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Plant Biotechnology

• improved productivity (increased yields and nutritional value) traditionally met by crossbreeding and hybridization methods
• biotech method of plant transgenesis (transferring genes to plants directly) now used to increase productivity
• products of plant transgenesis –
 plants produce own pesticides
 plants resistant to herbicide
 protection against pathogen (viral, fungal, bacterial)
 tolerance of environmental stress
 increased nutritional value
 increased shelf life
 plant vaccines – Dr. Bost and Dr. Piller from UNCC


3 reasons for producing transgenic plants

1. improves agricultural, horticultural, or ornamental value of the plant
2. plants can act as living bioreactors for the inexpensive production of economically important proteins
3. plants can be used to study the action of genes during development and other biological processes


How many different types of transgenic plants

• researchers have produced more than 140 different transgenic plants including tomatoes, tobacco, rice, corn, potatoes, beans, cranberries, papayas, petunias, and poplars


Methods Used In Plant Transgenesis

• cloning – growing plants from single cells


Protoplast Fusion

• when a plant is injured a mass of cells called a callus may grow over the site of the wound
• these callus cells have the potential to become the entire plant and they are ideal for genetic manipulation
• cellulase is added to these callus cells to form protoplasts
• protoplasts from different species of plant are fused to form a cell that will grow into a hybrid plant (brocoflower)


Leaf Fragment Technique

• soil bacterium Agrobacter can infect wounds in plants and transfer its DNA called T-DNA from a plasmid called Ti (tumor-inducing) plasmid into the plant genome
• T-DNA products are produced by plant – phytohormones which cause plant cells to have uncontrolled growth to form tumor (crown gall) and opines (used as carbon and nitrogen source for Agrobacter)
• Ti plasmid is an ideal vehicle for biotechnologists to use to transfer genes of interest into plants to create transgenic plants – one method is leaf fragment
• 3 major steps – produce recombinant DNA, introduce recombinant plasmid into Agrobacter, culture Agrobacter with plant cells


selectable marker gene

marker gene for plant, E. coli, and Agrobacter


. origin of DNA replication that allows plasmid to replicate in

E. coli and Agrobacter


VIR Genes

sequence of DNA that codes for the molecules needed for the recombinant plasmid to be integrated into the plant chromosomal DNA


1. Producing Recombinant DNA
Ti Plasmid need-

1. selectable marker gene – marker gene for plant, E. coli, and Agrobacter
2. origin of DNA replication that allows plasmid to replicate in E. coli and Agrobacter
3. sequence of DNA that codes for the molecules needed for the recombinant plasmid to be integrated into the plant chromosomal DNA – these genes are called vir genes (sometimes this is provided by helper plasmid)
4. cloning site – site where the gene of interest can be inserted in place of T-DNA


plasmid system

1. make plasmid with gene of interest,
selectable markers, origin (cloning vector)
2. put cloning vector in Agrobacter –
Agrobacter has helper plasmid with ONLY vir genes
3. incubate plant with Agrobacter –
vir genes products from helper plasmid allow
the gene of interest from the cloning vector
to be incorporated into the plant genomic DNA


Introducing Recombinant DNA into Agrobacter

• same methods used to introduce plasmids into E. coli
• both cloning vector and helper plasmid introduced into bacteria
• selectable markers used to select for bacteria that have both plasmids


Culture Agrobacter with plant cells

• this method can only be used with dicots (tomatoes, potatoes, apples, grapes, roses, and soybeans) NOT monocots (corn and wheat)
• when the Agrobacter is incubated with the leaf fragments it attaches to the leaf fragments and gains entry into the cells through breaches in cell wall
• the chemicals produced by the wounded leaf cause the vir genes to be expressed
• vir gen products induce the gene of interest in the to be incorporated into the plant chromosomal genome
• plant cells stimulated to grow
into transgenic plants
(shoots stimulated by cytokinin
and roots by auxin)


A Method For Introducing Genes Into Agrobacter-resistant Plants Is Using Gene Guns

