary as it is considered genotype-independent, technically simple (i.e. devoid of or with minimal tissue culture steps), affordable, and easy to implement in a broad range of experimental settings. In this article, we reviewed and categorized over 300 research articles, patents, theses, and videos demonstrating the applicability of different in planta transformation strategies in 105 different genera across 139 plant species. To support this review process, we propose a classification system for the in planta techniques based on five categories and a new nomenclature for more than 30 different in planta techniques. In complement to this, we clarified some grey areas regarding the in planta conceptual framework and provided insights regarding the past, current, and future scientific impacts of these techniques. To support the diffusion of this concept across the community, this review article will serve as an introductory point for an online compendium about in planta transformation strategies that will be available to all scientists. By expanding our knowledge about in planta transformation, we can find innovative approaches to unlock the full potential of plants, support the growth of scientific knowledge, and stimulate an equitable development of plant research in all countries and institutions.

Introduction

Although it has been more than 40 years since the first publications concerning transgenic plants, plant transformation remains a major bottleneck in most commercially important and underutilized crops [ 1 ]. The recalcitrant nature of many plant species and genotypes to in vitro regeneration is a significant barrier to plant improvement, thus slowing scientific progress and contributing to an overreliance on the same species and genotypes that are more easily amenable to transformation. However, several transformation strategies devoid of or with minimal tissue culture steps have been developed over the years. Altogether these methods offer a promising alternative to the laborious tissue culture steps associated with in vitro techniques. Such transformation strategies are loosely termed “in planta” and have been proven efficient in a breadth of monocot and dicot species. Generally, most in planta methods are also often considered genotype-independent since they do not rely heavily on hormone supplementation and often omit the callus regeneration step. As such, in planta strategies are less prone to somaclonal variations and offer an alternative to circumvent the challenges associated with these long-lasting genetic changes. The simple and affordable nature of these protocols in comparison to in vitro methods makes them particularly suited for minor crops. This feature can allow labs to manage simultaneous genetic transformation projects using various species, genotypes, and constructs with minimal financial requirements and trained personnel. On a global level, these aspects can guarantee an equitable development of plant research in all countries, institutions, and budgets. Moreover, the negligible financial inputs required by labs to undergo in planta projects signifies that riskier projects can be undertaken.

To this day, the only in planta method that has received widespread attention is the Arabidopsis thaliana floral dip method. The floral dip method is one of the most cited protocols in plant molecular biology and is one of the main factors that has contributed to propelling Arabidopsis to the honorable status of “most important model organism in plant biology” [ 2 , 3 , 4 ]. As a whole, the success of this technique clearly depicts the potential of development for universal in planta methods, particularly in the era of CRISPR-Cas9 and high-throughput genome editing. Over the years, several review papers have been written on the topic of in planta transformation, thus demonstrating the importance of the concept [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. Largely, papers focused on specific in planta methods, such as the floral dip and the shoot apical meristem (SAM) injury techniques, and do not include the most recent scientific developments in an area that is rapidly evolving. This article aims at complementing these past literature reviews and framing them into the bigger context of in planta transformation as a topic. Overall, we start this review by drawing the conceptual framework of in planta stable transformation and classifying the different in planta strategies. Subsequently, we describe several in planta experimental approaches with a focus on recent advances and finally discuss the future avenues and possibilities in this field of research.

Approaches for data collection and building of the in planta compendium

For the collection of data required to build our in planta transformation compendium, a systematic review was conducted using Google Scholar and Scopus search engines to identify the bulk of research articles. Following the use of these tools, we complemented the compendium using articles initially found on ResearchGate and several other online web references such as EuropePMC. Due to the large number of research articles available for specific techniques (e.g. floral dip and pollen-tube pathway), we focused on identifying research articles that demonstrate the efficiency of these approaches in understudied plant species (i.e. all plants that are not considered commercial or model crops) to improve our global understanding of the applicability of these transformation strategies. On the whole, we manually curated, annotated, and reviewed 323 references (research articles, thesis, patents, etc.) tackling the topic of in planta transformation using this classification scheme (Table S1 ). In total, this compendium includes a total of 139 different species, 105 genera, and a broad range of techniques for each type of explant (Fig.  1 ; Table S1 ). All of the sections referring to specific in planta transformation techniques de facto refer to this compendium to limit the number of in-text references. For visualization, ggplot2 package version 3.3.5 with R version 4.0.4 [ 12 ] was used to build Fig.  1 , whereas Figs.  2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 were created with www.BioRender.com .

Distribution of the publications found in the in planta compendium. This graph shows the distribution of the publications associated with each type of explant

Classification of the four de novo organogenesis pathways. Regeneration-dependent de novo organogenesis strategies can be performed under in vivo (in planta) or in vitro (not in planta) conditions. The direct regeneration mechanism has many advantages over the indirect mechanism as it is simpler and quicker to perform; however, it leads to the formation of chimeric T 0 mutants that require segregation in the T 1 generation to obtain non-chimeric offspring. Moreover, the direct regeneration mechanism does not suffer from somaclonal variation, unlike callus-based methods. Callus-based methods are generally more challenging to perform but can be useful for specific crops (e.g. plants with a long juvenile phase such as trees) that cannot be transformed efficiently using the direct regeneration mechanism. The in vitro indirect regeneration pathway is generally considered highly genotype-dependent due to the use of multiple growing media, whereas direct regeneration methods are more universal due to their use of simple cultivation medium that are suitable for a larger spectrum of genotypes. The classification of these pathways was inspired by the comparative scheme of bud regeneration avenues developed by Shi et al. [ 54 ]

Gamete-based transformation techniques. ( A ) Strategies targeting the female gamete (ovule). Several in planta techniques (e.g. the floral dip [ 59 ], vacuum-infiltration [ 60 ], floral spray [ 76 ], and floral painting [ 67 ]) targeting the female gametes have been developed and validated. In Arabidopsis , in planta strategies targeting the ovules often lead to the generation of hemizygous offspring in the T 1 generation as the male reproductive organs (i.e. pollen and pollen tubes) remain untouched [ 137 , 243 ]. A thorough screening must be performed in the T 1 generation and further to identify positive mutants using a selection marker or reporter gene [ 65 , 66 ]. ( B ) Male gametes-based in planta approaches. In these strategies, the pollen grains are transformed through various methods such as sonication [ 83 ], vacuum infiltration [ 82 ], magnetofection [ 85 , 86 ], Agrobacterium [ 82 , 84 ], particle bombardment [ 80 , 81 ], and electroporation [ 79 ]. Subsequently, these pollen grains are used to pollinate the recipient plant’s ovules and lead to the generation of putatively transformed T 1 offspring. Following this, screening is performed in the T 1 generation to identify positive transformants

Definition of in planta stable transformation

The act of generating stable plant transformants is a combination of two indissociable and interdependent steps: (i) the transformation of a plant cell; and (ii) the development of this cell into a whole plant [ 13 ]. In planta stable transformation, also called in situ transformation, techniques form a heterogeneous group of methods all aiming at performing the direct and stable integration of foreign T-DNA into a plant’s genome and regenerating the transformed cells into whole plants [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. Unlike in planta transient transformation strategies, such as agroinfiltration, in planta stable transformation aims at generating heritable modifications using exogenous genetic material. In opposition to in planta strategies, in vitro indirect transformation/regeneration techniques, often called conventional transformation/regeneration methods, aim at regenerating an explant that produces a callus (i.e. a more or less developed unorganized plant structure made of parenchyma cells) under strictly sterile conditions [ 8 ].

