ANS/PLSS 433:
Transgenic Plants

I.  Recombinant DNA Approaches to crop breeding

	Genetic engineering allows the transfer to plants of genes from 
widely different families and even bacteria or animals! Additionally 
protein engineering can substantially alter plant proteins in ways 
impossible by breeding and selection. All these transfers rely on plant 
transformation by foreign selectable DNA. Genetically engineered crop 
plants require a system for:

	1. Introduction of the genes of interest to germ line cells.
	2. Stable maintenance of transforming DNA.
	3. Transmission to subsequent generations.
	4. Appropriate expression levels and patterns.

Three systems are being explored bacterial, viral and naked DNA.

Plant viruses as vectors: 
	
	Viruses which give systemic infections might be useful vectors. 
They give high copy number and therefore high levels of gene expression. 
They can be disabled to reduce disease symptoms. Host range is limited and 
are oftentimes not seed transmitted, however they do pose containment 
problems in the eyes of industry regulators. They do serve as useful source of powerful plant promoters to express high levels of herbicide resistance.

Agrobacterium Ti plasmid vectors: 

	Crown gall is a growth of disorganized callus induced on dicot and 
gymnosperm plants by the bacterium A.tumefaciens. The callus grows 
independently of hormone application and produces opines characteristic of 
the infecting strain. It was discovered A.tumefaciens alters plant cell 
metabolism by transforming the plant cell with a fragment of the Ti 
plasmid called T-DNA by a process analogous to bacterial conjugation. The 
Ti plasmid carries the genes for DNA transfer the vir genes outside the 
T-DNA. All the T-DNA except for the 17-25 bp borders can be deleted and 
replaced  with unlimited amounts of rDNA. T-DNA vectors can be constructed 
to replicate in E.coli to facilitate in vitro manipulation then passed 
back to A.tumefaciens for plant transformation. Transformation is cellular 
not systemic so we must identify a transformed cell and induce it to give 
rise to a whole plant using plant tissue culture. Recombinant T-DNA always 
contains a herbicide resistance gene as a selectable marker. This is a 
bacterial gene with a promoter from a plant gene, plant virus gene or 
T-DNA gene. Transformed Plant cells need to be capable of forming callus, 
somatic embryos or organogenesis from single cells.   Agrobacterium 
transformation of cereals and legumes has not given transformed plants 
reproducibly as Agrobacteria transform the cells at low frequencies 
(1 in 10 million) and then inhibit regeneration by secretion of undefined 
factors.

Naked DNA Transformation: 

	Plant protoplasts will naturally take up DNA.  Alternately 
protoplasts can be immobilized and injected with DNA. Unfortunately plant 
regeneration from protoplasts is not easy with cereals although with 
legumes it may be possible using somatic embryogenesis from protoplast 
derived callus. Using the Gene gun we are able to transform organized 
tissues with naked DNA. DNA is coated on tungsten microprojectiles, 
shot 1/100 th the size of cells, which carry the DNA deep into cells and 
organelles. Transformed tissue is then induced to regenerate, usually by 
somatic embryogenesis. This technique has been successful in several 
cereal species including maize and rice. Many different tissues can be used.
The limit of its application is the availability of a regeneration system 
and low efficiency of stable transformation (1 in 1 million cells treated).


II.  Biotechnology and Plant Breeding 

(Molecular Biotechnology Chapter 13 and Molecular Approaches 
	to Crop Improvement Chapter 5)

Introduction:

	Herbicides generally affect processes found in plants but not 
animals (amino acid synthesis, photosynthesis) like antibiotics inhibit 
microbial processes not found in animals. These processes are shared by 
crops and weeds so finding useful selective herbicides is difficult.   
Two kinds of herbicides exist, BROAD SPECTRUM which kill all plants 
(Glyphosate, Basta) and SELECTIVE which kill some plants (eg all dicots by 
24D, corn not soybean by alachlor, soybean not corn by atrazine). An 
alternative approach to constantly searching for new selective herbicides 
is to transform crop plants with broad spectrum herbicide detoxifying 
genes so they become resistant to broad spectrum herbicides.

Weeds, Agriculture, and Herbicides.

