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# Summer School "Shape the future" - The many uses of plants

Cristel Carles

► To cite this version:

Cristel Carles. Summer School "Shape the future" - The many uses of plants. Doctoral. Université Grenoble Alpes (UGA) - Grenoble, Université Grenoble Alpes (UGA) - Grenoble, France. 2023. [hal-04933639](https://hal.science/hal-04933639v1)

HAL Id: hal-04933639

https://hal.science/hal-04933639v1

Submitted on 26 Feb 2025

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# Summer School

## « The many uses of plants »

### WHY ARE PLANTS FASCINATING?

### Overview on their biology and multiple usages

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Christel CARLES

Prof. UFR Chemistry Biology

Plant & Cell Physiology Lab

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LPCV
Plant uses in three main areas

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Used in alimentation/nutrition, health, sustainable development, energy...
# Outline of the conference

## I. Plants: subject of fascination

**Why study plants?**

**What is a plant?**

* Developmental stages

* Specificities of the plant cell: Chloroplast, Cell wall

* Botany, plant classification

* Secondary metabolites

Christel Carles parts
(in green)

## II. Plants: for what applications?

** Participation of students : examples they will have studied in the posters **

* Agriculture, Agronomy: Better crop yields, better nutrition, better environment

* Production of "green" molecules for medicine & industry

* Living w/ plants, from society to health care

* Sustainable development, phytoremediation, eco-responsability

## III. Key Technologies

* CIV & hormones

* Genetic transformation

* Extracting plant compounds

* Metabolite studies from LC/MS to MS/MS
# I. Plants: subjects of fascination

<figure><img src="image_11.png" /></figure>

## A. Why studying plants?

* For a better understanding of biology

* Because they are essential to our lives

* To conserve endangered species and threatened ecological niches

* To better exploit plants' capacity to provide us with food, medicine and energy
Plants, like most animals, are multicellular eukaryotes
+ unicellular algae

<figure><img src="image_12.png" /></figure>

Photo credits: Public Health Image Library;
NASA; © Dave Powell, USDA Forest Service; tom donald

80% of earth surface is covered with plants
# Plants make up a highly diversified kingdom

<figure><img src="image_13.png" /></figure>

Plants have developed the ability to thrive in a variety of terrestrial habitats

2000 new plant species recorded every year

Images courtesy [tom donald](https://www.example.com)
# Plants are fascinating living organisms

<figure><img src="image_14.png" /><figcaption>Largest flower (~1m;11kg)</figcaption></figure>

Rafflesia arnoldii

<figure><img src="image_15.png" /><figcaption>Longest living (~ 5000 years)</figcaption></figure>

Pinus longaeva
bristle cone pine

<figure><img src="image_16.png" /><figcaption>Largest organism (> 100m)</figcaption></figure>

Sequoiadendron giganteum
giant sequoia
A historical experiment from Joseph Priestley, 18th century

<figure><img src="image_17.png" /></figure>

Joseph Priestley (1772) showed that an animal kept in an airtight container would eventually faint and die.

He concluded that by breathing an animal "damages" the air.

Priestley also showed that plants have the ability to "restore" air.

We now know that they produce oxygen as a by-product of photosynthesis.

<figure><img src="image_18.png" /></figure>
Oxygen content of Earth's Atmosphere and the green lineage

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Courtesy of R. Blanvillain https://commons.wikimedia.org/wiki/File:Sauerstoffgehalt-1000mi2.png

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# Plants fix CO₂ in energy-rich molecules that animals use as food

<figure><img src="image_27.png" /></figure>

Plants convert CO₂ into sugars
through a process known as
**photosynthesis**

Carbon dioxyde
(in the air)

$$6 \text{ CO}_2 + 6 \text{ H}_2\text{O} \rightarrow$$

water

(underground)

Solar energy

chlorophylle

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

$$C_6H_{12}O_6 + 6 O_2 \rightarrow$$

Sugar

Oxygen
dioxyde
# Changes in atmospheric CO₂ concentration (march – july)

<figure><img src="image_30.png" /></figure>

GLOBAL CLIMATE CHANGE

Vital Signs of the Planet
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Capturing atmospheric CO2 by changing our farming practices