Historically, the most common definitions of in planta transformation have been (i) a means of transformation without tissue culture step [ 5 , 7 ] and (ii) a means of transformation of intact plants or plant tissues without callus culture or regeneration [ 14 , 15 ]. In our opinion, these definitions are incomplete and not nuanced enough to take into account the broad diversity of available in planta methods. The challenging aspect of most in vitro indirect transformation/regeneration techniques stems from the combination of the hard-to-maintain micropropagation conditions and the callus regeneration step, more than the singular features of each aspect taken alone. As such, multiple highly efficient in planta research articles performing callus regeneration under in vivo conditions have been published over the years [ 16 , 17 , 18 , 19 , 20 ]. Similarly, several effective in planta protocols using minimal in vitro steps have also been published [ 21 , 22 , 23 , 24 , 25 ]. Conceptually speaking, these published methods all fall within the scope of in planta transformation and were self-described as in planta by their authors; however, their methods do not strictly follow the definitions mentioned above. Furthermore, several articles with major in vitro (e.g [ 26 , 27 , 28 ]) components have been published and were also self-described as in planta by their authors. These contrasting definitions underline the grey zone concerning the use of micropropagation within the realm of in planta transformation. For the purposes of this article, we redefined the in planta concept as the following: a means of plant genetic transformation with no or minimal tissue culture steps. To be considered minimal, the tissue culture steps should meet the following pivotal criteria: (i) short duration with a limited number of medium transfers; (ii) high technical simplicity (i.e. simple medium composition with a limited list of hormones); and (iii) regeneration using a differentiated explant that does not undergo a callus development stage and thus relies on direct regeneration.

Classification of in planta transformation methods

In contrast to conventional transformation methods, in planta strategies are extremely heterogeneous in their modes of action and types of organ targeted. At present, there are hundreds of in planta protocols available in the literature. The classification of these protocols into a structured system is challenging due to numerous factors, including: (i) heterogeneous mode of action; (ii) skewed distribution of the publications between the methods (i.e. some methods have dozens of publications, while others have only one or a few); (iii) specific methods that have been reviewed thoroughly in the past while others are nearly absent from the literature; and (iv) the scientific pertinence/novelty versus the number of publications that are often uncorrelated.

This article has been written with the intent of finding a balance between all of these aspects, with an emphasis on techniques not thoroughly reviewed in the past. A large number of techniques presented in this paper were named/renamed by ourselves to distinguish them from similar techniques. As such, the names found in this paper might differ in other references. To build this review paper, we classified the references based on their explant of choice using the following nomenclature: (i) germline [female (ovule) and male (pollen) gametes]; (ii) embryo (aka zygotes); (iii) shoot apical meristem and adventitious meristems; (iv) vegetative tissues; and (v) novel systems (Table S2 ).

Germline transformation techniques are regeneration-independent strategies that target the haploid female (egg) or male (sperm) gametophytic cells before their fusion and the subsequent generation of a diploid zygote [ 29 , 30 ]. Germline-based transformation techniques can be divided into two categories based on the nature of the targeted sexual organ: (i) ovule (female organ) and (ii) pollen (male organ).

Plant zygotes are progenitor stem cells generated from the fusion of two haploid gametes, the egg and the sperm cells, from which all of the embryonic and post-embryonic organs are generated [ 31 ]. The zygote is divided into two parts, a small apical and a large basal cells [ 32 ]. Through the development of the embryo, the small apical part will give rise to the shoot meristem [ 32 ]. In this paper, the embryo section includes all the methods performed at the post-pollination stage until the emergence of the shoot apical meristem from the seed upon germination.

The shoot and root meristems are highly organized structures composed of proliferating embryonic-type cells involved in the continuous generation of aerial and underground plant organs through mitosis [ 33 ]. A portion of the stem cells present in these meristems are activated upon germination to produce primordia of lateral organs, while a pluripotent undifferentiated population is maintained at its center to ensure self-renewal and integrity [ 33 ]. Unlike the floral meristem, the stem cell features of the shoot apical meristem are maintained throughout the whole life cycle of the plant [ 34 ]. The protocols included in the pre-formed meristem sections include those that target different types of meristems (apical, axillary, or adventitious) upon their emergence from the seed until their senescence.

Callus refers to the accumulation of disorganized cell masses generally associated with the wounding of vegetative tissues [ 35 ]. These pluripotent cell masses either form roots or shoots through cellular reprogramming upon inductive cues (e.g. presence of light) [ 36 ]. Monocots and dicots have important biological differences that influence their respective abilities to form new meristems from a pluripotent callus mass [ 37 , 38 ]. In dicots, most anatomical organs display the ability to generate calluses during the whole life of the plant, whereas monocots do not have a true vascular cambium with the ability to undergo cell rearrangement [ 37 , 38 ]. Callus generation in monocots is limited to the base segment of leaves and the lateral and tip regions of roots [ 37 ]. As such, dicots are much more amenable to in vivo regeneration and propagation (e.g. grafting and cuttings) than monocots [ 37 , 39 ].

Two transformation techniques (i.e. grafting-mediated transformation and transformation using viral-based vectors) have been classified in the “ novel systems ” section because they harbor special features that limit their classification using the four other different types of explants. At present, the scope of these methods remains more limited than all of the other in planta strategies presented here due to specific experimental requirements.

Means of in planta transformation

Plant transformation techniques can be divided into two main gene transfer categories: (i) direct gene transfer; and (ii) indirect gene transfer [ 40 ]. The former transfer strategy aims at introducing naked DNA into a plant genome through chemical or physical means (e.g. biolistics, electroporation, and polyethylene glycol), whereas the latter involves the introduction of DNA using biological vectors (e.g. Agrobacterium spp ., Ochrobactrum haywardense , or viral vectors) [ 40 ]. Agrobacterium tumefaciens -mediated transformation is by far the most used method among the different in planta approaches as it is a simple and cost-effective option that generates few copy numbers in the generated transformants [ 11 ]. In addition, Agrobacterium is effective in a wide range of plant genotypes and species and can be used with various types of in planta strategies, thus making it a robust, reliable, and versatile transformation system [ 11 ]. Agrobacterium rhizogenes is generally used to perform in planta transformation that results in non-heritable changes through the formation of hairy roots in composite plants; however, the recently developed cut-dip-budding [ 41 ] and vine-cutting node inoculation [ 42 ] methods have demonstrated that A. rhizogenes can be used to perform stable transformation in asexually propagated plant species such as sweet potato ( Ipomoea batatas ). Although more marginal in their use, several other methods, such as direct DNA uptake [ 43 ] and biolistics [ 14 , 44 ], are now sometimes used in diverse in planta protocols and are alternatives to Agrobacterium -based methods.

Types of regeneration pathways

In most genetic transformation experiments, the regeneration of a positive somatic mutant cell into a whole plant is the rate-limiting step that is associated with the recalcitrant features of most hard-to-transform species [ 45 ]. In plants, this step can be undertaken using two strategies that are based on totipotency (i.e. a cell’s feature that enables it to dedifferentiate and redifferentiate into different tissues, organs, or whole organisms): (i) somatic embryogenesis; or (ii) de novo organogenesis [ 46 ]. Over the years, fertilization-based transformation techniques based on the transfer of exogenous DNA to male/female haploid gametes (e.g. floral dip) or fertilized diploid zygotes (e.g. pollen-tube pathway) have also been developed and are considered regeneration-independent [ 47 ]. In general, regeneration-independent techniques are often considered more efficient than their dependent counterparts due to their omission of the regeneration step; however, these approaches also have their own set of disadvantages including the generation of hemizygous (i.e. only one copy of a transgene at a given locus in an otherwise diploid cell) individuals when targeting haploid gametes [ 47 ].

Somatic embryogenesis

Somatic embryogenesis is a mechanism in which differentiated cells undergo dedifferentiation to become embryonic stem cells [ 48 , 49 ]. Following this step, embryonic stem cells can differentiate into meristematic cells to become a single and viable plant [ 48 ]. In the literature, the main difference between somatic embryogenesis and indirect de novo organogenesis is the presence of a somatic embryo formation step in the former, whereas the latter undergoes a callus generation step [ 48 ]. As such, both mechanisms require a regeneration step to form a new plant. To our knowledge, somatic embryogenesis, either through the direct or indirect pathways, is not a mechanism used for in planta transformation due to its extensive tissue culture requirements. In consequence, the term regeneration-dependent strategies will refer herein to only methods using a de novo organogenesis mechanism.

