Plant Biotechnology and Genetics: Principles, Techniques and Applications

(Brent) #1

The method that may hold the most promise for future increases in efficiency of
Agrobacterium-mediated transformation is to alter the response of infected plant cells to
the bacterium. During the interaction betweenAgrobacteriumand plant cells, elevated per-
oxidase activity and subsequent oxidation may cause tissue browning and cell death.
Improvements have been made in transformation frequencies following the addition of
reducing agents, which minimize the effects of oxidizing agents produced by infected
plant tissues. The most commonly used agents are cysteine, silver nitrate, and ascorbic
acid. In addition to reducing agents, enormous potential exists for using agents and
genes that eliminate or reduce programmed cell death (PCD) in target tissues. Although
PCD is a good natural defense mechanism for sequestering or localizing an infection and
preventing spread, a reversal of this defense leads to higher transformation efficiency.
Certainly, additional optimization strategies are possible, and the basic evaluation of com-
patible plant germplasm with differentAgrobacteriumstrains is always the best place to
start. Timing of coculture periods along with determination of media for optimum plant
tissue growth is always important.


10.3.4 Agroinfiltration

Certain situations exist where rapid manipulation of gene expression is needed, but it is not
necessary to transform a cell and take the time to recover a whole transformed plant. Why?
In some cases, the effects of introducing a new gene or lowering the levels of expression of
a native gene can be very quickly determined usingagroinfiltration(or agroinfection). For
agroinfiltration (Vaucheret 1994),Agrobacteriumis injected or infiltrated into leaves of a
suitable target plant, notablyNicotiana benthamiana, where large numbers of leaf cells
are transformed. For this method, anAgrobacteriumsuspension is forced into the internal
leaf airspace by tightly holding a syringe (without the needle) to the leaf and pushing the
plunger. A variation of this method requires dipping the plant into anAgrobacteriumsus-
pension to wet the leaves and then applying vacuum to force the bacterium into the internal
leaf airspace. To enhance the levels of gene delivery and spread, the T-DNA can be modi-
fied to contain viral gene components to launch the viral amplification and transfer machin-
ery, making this method very efficient for production of transgene product (Fig. 10.5) in
plants without transfer to the next generation.


10.3.5 Arabidopsis Floral Dip

Arabidopsishas become and remains the model for plant genomics. The genome and the
plant itself are small, the generation time is rapid, and it is ridiculously easy to transform.
The floral dip method was developed forAgrobacterium-mediated transformation of
Arabidopsis, and no other plant currently responds similarly, even after extensive research
efforts. Floral dip results in generation of independent transgenic seed, probably as a result
ofAgrobacterium-mediated transformation of the female gametophyte or the egg. For floral
dip,Arabidopsisplants are grown to the flowering stage or just prior to flowering. The
plants are simply immersed in a suspension ofAgrobacterium, containing the wetting
agent, Silwetw, a detergent that reduces surface tension and allows good access of the bac-
terial suspension to the cracks, crevices, and pores on the plant. After the dipping treatment,
plants are maintained under high humidity for a few days and allowed to eventually flower
and set seed. SinceArabidopsisproduces so many seed and the plants are so small, seeds
can be easily planted on selective media or seedlings/plants can be screened for a certain
characteristic or phenotype to recover whole transgenic plants.


254 TRANSGENIC PLANT PRODUCTION
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