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https://kisstheground.com/thebook/

https://kissthegroundmovie.com
Plants produce an extraordinary assortment of chemicals

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We couldn't live without plants

They produce:

* most of the **oxygen** we breathe

* most of the **stored chemical energy** we consume as **food** or burn as **fuel**

* an incredible assortment of **useful chemicals**

<figure><img src="image_39.png" /></figure>
**But also:** Studying plants tells us about our world

Cells were observed for the 1st time in plants, from bark
(R. Hooke, 1655)

<figure><img src="image_40.png" /><figcaption>Drawing of cork by **Robert Hooke,**
discoverer of "cells"</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>Photograph of cork cells</figcaption></figure>

Photo credit: ©David B. Fankhauser, Ph.D
Viruses were purified for the 1st time from plant material

<figure><img src="image_42.png" /><figcaption>Tobacco Mosaic Virus (TMV)</figcaption></figure>

RNA virus that infects tobacco and other members of the Solanaceae family. This virus was the first to be discovered and crystallized in 1935 by Wendell Meredith Stanley.

Viruses infect humans and plants, causing many diseases, including AIDS, hepatitis, SARS, swine flu, cervical cancer, chickenpox and polio
Mendel's studies on peas revealed the laws of genetic heritability...

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /><figcaption>G. Mendel, 1865</figcaption></figure>

... that help us understand human diseases such as sickle-cell anemia ...

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Mendel's studies on peas revealed the laws of genetic heritability...

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... and hemophilia, as well as countless other human diseases that have a genetic contribution.

<figure><img src="image_47.png" /><figcaption>Pedigree of family carrying hemophilia allele</figcaption></figure>
Mendel's discoveries form the basis of Borlaug's work on cereal improvement

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<figure><img src="image_51.png" /><figcaption>**Norman Borlaug**
1914-2009, Seed breeder</figcaption></figure>

« the father of the Green Revolution »

**Nobel prize in 1970**
for contributing saving over a billion people worldwide from starvation

Wheat improvement in Mexico:

* Bi-annual wheat

* Disease-resistant varieties (back-cross)

* Dwarfism

Extension to South Asia: **Green Revolution in the 60s**
# I. Plants: subject of fascination

## B. What is a plant?

* an **autotrophic** organism

* A **constantly developing** organism

Green plants obtain **energy from sunlight**, using **chloroplasts** derived from **endosymbiosis with cyanobacteria**
**Photosynthesis** uses chlorophyll = gives them their green colour

Some are parasitic + lost the ability to produce
<figure><img src="image_52.png" /><figcaption>The Plant Cell</figcaption></figure>
The Plant Cell

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<figure><img src="image_54.png" /><figcaption>Metabolism within a plant cell</figcaption></figure>
The Chloroplast:

→ production of organic matter and oxygen

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Developmental cycle of an Angiosperm

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No germ cells in the embryo
Developmental cycle of an Angiosperm

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# Growth in phytomeres

**Phytomere:** functional unit produced at the root or stem apex, throughout the plant's life cycle.

The whole of a Spermaphyte organism can be considered as a stack of phytomeres.

A phytomere (aerial) = node to which a leaf is attached + underlying internode + axillary bud at base of leaf.

Each component of a phytomer can continue to grow and differentiate.

**Plastochron:** Speed of leaf initiation.

Initially, a young plant will only produce phytomeres from its apical meristems. Later in development, secondary meristems will form and initiate lateral phytomeres.

<figure><img src="image_58.png" /><figcaption>Underground
Phytomeres</figcaption></figure>
Growth in phytomeres

**Phytomere:** functional unit produced at the root or stem apex, throughout the plant's life cycle.

The whole of a Spermaphyte organism can be considered as a stack of phytomeres.

Aerial
Phytomeres

<figure><img src="image_59.png" /></figure>

Underground
Phytomeres

<figure><img src="image_60.png" /></figure>
Plant growth is non homothetic

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## II. Plants: for what applications?

*Round tables & posters*
# II. Plants: for what applications?