De novo organogenesis

Plant regeneration occurs upon cell wounding and aims at repairing or replacing the damaged anatomical structures using totipotency and pluripotency, which will lead to the subsequent generation of adventitious organs [ 48 ]. Adventitious organs are defined as either root or shoot meristematic buds that arise from growing areas that typically do not contain such organs [ 50 ]. In the literature, no specific terms distinguish the adventitious organs which are obtained either from indirect or direct de novo organogenesis [ 48 ]. In addition, indirect and direct shoot regeneration events often occur simultaneously upon wounding [ 16 ], a phenomenon that can generate some confusion between the mechanisms in the literature. However, the distinction between both types of adventitious shoot formation pathways is important due to major differences in their underlying biological mechanisms and impacts on the transformation event. For instance, direct regeneration strategies, both under in vivo and in vitro conditions, can instigate a varying degree of chimerism in the transformants, thereby creating heterogenomic mutants that will require subsequent segregation to recover non-chimeric plants [ 51 , 52 ]. In plants obtained with indirect organogenesis, chimerism is less concerning because single-cell regeneration can be undergone using a selection marker (e.g. antibiotics or herbicides), but somaclonal variations are typically more prevalent [ 53 ].

Overall, techniques using a de novo organogenesis approach can be classified based on their use of tissue culture (i.e. in vivo/tissue culture-independent vs. in vitro/tissue culture-dependent) and methods of regeneration (i.e. direct regeneration vs. indirect regeneration) [ 54 ] (Fig.  2 ). In general, in planta strategies aim at limiting tissue culture to a minimum and consequently either use in vivo direct regeneration or in vivo indirect regeneration strategies. From a technical standpoint, the in vitro direct regeneration pathway can be considered a crossover between the in vitro direct regeneration and in vivo indirect regeneration concepts as the explants are micropropagated under sterile conditions but regenerated through direct organogenesis. Although not considered in planta per se , the protocols using the in vitro direct regeneration pathway generally have a faster regeneration rate (often between 4 and 8 weeks), lessened use of hormones, higher success rates, greater genotype-independency, and decreased technical skills requirements [ 55 , 56 ]. A short section of this paper will be dedicated to the methods using this pathway since those offer a promising alternative to the in vitro indirect regeneration pathway, particularly in monocots [ 57 , 58 ].

Germline transformation

Floral dip and similar methods (ovule).

The most important contributor to the spread of the in planta conceptual framework is undoubtedly the floral dip method in Arabidopsis [ 59 ]. At its essence, the floral dip method is a simple and reliable method that aims at performing germline transformation through the dipping of developing floral tissues into resuspended Agrobacterium inoculum [ 59 ] (Fig.  3 a). The first iteration of this method was developed by Bechtold and Pelletier [ 60 ] using vacuum-infiltration of the floral organs. Despite its high transformation rates, this protocol was largely supplanted by the protocol proposed by Clough and Bent [ 59 ] which removed the vacuum-infiltration step and replaced it with a simple dip into a solution containing Agrobacterium , sucrose, and a surfactant (i.e. Silwet L-77), thus streamlining the technical aspect of the method and increasing the speed of the procedure. As such, the approach developed by Clough and Bent [ 59 ] is now the mainstay for transforming Arabidopsis , a popularity largely due to its high transformation efficiency as rates between 0.1 and 3% are typical Footnote 1 [ 61 ]. Over the years, other iterations of the technique, such as the floral dip with low inoculum density [ 62 ], vacuum-infiltration of closed floral buds [ 63 ], and simplified floral dip [ 64 ], have been proposed to upgrade specific aspects of the method. Although the floral dip approach is a common technique for plant transformation, two factors still readily limit its development on a broader scale: (i) the generation of hemizygous offspring; and (ii) a narrow range of species amenable to the method.

In planta approaches targeting the embryos at an early stage of development. ( A ) Pollen-tube pathway [ 92 ]. To perform the pollen-tube pathway, the plant’s stigmas are removed and the styles are severed shortly after pollination. Subsequently, exogenous donor DNA is applied to the severed styles and delivered to the recipient plant’s ovaries via the growth of the pollen tube. Following the seed set, the putative transformants are screened to identify positive mutants. ( B ) Ovary-drip [ 92 ]. In this approach, the ovary sac is incised using a sterile scalpel, and exogenous DNA is directly delivered to the ovule drop-by-drop using a micropipette. ( C ) Pollen-tube agroinjection [ 113 ]. In this method, a solution of resuspended Agrobacterium is injected into the plant’s pollen tube using injector needles. To do so, the carina is punctured with the needles and the solution is injected until the wing petals are soaked. ( D ) Ovary injection [ 115 , 116 , 118 ]. To apply the ovary injection strategy, a solution of resuspended Agrobacterium is injected into the ovaries (i.e. soybean pods in this case) at an early stage of development to infect the developing embryos. Following this step, the mature seeds are further screened to identify positive mutants

Hemizygous offspring are generated with the floral dip method since the transformation event happens after the divergence of anther and ovary cell lineages in Arabidopsis [ 47 ]. In Arabidopsis , the stigmatic cap forms over the top of the gynoecium, enclosing the locules 3 days before anthesis [ 47 ]. As a consequence, the primary targets of the floral dip method are the female reproductive organs, the ovules, and embryo sacs, whereas the pollen or pollen tubes remain untouched [ 47 ]. To segregate all hemizygous progenies and recover only offspring with homozygous genotypes, a thorough screening must be performed until the T 3 generation as the progenies from the T 2 generation are not stable [ 65 , 66 ].

Although tremendous research has been pursued on the floral dip method, the number of species amenable to this technique remains modest in comparison to other techniques, such as the shoot apical meristem injury approach. At present, the bulk of the floral dip protocols have been developed for species belonging to the Brassicaceae family, but transformation procedures based on this approach have also been demonstrated to be efficient for 12 other families (e.g. Linaceae and Solanaceae) (Table S1 ). Still, the protocols targeting species belonging to families other than Brassicaceae are sparse and generally less efficient due to lower transformation rates, cumbersome manipulations, and complicated technical requirements (e.g. tomato/ Solanum lycopersicum [ 67 ]). Numerous biological and morphological factors have been suggested to explain the limited expansion of the floral dip technique to other plant species, including physical barriers associated with flower morphology [ 61 ], necrotic reaction to the presence of Agrobacterium causing abortions in the flowers [ 61 ], lower seed set [ 68 ], reduced susceptibility to Agrobacterium [ 68 ], and bigger size of the plant and/or flower structures [ 5 ]. Over the years, modifications to the floral dip method have been developed to increase its efficiency with plant species that are not members of the Brassicaceae, while retaining the core concepts of the strategy. Amongst these innovative strategies are the floral bud injection (tomato, poplar/ Populus sp. , chickpea/ Cicer arietinum and sunflower/ Helianthus annuus ) [ 69 , 70 , 71 , 72 ], floral bud painting (maize/ Zea mays and tomato) [ 67 , 73 ], and floral bud spray ( Arabidopsis , wheat/ Triticum aestivum , and Indian mustard/ Brassica juncea ) strategies [ 74 , 75 , 76 ].

Pollen transformation

In the pollen transformation method, the desired foreign gene is introduced into the pollen grains via Agrobacterium or directly with naked DNA [ 77 ] (Fig.  3 b). Following this step, the transformed pollen grains are subsequently used to pollinate the stigma and fertilize the recipient egg in vivo. Pollen grains are an interesting target for transformation as they can be easily isolated, occur in large numbers, and can be easily transformed [ 77 ]. Pollen grains harbor a coat derived from the anther tapetum (the pollenkitt/tryphine), an outer thick cell wall (the exine), and a thin inner cell wall (the intine), that block the integration of exogenous DNA [ 77 ]. In addition, germinating pollen grains release nucleases that catalyze the cleavage of phosphodiester bonds between nucleotides of nucleic acids [ 78 ]. In combination, the thick wall/coat and release of nucleases limit the use of conventional transformation methods to integrate the transgene into the pollen grain [ 77 , 78 ]. To circumvent this problem, various methods such as electroporation [ 79 ], particle bombardment [ 80 , 81 ], vacuum infiltration [ 82 ], sonication [ 83 ], Agrobacterium [ 82 , 84 ], and magnetofection [ 85 , 86 ] have been used to facilitate the introduction of transgenes into pollen grains or microspores, with varying degrees of success. Several transformation methods based on pollen incorporate a short in vitro period at the beginning of the experiment as in the case of the male germline transformation (MAGELITR) system [ 81 ], which can be a limiting factor for labs without access to micropropagation facilities. Overall, pollen transformation has been demonstrated to be efficient in several species, including tobacco [ 79 , 80 , 81 , 87 ], cotton ( Gossypium hirsutum ) [ 82 ], sorghum ( Sorghum bicolor ) [ 88 ], petunia ( Petunia x hybrida ) [ 89 ], Indian mustard [ 83 ], and maize [ 90 ], but its implementation remains challenging in a large number of species, with contrasting results between different labs (e.g. magnetofection was reported to be inefficient in monocots [ 91 ]).