	The US herbicide market of 200,000 metric tonnes is worth 
$4 billion/year, the largest agricultural chemical market, so many 
companies are competing. Existing Agro chemical companies look on this 
field of Plant Biotechnology as an opportunity to increase market share 
and increase use of particular herbicides so investment is heavy. 
In addition the companies can avoid the expense of developing and getting 
regulatory approval for new selective herbicide. Farmers who rely on 
herbicide for 35% of their annual yield (worth $14 billion), like the 
technology for the ease of rotation of crops. eg. Currently atrazine used 
on corn is damaging to soybean. Breeders like the technology for variety 
protection/identification. Environmentalists should like the technology 
because the broad spectrum herbicides are safer than the selectives 
because of their structures. However many fear herbicide resistant crops 
will mean increased herbicide use. Currently using selective herbicides 
10% of the US crop (worth $4 billion) is still lost to weed damage, this 
represents an economic break even point for farmers which will not shift 
unless herbicide or crop prices drop or new laws pass!

	Microbial degradation of Herbicides is important for two reasons. 
First to reduce persistence in the soil and contamination of ground water. 
Second as a source of herbicide resistance genes for transgenic plants.

Selective Aromatics:

	Acetamides; Alachlor, Metolachlor, Propachlor (Lasso, Dual, Ramrod)
	Carbamates; Chlopropham and Propham Dinitroanilines Treflan; 
	Diphenyl ethers; oxyfluorfen (Goal); Nitriles bromoxynil (Buctril);
	Imidazolinones (Pursuit); and Sulfonylureas (Accent, Beacon). 

	All give Selective control of weeds but disappear slowly 
(half lives of 4-6 weeks) and are incompletely mineralized by microbes 
leaving persistent benzene ring compounds in soil, groundwater, rivers, 
 
Broad spectrum, non aromatics:

	Organophosphorus glyphosate (Roundup) phosphinothricin (Basta)

	Both are completely degraded by soil bacteria though roundup 
degradation can be inhibited by the prescence of inorganic Phosphorous 
except  Flavobacteria.

III.	Genetic engineering of Plants to Herbicide resistance.

Phosphinothricin (Ignite) resistant crops:

Mode of action

	 PPT is a competitive inhibitor of Glutamine synthetase, the only 
enzyme capable of assimilating ammonia, turning sugar to aminoacid. Kills 
plants quickly especially foliar applications. PPT is an analog of 
glutamate, one substrate of GS, it inhibits GS but not other 
glutamate using enzymes. Resistance was engineered from a soil organism 
which produces phophinothricin (Streptomyces hygroscopicus). Fortunately a 
simple single gene (bar) product inactivates PPT by acetylation. To 
produce herbicide resistant plants the gene was tailored, the GTG 
initiation codon changed to ATG, the bacterial promoter replaced with a 
plant promoter. After plant transformation Expression of bar at 
0.001% to 0.01% of soluble protein gave total protection from herbicide. 
Weed control increased potato yield 11-51%. At four fold over recomended 
application concentration a yield loss of 20% was observed. PPT is being 
heavily used in Germany where atrazine is banned, Hoescht recently received 
a US label for corn (Ignite). PPTR is used as a selectable marker for corn 
and soybean transformation so all biotech companies already have PPT 
resistant plants "on the shelf". 

Glyphosate resistant crop plants:
 
Mode of action: 

	Glyphosate inhibits the enzyme EPSPS (5-enolpyruvyl-shikimate-3-phosphate 
synthase) an enzyme crucial for aromatic amino acid biosynthesis 
Tryptophane, Tyrosine, Phenyl alanine 3 of the 10 essential aminoacids. 
Essential aminoacids are not made by animals only microbes and plants so 
there is no toxic effect on humans. Glyphosate is an analog of phosphoenol 
pyruvate one substrate of EPSPS, it competitively inhibits EPSPS.

Resistant transgenic plants: 

	Were first produced from a bacterial mutant resistant to glyphosate.
Mutagenesis of Salmonella produced two classes of resistance mutants in 
aroA the gene encoding EPSPS. The first overproduced EPSPS, the second 
contained a point mutation which increases the specificity of EPSPS 
excluding glyphosate from the active site of the enzyme. Plants expressing 
these genes were incompletely Glyphosate tolerant even after modification 
to direct the bacterial EPSPS to the chloroplast, bacterial genes don't 
get expressed well in plant because of different codon usages. Plant EPSPS 
genes were isolated as cDNAs from a tissue culture cell line resistant to 
glyphosate. The enzyme was not a mutant just overexpressed. Transgenic 
plant overexpressing plant EPSPS were glyphosate resistant to 4 fold the 
killing dose. Point mutations in the plant gene gave even higher resistance 
when overexpressed but reduced catalytic efficiency. Unfortunately 
meristems can be more sensitive as glyphosate is phloem mobile and  
accumulates there. This problem can be overcome using promoters which give 
high levels of expression in meristems.