**Why improving and how benefitting the value of plants?**

→ Better **crop yields** (drought and pathogen resistance)

→ A better **environment** (better take of nutrients from the soil, for less fertilizer)

→ "Better" food

→ **Production of "green" molecules**, used in
- Medicine

- Industry

→ energy: biofuels

→ additives

→ **Sustainable development**
- phytoremediation

...
# THE STAKES OF HYDRIC STRESS

<table><caption>Physical and economical water scarcity</caption><thead><tr><th>Sources of abiotic stresses</th></tr></thead><tbody><tr><td>Cold/ excessive heat</td></tr><tr><td>Salinity</td></tr><tr><td>Drought /flood</td></tr><tr><td>Radiations (UV) / oxydative stress</td></tr><tr><td>Pesticides / Chemical pollutants</td></tr><tr><td>Winds /Dusts</td></tr><tr><td>Depletion of soil nutrients</td></tr></tbody></table>

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Abiotic stresses modify the physiology and development of plants

* increase plant production in an unfavorable climatic and pedological context

* find biological responses allowing plants to tolerate environmental constraints
Plant cultivation is often limited by drought

Areas of physical and economic water scarcity

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Source: IWMI report, Insights from the Comprehensive Assessment of Water Management in Agriculture, 2005 / p8
# Drought and global warming

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Data source: ERA5

<figure><img src="image_65.png" /></figure>

Copernicus
European Climate Change Service

IMPLEMENTED BY
ECMWF

Climate
Change Service
climate.copernicus.eu

Temperature change forecasts for the period 2071-2100, compared with 1961-1990.

<figure><img src="image_66.png" /></figure>

© European Communities, 1995-2009. Image Source: [The PESETA Project](https://www PESETA Project)

2020: hottest year on record: beats 2019, with an average of + 1.6°C
(compared with 2019)

Cumulative drought + global warming, a "viscious" circle
The drought vicious circle

In temperate regions, productivity drops by 3 to 5% for every 1° C rise in temperature.

<figure><img src="image_67.png" /></figure>

Even moderate droughts reduce photosynthesis efficiency and growth, vs. extreme droughts that kill plants.

Deforestation for agriculture increases CO₂ release into the atmosphere

<figure><img src="image_68.png" /></figure>

Need more surface area for the same production

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Heat and drought reduce plant productivity

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By manipulating a single gene, one can improve
drought resistance in plants

<figure><img src="image_71.png" /><figcaption>* Overexpression of *At*HDG11 Confers Drought Tolerance in Transgenic Tobacco Plants</figcaption></figure>
A more developed root system can contribute to
 drought tolerance

<figure><img src="image_72.png" /></figure>

Selecting plants for the size of
their root system can help them
grow in drought conditions.

Yu, H., et al. Plant Cell
2008;20:1134-1151

<footnote>* *enhanced drought tolerance1* (*edt1*) mutant: activation of the HD-START transcription factor (β more ABA); *EDT1=HDG11*</footnote>
The use of fertilisers depletes resources and causes huge environmental pollution

* Crops need fertilisers: potassium, phosphate, nitrogen...

* Potassium and phosphate are not renewable (extracted from mines)

* Nitrogen synthesis requires large quantities of energy

The leaching of fertilisers devastates entire regions and leads to over-proliferation of algae in the oceans, reducing the availability of oxygen for other species (including animals).

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Photo credits: Mining Top News; Library of Congress, Prints & Photographs Division, FSA-OWI Collection, LC-USW361-374
NASA/Goddard Space Flight Center Scientific Visualization Studio)
Nutrient uptake by plants can be improved

<figure><img src="image_75.png" /></figure>

Un meilleur système de transport racinaire peut réduire les besoins en engrais.
Perennial plants can better capture water and nutrients
in the soil

<figure><img src="image_76.png" /><figcaption>Photo: Jodi Torpey, westerngardeners.com</figcaption></figure>

Scientists are crossing cereals
with perennial plants to reduce
the dependence of crops on
water and fertilisers