• gold particles (about the size of some bacterial cells) are coated with DNA – usually plasmid DNA is precipitated onto the particles
• the coated particles are accelerated to high speed with a gene gun
• the first guns used gunpowder to provide the propelling force but now high-pressure helium is used
• the extent of particle penetration into the target plant cells can be controlled by – varying the intensity of the particle propulsion, altering the distance that the particles travel before reaching the target cells, and using different sized particles
• once inside the cell, the DNA is removed from the particles and may integrate into the plant genome
• considerations –
 using Linear DNA increases transformation efficiency
 large fragments can be fragmented by this technique
 YAC can be used as vectors – up to 150 kb of DNA can be integrated into plant genome (entire biosynthetic pathways may be introduced into plants in the future)
 recombinant DNA must also have marker gene (also called reporter gene) – allows you to select ONLY cells that have taken up DNA


Targeting Foreign DNA to the Chloroplast Genome
• 2 ways of getting the DNA into the chloroplast

1. use DNA gun to shoot plasmid with gene of interest into chloroplast
2. plasmid with gene of interest also contains DNA which encodes additional amino acids that direct transport to the chloroplast (fusion gene) – fusion gene is inserted into plant chromosomal DNA
Diagram of Fusion Method


fusion protein

green part tells the cell to take the protein to the chloroplast, then this part is cleaved off


How DNA Is Inserted Into The Chloroplast Genome

• most common method utilizes 2 plasmids – 2 plasmids are integrated into the chloroplast DNA by homologous recombination
• having foreign DNA and selectable marker on the same plasmid interferes with the expression of the chloroplast genes so using 2 plasmids is better
• steps


How DNA Is Inserted Into The Chloroplast Genome

1. an equal mixture of 2 different plasmids is inserted into chloroplast
 one plasmid has a selectable marker flanked by DNA from one region of the chloroplast DNA
 the other plasmid has the gene of interest flanked by DNA from a different region of the chloroplast genome
 both plasmids have promoters which allow transcription ONLY in the chloroplast
2. the 2 plasmids insert into the chloroplast genome by homologous recombination – insertion does not disrupt any chloroplast genes
3. cells that have incorporated plasmids are selected by incubating cells in specific antibiotic- about 30% plant cells incorporate both selectable marker and gene of interest
4. cells grow into mature plants – isolate plants that express gene of interest product
• benefits of inserting gene of interest into chloroplast genome


Antisense Technology

Flavr SavrTM tomato
• one of the first commercial transgenic plant procuts
• used Agrobacter to introduce antisense gene into plant


Vaccines For Plants

• viral infections can lead to reduced growth rate, poor crop yield. And low crop quality
• farmers can protect crops by vaccinating – dead or weakened strain of virus is injected into plants, plant’s immune system is induced to protect plant
• vaccinating entire field is very difficult
• biotech provides easier alternative – plant genetically engineered to have genes the code for vaccines (or a portion of pathogen which induces immune system)
• Example
Tobacco plants genetically engineered to have gene for protein on surface of Tobacco Mosaic Virus (TMV)


Genetic Pesticides

for 35 years farmers have relied on the natural bacterial pesticide, Bacillus thuringiensis (Bt)
• Bt cells produce a crystallized protein that kills harmful insects and their larvae – farmers spread spores of the bacterium across their fields
• now farmers can genetically engineer plants to produce Bt toxin - has been successfully introduced into tobacco, tomato, corn, and cotton
• controversy in 1999 – researchers found that Bt toxin in pollen of genetically engineered corn plants can also kill monarch butterflies in the lab
• in 2002 USDA announced that Bt toxin posed little risk to monarch butterflies in real-world situations – amount of Bt toxin in corn pollen transferred to nearby milkweed plants very little and butterflies consume very little toxin


Herbicide Resistance

• traditional weed killers have a fundamental flaw – they also kill desirable plants
• today crops can be genetically engineered to be resistant to common herbicides like glyphosphate (blocks an enzyme needed for photosynthesis)
• crops have been genetically engineered to produce an enzyme that is not affect by glyphosphate – especially successful in soybeans
• farmers who plant herbicide-resistant plants can control weeds with chemicals that are milder and more environmentally friendly


Enhanced Nutrition

• biotechnology providing solution for the world’s malnutrition induced health problems
• the most prevalent cause of blindness in children worldwide is due to vitamin A deficiency
• rice has been genetically engineered to contain a gene for beta carotene (provitamin which the body converts to vitamin A) – gene isolated from daffodils