Pollen-tube pathway

The pollen-tube pathway strategy aims at applying exogenous donor DNA onto the severed style of the recipient plant, which will be transported via the growth of the pollen tube to the ovary [ 92 ] (Fig.  4 a). Reaching the ovary, the foreign DNA will be integrated into the undivided recipient zygote, thus leading to the generation of a transformed embryo [ 92 ]. To improve the rates of transformation, researchers often cut the styles of the recipient plant [ 92 ]. The pollen-tube pathway transfer technique is one of the oldest transformation techniques that has been investigated, with reports dating back to 1983 in cotton [ 93 ] and 1989 in rice ( Oryza sativa ) [ 94 ]. Although beneficial in many aspects (e.g. no regeneration step and fast preparation), this method has also demonstrated some limitations in the past, such as poor transformation efficiency [ 95 , 96 ] and a lack of reproducibility [ 97 , 98 , 99 ], which led to a rise in skepticism regarding some of its claimed benefits (e.g. universal application) [ 92 ]. For instance, Li et al. [ 99 ] have observed many inconsistencies with soybean ( Glycine max ) plants treated with the pollen-tube pathway technique. In their experiments, all the plants exhibiting positive β-glucuronidase (GUS) activity were found to be untransformed when analyzed using polymerase chain reaction (PCR). Similarly, morphological variation was observed in the first generation of some plants, but not in the subsequent generations. As a consequence of these inconsistent results, there has been a disinterest in this transformation system in the Western hemisphere [ 92 ]. In the meantime, China continued to improve the procedure and has now developed broad expertise with this transformation strategy, resulting in a significant proportion of the research articles only being available in Mandarin [ 100 ]. When compiling the research articles for this review, we found that a broad selection of protocols is now available for this strategy with dozens of research articles published for major commercial crops, including cotton [ 101 , 102 , 103 ], maize [ 104 ], rice [ 105 ], and wheat [ 106 ], as well as for at least 24 other species.

In planta strategies targeting the embryos at a later stage of development. ( A ) Infection of pre-imbibed embryos with Agrobacterium . The seeds are imbibed with sterile water and either (i) kept uninjured [ 122 ] or (ii) injured using pricking, sonication, or vacuum infiltration [ 121 ]. Following this treatment, the seeds are infected with a solution of Agrobacterium and grown until the T 1 generation for selection. ( B ) Agro-imbibition [ 124 ]. In this approach, seeds are imbibed with a solution of Agrobacterium instead of sterile water and further selected in the T1 generation. ( C ) Imbibition of desiccated embryos [ 125 ]. To perform this method, seeds are first imbibed with sterile water and subsequently desiccated at room temperature for 9–36 h. The seeds are subsequently infected for 2 h with a solution of Agrobacterium and cultivated until the T 1 generation for selection

The ovary-drip method differs from the pollen-tube pathway as the exogenous DNA (i.e. which is supplied under the form of a minimal linear gene cassette) is directly delivered to the ovule after pollination with the complete removal of the style [ 107 ] (Fig.  4 b). Generally, the ovary-drip method has higher transformation rates than the pollen-tube pathway (e.g. 3.38% transformation frequency with the ovary-drip method vs. 0.86% with the pollen-tube pathway [ 108 ]), but requires careful manipulation to limit the risk of mechanical damage to the ovule [ 92 , 109 ]. This method has been used successfully to transform soybean [ 107 , 110 ] and maize [ 111 , 114 ]. One of the key factors influencing the success rate of this method is the length of the style. Liu et al. [ 112 ] investigated the optimal length of the soybean style and found that the complete removal of the style without ovary wounding generated the highest proportion of transformants, 11%.

Pollen-tube agroinjection

At its core, the pollen-tube agroinjection method combines the principles of the pollen-tube injection pathway with A. tumefaciens -mediated transformation (Fig.  4 c) [ 113 ]. In this method, carinas (i.e. two conjoined lower petals of a legume flower that enclose the stamen and style) of freshly opened flowers (in this case peanut) need to be punctured using injector needles and injected with 0.1 mL of resuspended Agrobacterium solution. The method was used to generate transgenic peanut lines encoding the peanut BAX INHIBITOR-1  gene with an overall transformation rate of 50%. To the best of our knowledge, only one research article using this approach has been published, but the high transformation rates suggest that it might be an efficient alternative to the conventional pollen-tube pathway technique.

Ovary injection transformation

The ovary injection method aims at injecting Agrobacterium directly into the locule of a plant’s ovary to reach the embryo using a micro-injector or a syringe after pollination (e.g. cotton [ 114 ]) (Fig.  4 d). This method has been used with success in about ten species, but has been demonstrated to be particularly effective in tomato [ 115 , 116 , 117 ] and, to a minor extent, soybean [ 118 ]. In tomato, Hasan et al. [ 116 ] developed a protocol in which mature and ripe fruits were injected with 1 mL of an Agrobacterium solution containing a GUS reporter and incubated at 28 °C for 48, 72, and 96 h. The highest number of stable transformed plants was obtained with a 48 h incubation period, with 88% being positive for the GUS assay. Using a similar protocol, Yasmeen et al. [ 115 ] obtained transformation rates of 35–42% in tomato depending on the construct. When injecting the Agrobacterium solution at stage I (i.e. 2–3 days after pod formation) in soybean, transformation efficiencies between 6.45 and 14.2% and 28.75–35.48% were respectively obtained using GUS assays on plants and seeds [ 118 ]. To improve the transformation rates of the ovary injection method, a similar method using micro-vibration was developed by Liou [ 119 ]. In this approach, the stigma of the flower is removed and exogenous DNA is injected through the cut-off position and toward the locule inside the ovary. Following this step, a micro-vibration treatment will be performed with an ultra-sonic device to favor the placement of DNA around the ovule and improve integration.

Infection of pre-imbibed embryos with agrobacterium

The infection of pre-imbibed embryos with Agrobacterium is a simple technique in which a seed is injured (e.g. seed pricking, tip cutting, sonication, or puncturation) and then imbibed to facilitate the infection of the embryo by Agrobacterium (Fig.  5 a). This technique was first developed by Graves and Goldman [ 121 ] by pricking four-day-old germinating maize seeds four times in an area extending from the scutellar node through the mesocotyl to infect the cells located in this zone with Agrobacterium . Subsequently, a method for the transformation of soybean was developed using a similar approach [ 121 ]. In the Chee, Fober, and Slightom [ 121 ] protocol, imbibed soybean seeds with one cotyledon removed were pricked at three different points into the plumule, cotyledonary node, and adjacent regions and injected with 30 µL of Agrobacterium culture at each injured point. The observed transformation rates obtained with this method were 0.7% in the R 0 plant and 0.07% in the R 1 generation. Although the rates of transformation were low for both of these protocols, they paved the way to more performing protocols in a large number of species. Following the development of the Graves and Goldman [ 120 ] method in maize, a variant involving the use of uninjured seeds was developed in 1987 using Arabidopsis [ 122 ]. In this protocol developed by Feldmann and David Marks [ 122 ], Arabidopsis seeds were imbibed for 6, 12, or 24 h following a one-step or two-step imbibition protocol, infected with 3 mL of an overnight culture of Agrobacterium and co-cultivated during 24 h before being washed with sterile water. Subsequently, the seeds were sown on vermiculite pre-soaked with a complete nutrient solution. Although the transformation efficiencies were rather low (0.0015-0.3200%), the protocol still demonstrated that it was possible to generate transformants without causing any injuries to the pre-imbibed seeds.