Perennial plant ≠ annual or biennial plant

<caption>Wes Jackson of the Land Institute
holding a perennial wheat relative
*Thinopyrum intermedium*</caption>
# Transgenic maize expressing the Bt* toxin gene resistant to the European corn borer (lepidoptera)

* Lepidopteran larvae attack the aerial parts, causing wounds to the ears

* Damaged grains more likely to be contaminated by fungi (produce toxins often dangerous to humans and animals)

* → Weakening of the plant

* → Major quantitative and qualitative crop losses

<figure><img src="image_77.png" /></figure>

## Use of *Bacillus thuringiensis (Bt) Cry1Ab toxin

Replaces conventional pesticides (production by the plant)

→ improves the environment?

<figure><img src="image_78.png" /></figure>
# Calgene's 'Flavr Savr' tomato, which won't rot

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

<figure><img src="image_79.png" /></figure>

RNA interference strategy inhibiting the production of ethylene (= fruit ripening hormone)

<figure><img src="image_80.png" /></figure>

ripening re-induced by ethylene spray

Controlling fruit ripening for better storage
## Supplementing plants with nutrients can alleviate problems of malnutrition

<figure><img src="image_81.png" /></figure>

Subsistence diets are often poor in nutrients (vitamins, minerals, calories) = Malnutrition is mainly a disease affecting poor populations.

<figure><img src="image_82.png" /></figure>

<figure><img src="image_83.png" /></figure>

Image sources: Petaholmes based on [WHO data](https://www.who.int); [WHO](https://www.who.int)

=> Supplementation with vitamins (e.g. folate, vitamin A) and micronutrients (iron, zinc, iodine) has remarkably reduced malnutrition worldwide.
Genetically biofortified foods

<figure><img src="image_84.png" /><figcaption>"Golden Rice": rice enriched in Vitamin A</figcaption></figure>

<caption>Tomatoes enriched in antioxidants (vs controls above)</caption>

Photos: Golden Rice Humanitarian Board © 2007; ETH Zurich / Christof Sautter; Butelli, E., et al., Nature Biotechnology.
# The case of Golden Rice

Created to make up for **Vitamin A deficiency** (antioxidant), which affects 400 million people
→ Blindness (0.5Mi children/year), increased susceptibility to infections

Transgenesis using genes from the β-carotene biosynthesis pathway (precursor of vitamin A)

<figure><img src="image_85.png" /></figure>

**Golden Rice v2** 4 genes introduced → produces up to 23 times more β-carotene than the v1

2018: first approvals as food in Australia, New Zealand, Canada and the United States
Plants provide more than just food...

→ Chemistry from plants

<figure><img src="image_86.png" /></figure>
Bioactives metabolites
from plants

* Alkaloids = basic nitrogenous heterocyclic organic molecules with pharmacological activity.

* They are found as secondary metabolites (derived from aa) mainly in plants, fungi and a small number of animal groups.

- Analgesics (morphine, codeine)

- Local anaesthetics (cocaine)

- Stimulants (caffeine, nicotine)

- Anti-infectives ((chloro)quinine, berberine)

- Anti-cancer drugs (taxol, vinblastine, vincristine)

Kutchan, Plant Cell 1995. Alkaloïds biosynthesis. The basis of metabolic engineering of medicinal plants.

<figure><img src="image_87.png" /></figure>
## Example of herbs and treatments

**Conext : Malaria kills millions of people**

<figure><img src="image_88.png" /></figure>

Regions of the world with the highest risk of malariae
The protozoan Plasmodium causes malaria

<figure><img src="image_89.png" /><figcaption>*Plasmodium in a*
*mouse cell*</figcaption></figure>

Image by Ute Frevert; false color by Margaret Shear.

<figure><img src="image_90.png" /><figcaption>The mosquito is the vector of
Plasmodium for humans</figcaption></figure>

CDC

CENTERS FOR DISEASE CONTROL AND PREVENTION
The bark of red cinchona contains quinine, which kills Plasmodium

<figure><img src="image_91.png" /></figure>

Image: Köhler; CDC

<figure><img src="image_92.png" /><figcaption>Arbre: ququina rouge
(Cinchona pubescens)</figcaption></figure>

<figure><img src="image_93.png" /><figcaption>Quinine
(alcaloïde)</figcaption></figure>
Gin Tonic?