IV.  Insect Resistant crops with Bacillus thuringiensis

	(Bio/Technology March 1992 p271 -275)

Introduction: 

	13% of crop yield is lost to insect pests. Innate resistance would 
eliminate the cost ($1bn in the US) and danger of pesticide application. 
Present chemicals are broad spectrum and kill useful insects, and can 
accumulate in the environment. Bt toxin was thought to be active only 
against caterpillars (Lepidoptera) but new strains have been discovered. 
Consequently the Bt toxin market worth $24m in 1980 and $107m in 1989 is 
projected to be worth $300m by 1999!

Bacillus Thuriniensis:

	Bacillus thuringiensis is a gram positive spore forming bacterium 
characterized by a parasporal crystalline protein inclusion. They are 
often sprayed on crops at 10-50 g/acre and are 300 fold more active than 
the best chemicals. The crystalline inclusion dissolves in the insect 
midgut releasing several protoxins which are proteolytically cleaved by 
the insect to release active toxin. Activated toxin binds to a receptor 
molecule on the midgut lining creating pores that interrupt osmotic balance 
leading to cell lysis, cell lysis caused by each crystal is specific to one 
or a few species. The receptor molecules differ in sensitive and resistant 
insects. When insect Bt resistant mutants arise they usually alter the 
shape of the receptor.

Diversity in toxins:

	Most of the 3000+ strains characterized are active against 
Lepidoptera (caterpillars, armyworm), Diptera (mosquito, blackfly, housefly), 
Coleoptera (beetles, corn rootworm).

	Recently discoveries of nematode and flatworm active strains suggests any eukaryote is a potential target for a particular strain! Toxin genes are encoded by self transmissible plasmids which direct 33-45% of total protein synthesis during spore formation. Engineered toxins might replace strain screening in the future.
Delivery systems.  Traditional systems relied on spore sprays which did not
persist. Recently Pseudomonas fluorescens strains were found to provide 
enhanced delivery in killed cells (M-trak Mycogen Inc.). Bacterial delivery 
system will allow rapid cycling of pesticides and avoid pest resistance. 
The use of dead cells reduces the possibility of gene escape. However at 
least 5 companies are putting Bt genes into plants to eliminate application
costs. Problems in plant expression include codon usage, mRNA destabilizing 
sequences which prevent high expression. Whole genes have been resynthesized
to be more like plant genes. Also each gene confers resistance to one pest, 
often chemical pesticides will control several pests. In addition farmers 
are used to fast knock down of pests, whereas Bt poisoned insect live on, 
just cease feeding.

V.  Insect Resistance with Proteinase inhibitors

	(Chapter 4  Molecular Approaches to crop improvement)

	Protease inhibitors are 8Kd to 20 Kd plant proteins which inhibit 
1 or 2 of the 5 known classes of proteases, usually inhibiting all members 
of a class by blocking the protease binding site. They represent a natural 
plant defense to insect feeding which can be enhanced by biotechnology. 
Inhibition is lost at low pH (mammalian gut) but does deter insect feeding.
Cowpea typsin inhibitor has been used to create transgenic plants 
simultaneously resistant to  several insect pests. However very high 
expression levels (1-2% total protein) and very effective inhibitors will 
be necessary to match Bt and chemical performances. Resistant mutant insects
are very unlikely to arise as they would need to alter very many enzymes 
in their gut. Pyramiding resistance genes in transgenic plants is possible.

VI.  Virus Resistant Plants

Introduction: 

	Major crop losses occur to viruses, perhaps 10% of yields per year. 
Currently the are few control agents so the farmer relies on the breeding 
of resistant varieties and the production of virus free seed. Unfortunately 
useful resistance genes are not always available in every crop.

	Virus coat protein genes isolated, linked to the CaMV35S promoter 
and transferred to plant cells. Regenerated plants were observed to be 
resistant to infection with the corresponding virus. In fact, virus symptoms
will develop at high infection titre but it is significantly delayed 
(14-28 d) often enough to prevent widespread crop losses. Some cross 
protection is observed so that a second type of virus is also delayed in 
infection! In field tests transgenic lines can be selected which perform 
as well as their progenitor varieties but give 40% better yield after early
disease inoculation. However concerns arise as to whether plant cells 
expressing these proteins would serve as incubators for new viruses, 
supplying coat to viroids for example. Recombination between virus already 
occur, (eg the human flu and duck flu in pigs). The plant genome is awash 
in cryptic virus like sequences. However packaging events are rare and 
very unlikely to also pick up the coat protein gene from the plant 
chromosome. Encouragingly mutant coat protein genes are more effective 
than the normal gene in cross-protection but are ineffective in virus 
particle formation.