Transformation approaches targeting the apical and adventitious meristems. ( A ) Shoot apical meristem injury under in vivo conditions [ 129 ]. The apical meristematic region is pricked with a needle and subsequently infected with resuspended Agrobacterium . Chimeric T 0 plants are grown under in vivo conditions until seed set. Non-chimeric lines are further selected in the T 1 generation. ( B ) Plumular meristem approach [ 22 , 148 ]. In the plumular meristem approach, young seedlings are decapitated and their radicules excised with a sterile scalpel. Following this treatment, the explants are infected with Agrobacterium and co-cultivated on a sterile medium under in vitro conditions. After co-cultivation, the seedlings are moved to greenhouse conditions and allowed to set seeds. The T 1 offspring are then screened to identify positive mutants

Agro-imbibition

The agro-imbibition technique is a relatively new approach that aims at fully imbibing whole seeds with an Agrobacterium solution to infect them (Fig.  5 b). The method is simple and has a reduced workload; however, seven patents have been deposited for this method, suggesting that a license might be required to use it [ 123 ]. In their recent article, Kharb et al. [ 123 ] detailed the core principles of this genotype-independent in planta strategy. In their protocol, seeds are surface sterilized using a 0.1% HgCl2 solution for 10 min, imbibed in a resuspended culture of Agrobacterium (O.D. = 0.6) with shaking at 100 revolutions per minute (RPM), and then germinated on a simple germination medium containing 250 mg/L cefotaxime or on soil. According to the authors, many species (e.g. chickpea, pigeon pea/ Cajanus cajan , wheat, soybean, and rice) are amenable to this approach, with efficiencies ranging from 14.3% in chickpea up to 93.8% in rice.

Imbibition of desiccated embryos

This approach aims at rehydrating desiccated zygotic embryos with an Agrobacterium solution [ 125 ] (Fig.  5 c). Upon desiccation, several physiological modifications (e.g. bursting of the cell walls) occur which facilitate the integration of DNA in the zygotic embryo [ 126 ]. Consequently, dry cells become permeable to large plasmid DNA molecules and transformation can happen without relying on Agrobacterium [ 126 ]. In addition, cellular permeabilization agents (e.g. toluenes) can be used to improve the proportion of DNA intake [ 127 ]. Arias et al. [ 125 ] developed a protocol in which soybean embryonic axes (i.e. zygotic embryos) were imbibed in an aqueous solution for 18 h and subsequently desiccated at room temperature until reaching a moisture content of 10–25%. After desiccation, the zygotic embryos were imbibed again with an Agrobacterium solution for approximately 2 h at room temperature. Arias et al. [ 125 ] indicated transformation rates between 0 and 80% in T 0 mutants using GUS assays and mentioned that T 3 transformants were generated for the pBPSLM003 and pCAMBIA3301 plasmids with this method, thus indicating that the method can be efficiently used to generate stable transformants. In addition, the method has also been proven to be compatible with Arabidopsis [ 125 ].

Shoot apical and adventitious meristems

Shoot apical meristem injury under in vivo conditions.

The shoot apical meristem is one of the primary targets of in planta transformation, and an extensive literature targeting this organ under in vivo growing conditions is available. All plant species display at least one form of shoot apical meristem [ 128 ], and the transformation of this organ can be performed at almost any stage of a plant’s life, from the seedling to the adult stages [ 8 ]. Together, these two characteristics (i.e. all stages of growth and all plant species) contribute to the universal applicability of the shoot apical meristem injury transformation approach [ 8 , 128 ]. On the whole, the strategies grouped under this approach loosely share four core concepts that are: (i) wounding the apical meristem region using a needle, scalpel, syringe, or another method (e.g. sonication); (ii) infecting the meristem with Agrobacterium ; (iii) growth of the seedlings under in vivo conditions for most of their lifecycle; and (iv) chimeric T 0 generation with selection in the T 0 (rare) or T 1 (standard) generation [ 129 ] (Fig.  6 a). A standardized protocol named apical meristem targeted in planta transformation, which was first validated in safflower ( Carthamus tinctorius ) and peanut respectively by Rohini and Sankara Rao [ 130 ] and Rohini and Sakanra Rao [ 131 ], was proposed as a low-tech efficient transformation method that can be applied to both dicots and monocots. In this standardized method, the differentiating apical meristem region of two-day-old seedlings is injured using a needle and subsequently infected using an Agrobacterium solution supplemented with Winans’ AB minimal and wounded tobacco leaf extract [ 129 , 132 ]. After the infection, the plants are transferred to autoclaved soilrite and allowed to grow for ≈ 1 week under a 16 h photoperiod [ 129 ]. Following this step, the plant is transferred to pots and allowed to set seed. The T 1 offspring of these chimeric plants are subsequently screened using a selectable marker such as antibiotic resistance and/or PCR amplification [ 129 ]. Overall, the transformation efficiencies can be quite high considering the simplicity of the approach. For example, the transformation efficiencies were respectively evaluated to be 5.3% and 1.3% in the cultivars ‘A-1’ and ‘A-300’ using histochemical assays, PCR amplification, and Southern blot analyses in T 0 and T 1 safflower plants [ 130 ]. In peanut, the transformation frequencies were evaluated to be 3.3% based on histochemical assay and by PCR analysis of the GUS gene [ 131 ].

Additional in planta techniques targeting the shoot apical and adventitious meristems. ( A ) Direct organogenesis of propagules (cut-dip-budding technique) [ 41 ]. To perform this method, plants with a high asexual reproduction capacity (e.g. sweet potato) are decapitated and their wounds are treated with a solution of resuspended Agrobacterium rhizogenes . Due to the root-suckering features of these plants, transgenic hairy roots will slowly develop and generate a newly transformed plant. ( B ) Direct organogenesis of propagules (Regenerative activity-dependent in planta injection delivery technique) [ 150 ]. In the RAPID method, a solution of resuspended A. tumefaciens is injected into the stem of plants with a high asexual reproduction capacity such as sweet potato. The plant is subsequently transplanted and transformed roots (pathway #1) or shoots (pathway #2) will subsequently emerge from the wound sites. ( C ) Direct delivery of exogenous morphogenic regulators [ 175 , 244 ]. In the Direct delivery approach, the recipient plants’ meristems are removed using a sterile scalpel, and developmental regulators (e.g. WUSCHEL/WUSCHEL2 ) are subsequently delivered by injecting a solution of resuspended A. tumefaciens into the wound sites. Following this, the wild-type abnormal transgenic offshoots are culled, whereas the normal transgenic shoots are identified for further propagation

Over the years, several variations have been incorporated into this standard protocol to improve the rate of transformation. For example, the generation of mosaic plants in the T 0 generation requires a stringent screening of the transformants to be performed in the T 1 generation. In some protocols, a selection step under in vivo conditions (e.g. maize [ 133 ]) or in vitro conditions using soilrite as a medium (e.g. roselle/ Hibiscus sabdariffa [ 134 ]) has been added after inoculation to select the best-performing T 0 chimeric plants. The addition of this selection step limits the number of plants that will be cultivated until the T 1 generation and improves the overall rate of transformation. Similarly, some protocols have incorporated steps to improve the injury step by adding sonication (e.g. horse gram/ Macrotyloma uniflorum [ 25 ]), electroporation (e.g. pea/ Pisum sativum , soybean, cowpea/ Vigna unguiculata , and lentil/ Lens culinaris [ 135 , 136 ]), and/or vacuum infiltration (e.g. Arabidopsis [ 137 ], barrel clover/ Medicago truncatula [ 138 ], cumin/ Cuminum cyminum [ 139 ], mung bean/ Vigna radiata [ 140 ] and horse gram [ 25 ]) procedures. Additional modifications include: (i) optimization of the Agrobacterium inoculum optical density (e.g. pigeon pea [ 141 ]); (ii) optimization of the acetosyringone concentration (e.g. tuberose/ Polianthes tuberosa [ 142 ]); (iii) addition of a pre-culture step on Murashige and Skoog (MS) medium before inoculation (e.g. chickpea [ 143 ]); (iv) addition of a co-cultivation step on MS medium after inoculation (e.g. radish/ Raphanus sativus [ 144 ]); and (v) use of a germination medium under in vitro conditions (e.g. sesame/ Sesamum indicum [ 145 ]).