* British soldiers in tropical regions took quinine tablets to treat malaria. To hide its unpleasant bitter taste, it was mixed with carbonated sugar water ("tonic") and also often with gin.

= origin of "gin and tonic".

<figure><img src="image_94.png" /></figure>

<figure><img src="image_95.png" /><figcaption>Photograph of the Imperial War Museum, London</figcaption></figure>
The bark of red cinchona contains quinine, which kills Plasmodium

<figure><img src="image_96.png" /></figure>

Plasmodium has developed resistance to quinine
⇒ need to find other compounds

Image: Köhler; CDC
*Artemisia annua* is a plant with new anti-malaria properties

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /><figcaption>Artemisinin</figcaption></figure>

Artemisia has been used by Chinese herbalists for thousands of
years. In 1972 the acitf compound was discovered and purified
= **artemisinin**.
In 2015, Tu Youyou was awarded the Nobel Prize for his discoveries concerning a new malaria treatment: artemisinin.

At the age of 84, Chinese researcher Tu Youyou, who had long been considered for the prize, was rewarded for a particularly effective treatment for malaria using an extract from the annual mugwort plant (Artemisia annua).

* An extract from the wormwood plant (Artemisia absinthium) had shown promise in mice. Inspired by an ancient document, Dr Tu modified the extraction process to make it more effective before isolating the active ingredient in wormwood, artemisinin, in the early 1970s.

Artemisinin is the safest and most effective treatment for malaria, a disease that affects nearly 200 million people a year and kills more than 500,000, mainly African children.

<figure><img src="image_99.png" /></figure>
# Researchers have developed Artemisia plants that produce greater quantities of artemisinin

<figure><img src="image_100.png" /></figure>

CNAP Artemisia Research Project

(YORK University):

Artemisinin yield increase

Artemisia produces and stores artemisinin in glandular trichomes on the surface of its leaves.

Artemisinin production can be increased by increasing:

- the number of leaves on a plant

- the number of glands on a leaf

- the amount of artemisinin produced by each gland

<figure><img src="image_101.png" /></figure>

www.york.ac.uk/org/cnap/artemisiaproject/
# Phytoremediation

<figure><img src="image_102.png" /></figure>

## Phytoremediation of heavy metals from soils

Terry McIntyre¹

Affiliations + expand

PMID: 12674400 DOI: 10.1007/3-540-45991-x_4

## Phytoremediation of Soils Contaminated with Metals and Metalloids at Mining Areas: Potential of Native Flora

March 2014

DOI: 10.5772/57469

In book: Environmental Risk Assessment of Soil Contamination. Chapter: Phytoremediation of Soils Contaminated with Metals and Metalloids at Mining Areas: Potential of Native Flora

<figure><img src="image_103.png" /></figure>

*Miscanthus* sp. is a plant widely used for soil decontamination. However, we need to be careful about what we do with the harvested material.
III. Key technologies
Advances in fundamental science: a powerful driver of plant improvement

<figure><img src="image_104.png" /></figure>

Découverte du rôle des organes sexuels chez les végétaux

<figure><img src="image_105.png" /></figure>

Millington -Grew

<figure><img src="image_106.png" /></figure>

Visualisation des chromosomes

→ mise en évidence de la division cellulaire

<figure><img src="image_107.png" /></figure>

Strasburger -Boveri

<figure><img src="image_108.png" /></figure>

Mise en application des lois de Mendel sur l'hérédité

→ règles de base de la génétique

<figure><img src="image_109.png" /></figure>

Mendel

<figure><img src="image_110.png" /></figure>

Découverte de la totipotence des cellules végétales

→ régénération

<figure><img src="image_111.png" /></figure>

Haberland

<figure><img src="image_112.png" /></figure>

Découverte de l'intérêt des hybrides sur le maïs

Hétérosis

<figure><img src="image_113.png" /></figure>

Shull

<figure><img src="image_114.png" /></figure>

Description de la structure en double hélice de l'ADN

<figure><img src="image_115.png" /></figure>

Watson & Crick

<figure><img src="image_116.png" /></figure>

Découverte du code génétique

<figure><img src="image_117.png" /></figure>

Crick et al.