	Ribozyme RNA or antisense RNA or satellite RNA can also be targeted
to reduce virus symptoms. Ribozymes can be designed to chop up the incoming 
viral RNA molecule during infection at almost any sequence, several such 
ribozymes in a single plant would confer great selective advantage. 
Antisense genes are often targeted to essential viral functions. Since 
most plants are RNA viruses and require the production of large amounts of 
DNA from the RNA genome many encode a powerful reverse transcriptase, 
often an antisense target. The virus proteins that cause systemic spread 
through plasmodesmata are also common targets. Antisense RNA targets the 
same genes but seeks to inhibit translation or stability of the mRNA 
transcript. Because they are sequence specific cross protection might not 
be as effective and resistance not durable. Viral DNA replication is very 
error prone as is their transcription leading to high mutation rates.

	Satellite RNAs are present in many virus particles. These RNAs are 
parasitizing the virus! Competing for replication and reducing infective 
viral transmission. Engineering plant cells to produce these satellite 
RNAs can confer virus resistance. However the satellite mRNAs can be 
pathogenic or can mutate to become pathogenic.

VII.  Flower color 

	Flower color can be altered by over or under expressing any of the 
genes involved in anthocyanin biosynthesis. About 20 cytochromes are 
involved along with Chalcone isomerase, chalcone synthase and 
Phenylalanine ammonia lyase. Overexpression or antisense expression of any 
of these can cause unusual color pattern development by cosupression of 
gene expression. The same mechanism explains viral resistance mediated by 
coat proteins.


VIII.  Engineering Post-harvest Physiology

Introduction: 

	A major concern in the soft fruit and vegetable industry is 
spoilage caused by imperfections that develop in the crop after harvest. 
Some spoilage is caused by infection but most is caused by self digestion 
and then infection!  If this could be stopped spoilage could be reduced 
and maybe refrigeration would be unnecessary!

Flavr-Savr Tomatoes: 

	One of the enzymes that softens fruit is polygalacturonase (PG) 
which act to break down pectin in cell walls to simple sugars 
simultaneously softening and sweetening fruit. PG is synthesized de novo 
during ripening and was cDNA cloned by protein purification, sequencing 
and oligomer probe synthesis. PG expression in plants was reduced by 
expressing the PG cDNA backwards in transgenic tomatoes and produce the 
Flavr -Savr tomato that is slow to rot so that the tomato can be allowed 
to ripen on the vine. During vine ripening the tomato acquires flavor, 
organic acids and various volatiles that are absent from supermarket 
tomatoes which are presently picked green. 

The next Generation - Ethylene less fruit: 

	PG is only one of 20 or so enzymes which cause fruit ripening all 
are controlled or switched on by the build up of ethylene in plant tissues. 
Ethylene is produced from S-adenosyl-L-methionine by ACC synthase and then 
ACC oxidase (ACC=aminocyclopropane-1-carboxylic acid). cDNA clones for 
both enzymes have been isolated and used in antisense to prevent ripening 
and spoiling. Particularly useful in the tomato processing industry 
(Ragu etc.). However the gene sequences evolve so fast that the gene must 
be reisolated from each species targeted for antisense. ACC deaminase, an 
enzyme which destroys ACC and thus prevents ethylene synthesis was cloned 
from bacteria and expressed in plants where ripening and spoiling were 
greatly retarded at room temperature! The deaminase is not sequence 
specific so can be used in all crops. 

Fungal Resistance:

       Tomatoes are also targets for general fungal resistance genes. 
Chitinases are produced by many plants to ward off chitin containing fungi. 
In tomato these may prevent rot after harvest. 

IX.  Modification of food and feed proteins

Introduction: 

	Increased demand for animal protein in human diet caused a shift 
to intensive rearing of farm animals and the use of soybean and corn as 
animal feeds which are deficient in Methionine Cysteine and Lysine.

	Seed storage Proteins containing lots of these aminoacids are 
found in several other plants and the genes cloned. Transgenic tobacco 
plant accumulate upto 8% soluble protein as the transgene. Soybean 
accumulated only 0.1% however presumably because it makes too little S 
aminoacids. The aminoacid synthesis is subject to endproduct inhibition. 
Cloning genes which are inhibited and overexpressing them may produce 
large seeded crops with high yields and high essential aminoacid contents.