Plumular meristem strategy

Amongst the different protocols using direct de novo shoot organogenesis, the plumular meristem strategy was proposed as a time-efficient direct regeneration-based transformation approach with high transformation rates for chickpea [ 22 , 146 ] and pigeon pea [ 147 , 148 ]. In this system, three-day-old seedlings are decapitated at the shoot apex and pricked in the apical portion and cotyledonary nodes [ 22 , 147 ] (Fig.  6 b). After co-cultivation with A. tumefaciens , multiple shoot induction is performed through the transfer of the explants on a sterile MS medium containing 6-benzyl amino purine (BAP) and 1-naphthaleneacetic acid (NAA) for three days. Following this step, the plants are moved to pots and grown under greenhouse conditions until reaching the T 1 generation. The transformation rates using the plumular meristem strategy method were 44% and 72% in the T 1 generation of chickpea [ 22 ] and pigeon pea [ 147 ], respectively. A similar protocol to the plumular meristem method was developed for alfalfa ( Medicago sativa ) [ 149 ]. In this protocol, three-day-old alfalfa seedlings are excised at the cotyledonary attachment region of the hypocotyl and wounded by vortexing with sterile sand. Following the excisions, the plants are transferred to a hormone-free medium for a short recovery time and cultivated in vitro for 14 days in a half-strength MS medium containing timentin. After this cultivation step, plants are transferred to greenhouse conditions for further growth. When performing this protocol, Weeks et al. [ 149 ] observed that excisions performed below the unifoliate leaf base eliminated the potential for shoot recovery, whereas those performed at or above the apical node resulted in the growth of new shoots in 95% of the cases. Using this protocol, about 7% of the seedlings produced progenies segregating for the T-DNA [ 149 ].

Propagule transformation

Several specialized vegetative plant organs involved in asexual reproduction, often called vegetative propagules, are ideal targets for in planta transformation due to the rapid development of growing permanent plant tissues from actively dividing meristematic cells through mitosis [ 150 ]. Propagules include stem tubers (e.g. potato and yams), tuberous roots (e.g. sweet potato and dahlia), root suckers (e.g. apple, pear, blackberries, and raspberries), runners (e.g. strawberries), bulbs (e.g. onions, tulips, and lilies), and plantlets (e.g. mother of thousands/ Kalanchoe daigremontianum ) [ 151 ]. The cut–dip–budding delivery approach aims at actively regenerating shoots from adventitious buds developed from root suckers transformed with Agrobacterium rhizogenes under in vivo conditions [ 41 ] (Fig.  7 a). This strategy has been demonstrated to be efficient with ten cultivars of sweet potato, two herbaceous plants (i.e. rubber dandelion/ Taraxacum kok-saghyz and crown vetch/ Coronilla varia ), and three woody plants (i.e. Chinese sumac/ Ailanthus altissima , Japanese angelica tree/ Aralia elata , and glorybower/ Clerodendrum chinense ). Using this approach, the observed transformation efficiencies were 10–47% for sweet potato, 40–50% for T. kok-saghyz , 3% for C. varia , 39% for A. altissima , 2% for A. elata , and 48% for C. chinense [ 41 ]. A similar in vitro protocol based on the regeneration of shoots from A. rhizogenes -infected hairy roots has been demonstrated to be efficient in apple ( Malus pumila ) and kiwi ( Actinidia chinensis ), with a short regeneration time of about 9–11 weeks [ 152 ]. The Regenerative activity-dependent in planta injection delivery (RAPID) method aims at generating transformants from infected renascent tissues of sweet potato, potato ( Solanum tuberosum ), and bayhops ( Ipomoea pes-caprae ) under in vivo conditions [ 150 ] (Fig.  7 b). In this protocol, stable transformation is obtained through the delivery of A. tumefaciens to the stem by injection and subsequent vegetative propagation of the emerging positive tissues from the wound site. Selection of the positive tissues is performed through molecular detection and/or phenotypic analysis if using a visual selection marker. Overall, the RAPID protocol displayed a short transformation time, between three to ten weeks, with a high transformant acquisition rate of 28–40%. Additional systems for propagule transformation have been developed for banana ( Musa. sp.) suckers [ 153 ], gemmae of umbrella liverwort ( Marchantia polymorphya ) [ 154 ], leaf notches of cathedral bells ( Kalanchoe pinnata ) [ 155 ], sugarcane ( Saccharum spp. ) setts [ 156 ], and bulbs of the Notocactus scopa and Hylocereus trigonus cacti [ 157 ].

In planta transformation using in vitro direct organogenesis and in vivo callus-based approaches. ( A ) In vitro direct organogenesis. The shoot apical meristems (SAM) are excised from the growing seedlings and inoculated with resuspended Agrobacterium [ 57 , 58 ]. Following inoculation, the putatively transformed shoot apical meristems are grown and screened under in vitro conditions to identify positive T 0 mutants. Following the screening process, mutants are rooted and then transferred to in vivo conditions for seed setting. Optionally, embryonic axes from imbibed seeds can be used similarly to shoot apical meristems (details not shown in the figure) [ 55 , 56 , 245 ]. ( B ) In vivo callus regeneration [ 16 , 17 , 19 , 210 ]. Dicot plants are decapitated and their wound sites are injected or rubbed with a solution of Agrobacterium . Subsequently, the wound sites are covered with parafilm and/or aluminum foil to retain moisture and keep the sites under dark conditions to favor callus formation. Optionally, the wounds can be treated with different hormones to promote the formation of a callus. Before or after callus formation, the sites can be treated with a selection marker such as an antibiotic or herbicide to eliminate untransformed calli cells. After the callus is formed, shoot formation is favored by cultivating the callus site under a regular photoperiodic regime. Under these conditions, transformed shoots will emerge from the calli cells surviving the screening process. ( C ) Shoot apical meristem removal and direct regeneration of adventitious meristems [ 16 , 19 , 210 ]. Plants are decapitated and the wound site is inoculated with Agrobacterium through injection and/or rubbing. The wound site is subsequently covered with parafilm and/or aluminum foil to retain moisture and keep it under dark conditions. Chimeric plants regenerate from the wound site and the adventitious shoot can be maintained on the same plant, grafted on another plant, or rooted in a separate container. Selection is performed in the T 1 generation to retrieve non-chimeric offspring

Exogenous morphogenic regulators and direct delivery

In recent years, the use of exogenous morphogenic regulators has also been explored as an efficient option to induce de novo shoot organogenesis. Morphogenic regulators, such as LEAFY COTYLEDON 1 [ 158 , 159 ], LEAFY COTYLEDON 2 [ 160 ], BABY BOOM [ 161 ] and WUSCHEL [ 162 ], are key genes involved in a plethora of functions such as plant morphogenesis and regeneration [ 163 ], de novo establishment of shoot stem cell niche [ 164 ], shoot and root meristem homeostasis [ 165 ] and shoot apical establishment [ 166 ]. As such, their expression is critical for de novo shoot organogenesis. Morphogenic regulators promote the production of somatic embryos or embryo-like structures on vegetative or callus explants, an effect that is increased upon overexpression [ 167 , 171 , 169 ]. Current reports have demonstrated that ectopicly expressed morphogenic regulators can be harnessed to improve the in vitro recovery rates of transgenic calli from hard-to-transform genotypes of at least 12 commercially important monocot species (e.g. rice) [ 170 , 171 , 172 , 173 , 174 ]. Despite the observed increase in the regeneration rates of transgenic calli [ 170 ], the in vitro use of ectopically expressed morphogenic regulators still remains challenging on a technical level.