<figure><img src="image_118.png" /></figure>

Découverte des enzymes de restriction

<figure><img src="image_119.png" /></figure>

Aber et al.

<figure><img src="image_120.png" /></figure>

Découverte du transfert de gènes par des agrobactéries

<figure><img src="image_121.png" /></figure>

Schell
Advances in fundamental science: a powerful driver of plant improvement

<figure><img src="image_122.png" /></figure>

Découverte du rôle des organes sexuels chez les végétaux

<figure><img src="image_123.png" /></figure>

Millington -Grew

<figure><img src="image_124.png" /></figure>

Visualisation des chromosomes

→ mise en évidence de la division cellulaire

<figure><img src="image_125.png" /></figure>

Strasburger -Boveri

<figure><img src="image_126.png" /></figure>

Mise en application des lois de Mendel sur l'hérédité

→ règles de base de la génétique

<figure><img src="image_127.png" /></figure>

Mendel

<figure><img src="image_128.png" /></figure>

Découverte de la totipotence des cellules végétales

→ régénération

<figure><img src="image_129.png" /></figure>

Haberland

<figure><img src="image_130.png" /></figure>

Découverte de l'intérêt des hybrides sur le mais

Hétérosis

<figure><img src="image_131.png" /></figure>

Shull

<figure><img src="image_132.png" /></figure>

Description de la structure en double hélice de l'ADN

<figure><img src="image_133.png" /></figure>

Watson & Crick

<figure><img src="image_134.png" /></figure>

Découverte du code génétique

<figure><img src="image_135.png" /></figure>

Crick et al.

<figure><img src="image_136.png" /></figure>

Découverte des enzymes de restriction

<figure><img src="image_137.png" /></figure>

Aber et al.

<figure><img src="image_138.png" /></figure>

Découverte des séquences de gènes CRISPR chez E. coli (Ishino et al., 1987)

<figure><img src="image_139.png" /></figure>

<figure><img src="image_140.png" /></figure>

Ishino & Schell Nakata
Advances in fundamental science: a powerful driver of plant improvement

<figure><img src="image_141.png" /></figure>

Découverte du rôle des organes sexuels chez les végétaux

<figure><img src="image_142.png" /></figure>

Millington -Grew

<figure><img src="image_143.png" /></figure>

Visualisation des chromosomes

→ mise en évidence de la division cellulaire

<figure><img src="image_144.png" /></figure>

Strasburger -Boveri

<figure><img src="image_145.png" /></figure>

Mise en application des lois de Mendel sur l'hérédité

→ règles de base de la génétique

<figure><img src="image_146.png" /></figure>

Mendel

<figure><img src="image_147.png" /></figure>

Découverte de la totipotence des cellules végétales

→ régénération

<figure><img src="image_148.png" /></figure>

Haberland

<figure><img src="image_149.png" /></figure>

Découverte de l'intérêt des hybrides sur le maïs

Hétérosis

<figure><img src="image_150.png" /></figure>

Shull

<figure><img src="image_151.png" /></figure>

Description de la structure en double hélice de l'ADN

<figure><img src="image_152.png" /></figure>

Watson & Crick

<figure><img src="image_153.png" /></figure>

Découverte du code génétique

<figure><img src="image_154.png" /></figure>

Crick et al.

<figure><img src="image_155.png" /></figure>

Découverte des enzymes de restriction

<figure><img src="image_156.png" /></figure>

Aber et al.

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Découverte du transfert de gènes par des agrobactéries

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Schell

2012

Découverte de la technologie CRISPR (束子毅, 1996). *coli* (Ishino et al., 1987)

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Douglas & Charpentier
The discovery of totipotency has been the guiding principle behind the development of plant biotechnologies.

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<figure><img src="image_161.png" /></figure>

Plant cells, taken from any organ of a plant, have the capacity to regenerate a complete individual identical to the mother plant.