To overcome these limitations, Maher et al. [ 175 ] and Cody et al. [ 176 ] developed an exogenous morphogenic regulator-based in vivo transformation method called Direct Delivery. In opposition to the Fast-treated Agrobacterium co-culture (Fast-TrACC) method (i.e. a similar method with an in vitro phase), the Direct Delivery entirely sidesteps tissue culture [ 176 ]. In the Direct Delivery method, developmental regulators, such as maize WUSCHEL/WUSCHEL 2 ( Wus2 ), cytokinin ISOPENTYL TRANSFERASE ( ipt ), and A. thaliana SHOOT MERISTEMLESS ( STM ), and gene-editing reagents are directly delivered with Agrobacterium to somatic cells of whole plants to induce the formation of de novo meristems [ 175 , 176 ] (Fig.  7 c). Following the injection of Agrobacterium , visible meristems are removed and shoot formation occurs at the wound sites after 38–48 days [ 175 , 176 ]. Maher et al. [ 175 ] demonstrated that this approach generates high transformation rates with tobacco/ Nicotiana benthamiana (i.e. gene editing efficiencies ranging from 30 to 95%) and observed positive results with potato and grapevine ( Vitis vinifera ) under in vitro conditions. Lian et al. [ 177 ] successfully regenerated snapdragon ( Antirrhinum majus ) and tomato shoots using a protocol similar to Direct Delivery under in vivo conditions but with the PLETHORA (PLT5) developmental regulator. With this ectopic expression approach, transformation efficiencies up to 11.25% and 13.3% were obtained for snapdragon and tomato, respectively [ 177 ]. The same test was performed on cabbage ( Brassica rapa ) and sweet pepper ( Capsicum spp. ) in vivo, but possibly failed due to the rapid deposition of suberin and lignin in response to wounding [ 177 ]. Direct delivery was also performed on apple ( Malus pumila ) and grapevine by Spicer [ 178 ], but without observing gene edits in the generated shoots.

Nodal agroinjection

The nodal agroinjection approach is a simple method that aims at injecting resuspended Agrobacterium in the first and second nodes of cotyledonary branches. This strategy was first validated by Wang et al. [ 179 ] in peanut and subsequently used by Han et al. [ 180 ] to generate CRISPR-Cas9 knockout peanut mutants for the FATTY ACID DESATURASE 2B ( AhFAD2B ) gene. In the original protocol, 5 µL of Agrobacterium was injected into the nodal sections of 30-day-old peanut plants. From the 820 plants recovered with this method, a total of 371 (45.24%) were PCR-positive.

Direct regeneration of embryos and shoot apical meristems under in vitro conditions

This direct regeneration strategy aims at regenerating the meristematic cells of a differentiated explant under in vitro conditions (Fig.  8 a). Both embryonic axes and developed shoot apical meristems have been demonstrated to be suitable explants for direct organogenesis under in vitro conditions. The use of a differentiated explant typically hastens the shoot regeneration rate, diminishes the requirements in hormones, simplifies medium composition (i.e. often only sucrose), and increases the resilience of the explant toward Agrobacterium overgrowth [ 55 , 56 , 181 ]. A large literature search has demonstrated the efficiency of several transformation/regeneration systems for the embryonic axes of watermelon [ 182 ], field bean [ 183 ], cowpea [ 184 ], chickpea [ 185 , 186 ], common bean [ 184 , 187 ], black gram ( Vigna mungo ) [ 188 , 189 ], purslane [ 190 ], eggplant [ 191 ], and snake gourd ( Tricosanthes cucumerina ) [ 27 ]. Two of the most commonly transformed species using the in vitro embryonic axis method are soybean and cotton, sometimes with innovative technical aspects. For example, Paes de Melo et al. [ 55 ] and Ribeiro et al. [ 56 ] have respectively proposed protocols in which soybean and cotton embryonic axes are injured using biolistics and subsequently infected with Agrobacterium . In their protocols, shooting, rooting, and selection are subsequently performed simultaneously in a medium containing 6-benzylaminopurine (BAP) and activated charcoal. In this system, transformants are selected with the selection marker gene AHAS which confers resistance to the systemic herbicide Imazapyr. Using these protocols, Paes de Melo et al. [ 55 ] and Ribeiro et al. [ 56 ] have obtained transformation efficiencies averaging 9.84% for soybean and 60% for cotton. Similarly, several shoot apical meristem-based transformation/regeneration systems have been demonstrated in many dicots (e.g. cucumber [ 192 ], petunia [ 193 ], camelina/ Camelina sativa [ 194 ], Dalmatian chrysanthemum/ Tanacetum cinerariifolium [ 195 ] and cotton [ 196 , 197 ]) and monocots (e.g. wheat [ 14 , 44 , 198 ], finger millet/ Eleusine coracana [ 199 ], foxtail millet/ Setaria italica [ 200 ], pearl millet/ Pennisetum glaucum [ 201 ] and rice [ 57 , 58 , 202 , 203 ]). In addition, an extensive literature dedicated to the in vitro regeneration of embryonic axes or excised shoot apical meristem without transformation is available for a large number of species (e.g. finger millet [ 204 , 205 ], maize [ 206 ] and rice [ 207 ]). These regeneration protocols serve as a basis for the development of new transformation methods as those could be converted with minimal effort. Overall, these in vitro systems offer numerous benefits over many of the in planta systems and are one of the most interesting alternatives to streamline transformation in monocots. However, these methods require access to micropropagation facilities and are technically more challenging than most in planta techniques.

Novel transformation techniques used for in planta transformation. ( A ) Grafting-mediated transformation [ 227 ]. Wild-type scion is grafted to a transgenic rootstock containing Cas9 and gRNA sequences. The grafting procedure leads to the formation of chimeric scions containing Cas9 and gRNA sequences due to the movement of tRNA-like sequence motifs that ensure transcript mobility across the plant. The rootstock to scion movement of these sequences causes heritable edits in the germline cells and edited offspring can be retrieved upon selection in the T 1 generation. ( B ) Viral-based vector using a mobile FT cassette [ 238 ]. The leaves of mutant plants overexpressing Cas9 are agroinfiltrated with a viral vector (e.g. tobacco rattle virus vector) containing a gRNA sequence fused to mobile FT sequences. The gRNA sequence reach the germline cells of the Cas9 overexpressing mutants upon the transcription of FT due to its endogenous natural movement to the shoot apical meristem and the edited offspring are retrieved in the T 1 generation upon selection

Vegetative tissues

Callus-based transformation system.

In transformation systems using an in vivo callus-based approach, seedlings or mature plants are injured and their wounds are treated using a solution of Agrobacterium [ 16 , 54 ] (Fig.  8 b). Following this step, the injuries are subjected to hormone treatment, if necessary, to promote the development of a callus and/or adventitious buds [ 16 ]. In some cases, selection by treating the wounded area using a selection marker (i.e. antibiotic or herbicide) is performed to identify the putative transformants [ 16 ]. In transformed tomatoes, Pozueta-Romero et al. [ 208 ] observed that proper kanamycin selection favors the competition of transformed over untransformed cells during de novo shoot organogenesis, thus increasing significantly the number of regenerated transformed shoots. To promote callus growth, inoculated wounds can be covered with parafilm, aluminum foil, mud, or plastic to maintain proper humidity, and adequate temperature and to provide a dark treatment, as darkness has been demonstrated to favor the development of callus mass [ 16 , 17 ]. Over the years, in vivo callus transformation and/or regeneration has been demonstrated to be feasible in a broad range of fruit trees (e.g. orange/ Citrus sinensis [ 20 , 209 ], longan [ 19 , 210 ], and pomelo/ Citrus maxima [ 17 , 209 ]), vines [passionfruit [ 16 ]], shrubs/trees (e.g. poplar [ 211 , 212 , 213 ] and eucalyptus/ Eucalyptus sp. [ 211 ]) and perennial dicots cultivated as annual (e.g. tomato [ 18 , 208 ]). In their patent, Mily et al. [ 18 ] also mentioned that soybean and coffee ( Coffea sp. ) generate new shoots upon decapitation and that chili pepper, eggplant ( Solanum melongena ), and common bean also display excellent regeneration and GUS expression abilities. Often, plants regenerated using this system will concomitantly undergo direct regeneration events (e.g. tomato and several relatives [ 214 , 215 , 216 , 217 ], soybean [ 218 ], and peanut [ 51 ]) which can lead to some form of mosaicism in the transformed plant (Fig.  8 c). Although the literature for this technique is relatively sparse in comparison to other transformation strategies, a plethora of protocols using indirect de novo shoot induction without transformation are currently available for species such as poinsettia ( Euphorbia pulcherrima ) [ 219 ], tomato [ 216 , 220 , 221 , 222 ], and chili pepper [ 208 ]. In addition, indirect de novo shoot induction without transformation has been validated in lignified woody jujube [ 54 , 223 , 224 , 225 ] and pomegranate ( Punica granatum ) [ 226 ] trees under field conditions for colchicine mutagenesis treatments, thus demonstrating its versatility and potential.