**Totipotency** = property of plant cells to regenerate an individual when placed in appropriate conditions, possibly passing through a stage of dedifferentiation.

Gottlieb Haberlandt, Hungarian botanist (1902)

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## Discovery of phytohormones: auxins & cytokinins

<figure><img src="image_167.png" /></figure>

* **Went (1926):** acide indole acetic (IAA) = Auxin, derived from Tryptophane

* Stimulates cell elongation, rhizogenesis (effect on cell plasticity and gene networks)

* Synthesis in meristematic stem apices, young leaves and apical buds

* Polarized transport from apex to base of plant

<figure><img src="image_168.png" /></figure>

<figure><img src="image_169.png" /></figure>

* **Blakeslee (1941)** saves Datura hybrid embryos using coconut milk (liquid albumen) 1962: the active molecule has nucleic base characteristics.

<figure><img src="image_170.png" /><figcaption>Datura</figcaption></figure>

Independently:

* **Isolation of ribosyl zeatin from coconut milk (1974), zeatin from maize albumen, kinetin from yeast extract = cytokinins**

* Stimulates cell elongation and organogenesis of aerial parts

* Synthesis in roots

* Polarized transport to apex via raw sap (xylem)

* **Skoog and Miller (1957):** DNA degradation products stimulate cell proliferation

<figure><img src="image_171.png" /></figure>

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The Auxin/Cytokinin ratio matters for the type of organogenesis

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Exercice: auxine vs cytokinines

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a.

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b.

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c.

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
# Plant transformations

Goals, history and strategies
# Field of application of transgenesis

## Agronomy

- Stress tolerance (biotic/Abiotic)

- Seeds dormancy

- Disease resistance (bacterial/fungi)

- Nematode resistance

## FOOD

- Nutritional value

- Fruit ripening

- Vitamin boost

<figure><img src="image_177.png" /></figure>

Applications

## Industry

- Paper industry

- High value Oils

- Pigments/ dye

## Health

- antibodies

- Vaccines

- Therapeutic proteins
History of plant genetic transformations

<figure><img src="image_178.png" /></figure>
Overview of improving plant traits

<figure><img src="image_179.png" /></figure>

Adapted from Choudhury et al., 2021
Agrobacterium tumefaciens

* Crown gall disease caused by A. tumefaciens affects more than 600 species including grapevine, rice, beet...

* Collar: junction zone between the root apparatus and the aerial vegetative apparatus

* Widespread pathogen in the soil

* Gram-negative Bacteria

* Agrobacterium is a bacterial type close to Rhizobia involved in nitrogen-fixing symbiosis

* Infection process studied in 1977

* *Agrobacterium rhizogenes* causes a tumor enlarging the root hair

<figure><img src="image_180.png" /></figure>

Both *A.tumefaciens* and *A. rhizogenes* autonomously produce auxins and cytokinins
Plant transformation by *A.tumefaciens*

* Tumourisation involves plasmid (pTi or pRi for A.Rhizogenes)

* T-DNA integration within the host cell genome (transformation)

* Hijacking plant metabolism for bacterial benefit

* Plant develop tumerised tissues where bacteria can developed

* pTi was modified for plant engineering and human uses

<figure><img src="image_181.png" /></figure>
# Infection and tumourisation

* pTi plasmid contents:

- Double strand circular DNA of 190Kb

- T-DNA, the part integrated in the plant genome containing **genes for hormones synthesis and opine catabolism** (C&N)

- **Virulence genes**, necessary T-DNA single strand release (endonuclease) and export within the cell.

- **Origine of replication** for the plasmid

<figure><img src="image_182.png" /></figure>
# Specifications to engineer pTi for plant research?

* pTi is large and thus delicate to use for cloning (restriction sites)

* Most of the genes present on T-DNA are <u>not</u> necessary for T-DNA transfer and the functions encoded by « Vir » genes can be relocated on an another plasmid (« disarmed plasmid ») → birth of binary vectors

* Then a large collection binary vectors have been developed, they include:

* Right and left border for T-DNA integration in plants

* Gene selection in plants (Kanamycin, hygromycin, Basta)

* multiple clonalage site (MCS)

* Origine of replication (bacteria)

* Resistance to selection agent in bacteria (antibiotics)
Physical transformation method

1) « gene gun ».