Novel systems

Grafting-mediated transformation.

At present, only one technique, named grafting-mediated genome editing, has been developed as a systematic in planta transformation tool to induce precise modifications in the genome [ 227 ] (Fig.  9 a). In grafted plants, the formation of a successful graft union requires several steps, including the (i) lining of the vascular cambium; (ii) wound healing; (iii) formation of a callus bridge between the rootstock and the scion; (iv) generation of vascular cambium; and (v) development of the secondary xylem and phloem [ 228 ]. The formation of a callus bridge enables the horizontal gene transfer of phloem-mobile protein-coding RNAs through the phloem vasculature of grafted plants [ 229 ]. In 2016, Zhang et al. [ 230 ] demonstrated that transcripts harboring distinctive tRNA-like structures can move from a transgenic rootstock to a wild-type scion and be translated into proteins after transport. Taking advantage of this discovery, Yang et al. [ 227 ] investigated the generation of stable gene-edited plant lines using intraspecific and interspecific grafting in wild-type Arabidopsis and Brassica rapa to generate heritable modifications. To do so, phloem-mobile tRNA-like sequences were fused to Cas9 and guide RNA (gRNA) sequences to induce transport from the provider transgenic rootstock to the recipient scion through root-to-shoot movement. Using this system, the inheritance of deletion edits was 1.6% for heterozygotic and 0.1% for homozygotic genotypes, although the authors underline that these numbers were probably underestimated because the seedlings were screened in pools using PCR. As the T 0 generation is chimeric, segregation must be performed in the subsequent generation to recover non-chimeric lines. To circumvent the step involving the generation of the mutant rootstock in recalcitrant species, the authors suggest using A. thaliana and Nicotiana sp. as rootstocks due to their simple and reliable transformation protocols and their very wide range of compatible distantly related species, including soybean and fava bean [ 39 ].

Viral-based vectors

Virus-induced gene silencing (VIGS) is a method that uses modified viral vectors to induce transient gene silencing in plants [ 231 , 232 ]. This technique allows for efficient gene function analysis but is generally not considered as a reliable method to generate stable mutations in plants although some shreds of evidence suggest that the silencing effect can be transmitted to the next generation [ 233 ]. To circumvent this issue, the virus-induced genome editing (VIGE) method was developed as a means to generate permanent mutations for the production of true-breeding lines [ 234 ]. The scope of action of viral-based vectors significantly increased with the development of genome editing technologies as the expression of short RNA sequences (e.g. gRNA) can be readily performed with the use of in planta Agrobacterium transient transformation strategies (e.g. agroinfiltration, agroinjection, agrospray, agrodrench, and rub inoculation) [ 235 , 236 ]; however, heritable mutations are challenging to generate due to the seclusion of viruses from the meristematic cells of the shoot apical meristem but have been reported on rare occasions (e.g. Tobacco rattle virus [ 234 ] and Barley stripe mosaic virus [ 237 ]). To obtain a greater efficiency at generating heritable genome editing events, Ellison et al. [ 238 ] fused gRNA sequences to mobile FLOWERING LOCUS T ( FT ) sequences and cloned them into a Tobacco rattle virus vector (Fig.  9 b). The resulting vector was subsequently inserted into the cells of Cas9-overexpressing tobacco plants via agroinfiltration. In its natural state, endogenous FT sequences move to the shoot apical meristem to induce flowering via the phloem upon transcription in the leaf tissues [ 239 ]. This characteristic enables the gRNAs to enter the shoot apical meristem upon the transcription of  FT , thus generating stable mutations in the future offspring without relying on tissue culture. Following the publication of Ellison et al. [ 238 ], this versatile editing system has been confirmed to be also compatible with the Barley yellow striate mosaic virus [ 240 ] and Cotton leaf crumple virus [ 241 , 242 ].

Since the first reports of in planta transformation in the 1980s [ 122 ], hundreds of in planta protocols have been developed for a large number of species. The classification of these protocols into a structured system is challenging due to the broad range of approaches. However, much of the strength of the in planta concept lies in this heterogeneity and high diversity since it aims to work with the natural biological and morphological features of each species instead of trying to “force” the transformation process through challenging regeneration steps. The high level of versatility, decreased upfront cost, and reduced technical requirements of many of these techniques demonstrate the importance of this field of research for the progress of plant science. Still, many of these techniques require more extended research to validate their use in a broad range of species. For instance, de novo shoot induction using tissue culture-independent approaches seems to be a promising strategy for dicot transformation, particularly for species with a long juvenile period (e.g. fruit trees). The methods are simple, cost and time-efficient, mostly genotype-independent, reliable, and based on prior knowledge from tissue culture-based de novo shoot induction methods. Furthermore, the protocols can be adapted for a wide range of experimental settings (e.g. lab vs. field conditions) and plant developmental stages (e.g. younger seedlings vs. lignified woody plants). Theoretically, this approach boasts all the most important features for a transformation method; however, it seems largely unexplored in the literature in comparison to its in vitro counterpart. The same observations can be made for several methods cited in this article such as embryo desiccation or the shoot apical meristem methods.

At present, the specific reasons slowing a wider adoption of these in planta approaches in the scientific community remain elusive as many of these techniques were demonstrated to be efficient in a large number of species. On the whole, this paper tried to review as many sources as possible, including those hard-to-access research articles, to build a compendium of references and provide the most accurate picture of a field that is rapidly evolving. In their reviews, Kaur and Devi [ 5 ] suggested that the field of in planta research is still in its early stages of development. While we understand the reasons underlying this standpoint, we would like to add some nuances. In planta research has always been at the core of transformation research since its beginning, and the floral dip approach in Arabidopsis is still a major propeller for the development of plant molecular biology. Several approaches are now in their mature phases, especially for dicots, with standardized protocols for a large number of species. In the longer term, many strategies targeting dicots, such as the tissue culture-independent de novo shoot induction method, clearly have the potential to become a mainstay of plant transformation. On the other hand, in planta techniques for monocots are less advanced, less diversified, and often more challenging to operate. Nonetheless, several approaches (e.g. pollen-tube pathway and shoot apical meristem injury methods) have already demonstrated their potential and are used regularly by several labs across the world. In conclusion, the in planta transformation concept offers important contributions to plant biotechnology by offering an alternative to traditional transformation/regeneration techniques and will surely become an increasingly important player in the field of plant transformation in the future.

Online compendium

To further strengthen the content of this compendium, we solicit the support and help of the community to add additional references to the online version of this document available at https://github.com/Inplanta/In_planta_transformation . To do so, people can send their annotated references to the ‘’Issue’’ section of the GitHub page under the following format: (i) Family; (ii) Genera; (iii) Species; (iv) Common name; (v) Type of explant; (vi) Method; (vii) Notes; and (viii) Complete reference. The references should be in an Excel format and need to be submitted along with the original document. The compendium was built to limit in-text citations and provide a user-friendly versatile document to group and annotate in planta references. Overall, video footage showing specific methodological aspects is considered to be particularly helpful for the understanding and replicability of the techniques. To maximize the understanding of this paper, readers are invited to consult the compendium as they are reading.

Data availability

A free, online, and up-to-date version of the in planta compendium is available at https://github.com/Inplanta/In_planta_transformation .

The transformation rates provided in the article have been often calculated using different methods and cannot, in a large number of cases, be directly used for comparison.

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Acknowledgements

Author JGB is thankful to Éric Fortier for the review of the article.

JGB was supported by the Natural Sciences and Engineering Research Council of Canada, les Fonds de recherche du Québec volet Nature et Technologie, Centre SÈVE, MITACS, and Seed World Group.

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Bélanger, J.G., Copley, T.R., Hoyos-Villegas, V. et al. A comprehensive review of in planta stable transformation strategies. Plant Methods 20 , 79 (2024). https://doi.org/10.1186/s13007-024-01200-8

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Received : 05 March 2024

Accepted : 01 May 2024

Published : 31 May 2024

DOI : https://doi.org/10.1186/s13007-024-01200-8

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• In planta transformation
• In situ transformation
• Direct organogenesis
• Indirect organogenesis
• Recalcitrant species
• In vivo regeneration

Plant Methods

ISSN: 1746-4811

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