* Gold particles are coated with transformant DNA (vector), and bombarded over plant tissue

* (+) Universal method (any plants, any type of cells)

* (-) requires specific equipments

* (+/-) Large DNA quantity necessary

* Transient transformation (stable if CIV)

<figure><img src="image_183.png" /></figure>

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Plant protoplast transformed by Electroporation or PEG

* Physical or chemical agent provokes membrane permeabilisation and thus vector DNA entry in the cells.

* (-) requires protoplast preparation (from leaf, cell culture, any plant: tobacco, Arabidopsis, rice...)

* (-) Requires a DIY process

* (+/-) Needs large quantity of DNA

* Transient transformation (stable if CIV)

<figure><img src="image_186.png" /></figure>
# CRISPR-Cas9: towards genome editing without transgenesis...

<figure><img src="image_187.png" /></figure>

<figure><img src="image_188.png" /></figure>

## the principle:

<figure><img src="image_189.png" /></figure>

Source: The Economist

Economist.com
<figure><img src="image_190.png" /></figure>

ARTICLE

Received 29 Aug 2016 | Accepted 16 Sep 2016 | Published 16 Nov 2016

DOI: 10.1038/ncomms13274

OPEN

# Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes

Sergei Svitashev¹, Christine Schwartz¹, Brian Lenderts¹, Joshua K. Young¹ & A. Mark Cigan¹,†

Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

**Simple injection of a Ribonucleoprotein cocktail !**

It works:

* in plants: e.g. corn embryo

* in animals: e.g. mouse zygote, and...

* human...

No T-DNA nor transgenesis required!

**The current debate: GMO or not GMO? (in legislative semantics)**
How should CRISPR-modified organisms be labelled?

July 25th 2018: the European Court of Justice classified the Genome Editing and Crispr selection techniques as GMO techniques. This ruling runs counter to developments elsewhere in the world: USA, Canada, Argentina, Israel and Australia did not take this kind of precaution.

**Expression of opinion**
in scientific publications

<figure><img src="image_191.png" /></figure>

frontiers
in Plant Science

# Development of Wheat With Hypoimmunogenic Gluten Obstructed by the Gene Editing Policy in Europe

Aurélie Jouanin<fnref n="1" />,²*, Lesley Boyd², Richard G. F. Visser<fnref n="1" /> and Marinus J. M. Smulders<fnref n="1" />*

Coeliac Disease (CD) is an auto-immune reaction to gluten in 1-2% of the human population. A gluten-free (GF) diet, excluding wheat, barley, and rye, is the only remedy. This diet is difficult to adhere to, partly because wheat gluten is added to

**Petition** from European research organizations

Regulating genome edited organisms as GMOs has negative consequences for agriculture, society and economy

On July 25th, the Court of Justice of the European Union (ECJ) ruled that organisms obtained by modern forms of mutagenesis such as CRISPR are not exempt from the EU GMO legislation. Consequently, genome edited organisms must comply with the strict conditions of the EU GMO legislation. This is in stark contrast with the opinion of the Advocate-General of the Court, which was published in January of this year and advised ruling otherwise. We regret the purely process-based interpretation of the legislation by the Court and conclude that the EU GMO legislation does not correctly reflect the current state of scientific knowledge. Organisms that have undergone simple and targeted genome edits by means of precision breeding and which do not contain foreign genes are at least as safe as if they were derived from classical breeding techniques. Therefore, we call upon all European authorities to quickly respond to this ruling and alter the legislation such that organisms containing such edits are not subject to the provisions of the GMO Directive but instead fall under the regulatory regime that applies to classically bred varieties. In the longer term, the GMO Directive should be thoroughly revised to correctly reflect scientific progress in biotechnology.

<footnote n="1">¹Plant Breeding, Wageningen University & Research, Wageningen, Netherlands, ²Genetics & Breeding Research, National Institute of Agricultural Botany, Cambridge, United Kingdom</footnote>
The end...

of this introductory conference