Lectut btn-202-ppt-l36. genetic transformation of plants

Genetic Transformation of Plants

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Lecture- 36

Transformation methods in plants

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•Uptake of foreign DNA by seeds and seedlings

•Uptake of DNA by pollen

•Transformation of protoplasts

•Agrobacterium-mediated transformation

•Gene gun method

Uptake of foreign DNA by seeds and seedlings

• Most of the early plant transformation experimentswere done with whole plants system such as seedsand seedlings.

• It was shown that exogenous DNA was not onlytaken up by plants but also integrated into the hostgenome.

• The evidence for integration and replication of theexogenous DNA was based mainly on isopyniccentrifugation of cesium chloride(CsCl).

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Experiments to testify Integration and replication

• Radioactive labeled DNA of density different fromthe host DNA was administered to the seedlings.

• DNA from seedlings (after a period of metabolism)was isolated for analysis on CsCl gradients.

• Evidence for integration of the radioactive DNA wasascertained by the occurrence of a radioactive bandapproximately intermediate in density between thehost and donor DNA.

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• This band was further analyzed by sonicationand denaturation.

• Replication follows the same procedure but differs only in respect of the donor DNA, which is non-radioactive here.

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Uptake of DNA by pollen

• In 1976, Hess and co-workers incubated Nicotiana glaucapollen with DNA isolated from Nicotiana langsdorffii andused this pollen to pollinate emasculated N. glaucaflowers.

• This led to the production of the transformed plants whichdeveloped tumors at the wound site of the stem.

• This is a characteristic feature exhibited by the sexualhybrids arising between these two species.

• This suggests that pollen can act as a vehicle for thetransfer of foreign genes into plants.

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Testifying uptake of DNA by pollen

• De la Pena et al. (1987) manually injected young floraltillers of rye plant with a solution of plasmid DNAcarrying a chimeric gene consisting of T-DNA nopalinesynthase gene flanking coding sequence of the Tn5 neogene.

• Injected flowers were pollinated with normal pollenand progeny seeds plated on kanamycin medium.

• Of the 300 seeds only two seedlings showing traits ofkanamycin as well as nos-neo gene were isolated.

• Thus it was concluded that the injected DNA was takenup. 7

Transformation of protoplasts

• The isolated protoplasts system has provedmost promising for genetic modification ofcells.

• In the absence of a wall around the plasmamembrane, protoplasts are not only able tofuse but also can take up chloroplasts, nuclei,micro-organisms and isolated DNA.

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METHODS OF PROTOPLAST TRANSFORMATION

UPTAKE OF ORGANELLES

UPTAKE OF VIRUSES/MICROORGANISMS

TRANSFORMATION USING AGAROBACTERIUM SYSTEM

DIRECT DNA TRANSFER METHODS

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Uptake of organelles

Chloroplast

•Uptake of chloroplasts by albino protoplastsisolated from Petunia, tobacco and carrotplants was reported in during 1973-74.

• This required exposing a mixture of albinoprotoplasts and chloroplasts to PEG in amanner similar to protoplast fusion.

• EM studies revealed that PEG caused thefusion of two external membranes of theplastids and protoplasts. 10

Mitochondria

• No evidence supports the direct uptake of isolatedmitochondria by protoplasts however there arereports on incorporation of mitochondrial DNAthrough protoplasts fusion.

• This leads to the formation of cybrids.

• It may also be possible to transfer traits such astoxin sensitivity of Texas male sterile cytoplasmencoded by mitochondrial DNA in maize.

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Nucleus

• Potrykus and Hoffman reported uptake of isolated nuclei by mesophyll protoplasts of Petunia, tobacco and maize.

• Transplantation success improved from 0.5% to 5% in the presence of PEG and Ca++.

• The uptake of isolated chromosome instead of complete nuclei is yet to be explored.

• Transfer of micronuclei into protoplasts has also been attempted. 12

Uptake of viruses/microorganisms

• Viruses have been used as vectors for genetic modification.

• Experiments on uptake of tobacco mosaic virus (TMV) by isolated protoplasts were initiated in 1969.

• Most plant viruses have encapsulated genomes and it is assumed that no specific virus receptor site exists on the exterior of the plasma membrane.

• Factors to be considered while selecting progenitor (virus) are : 1. amenability to genetic engineering techniques, 2. packaging constraints in the particle and 3. development of a system in which expression can be studied.

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Contd…

• The cauliflower mosaic virus (CaMV) is the most amenable and widely chosen progenitor.

• The ssDNA genome in CaMV has appropriate restriction sites compatible with the methodology developed for A. tumefaciensplasmid.

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Transformation using Agrobacteriumsystem

• Agrobacterium tumefaciens is a soil bacterium, has the ability to infect most dicotyledonous plants usually at a wounded site.

• The tissue around the infected wound develops a neoplastic growth known as gall tumor.

• The tumor tissue excised from the plant is capable of growing on hormone free medium.

• In culture, the crown gall tissue produces a set of metabolites termed as opines

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Contd…

• Agrobacterium harbors the Ti Plasmid, which is directly responsible for tumor induction.

• The transfer of small DNA segments from this plasmid and their integration into the genome of a host cell induces tumor formation in the plants.

• Agrobacterium rhizogenes is another bacterium used in plant genetic manipulations.

• The infections by this bacterium causes hairy roots which are of clonal origin.

• The tumor-inducing properties of rhizogenes strains are also carried on a large Ri plasmid DNA(Ri T-DNA).

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Infection of plant cells with Agrobacterium

• Agrobacterium engineered with a desired foreign geneis directly inoculated at the wound site after removingthe top leaves and apical meristem of plant.

• Non-oncogenic vectors do not induce tumourformation and instead a wound callus proliferates in 3-4 weeks.

• The transformed callus tissue is excised and cultured inthe medium supplemented with an appropriatecombination of growth regulators to regeneratetransformed fertile plant.

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Other approaches

Co-cultivation with protoplast

• Protoplasts are co-cultivated with Agrobacterium for few days, washed and then cultured in antibiotic containing medium (to eliminate bacteria).

• These protoplasts are regenerated in a suitable medium enabling the development of transformed callus, shoots and plants.

Leaf disc infection method

• A leaf disc of a plant is inoculated with A. tumefaciens strain having modified tumour inducing plasmid and cultured for 2 days.

• The leaf disc which develops infection is transferred to a selection media enriched only with kanamycin and further cultured to form plants.

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Contd…

Floral dip method

• Used for genetic transformation of Arabidopsis thalianaplants.

• Primary and secondary inflorescence shoots are cut attheir bases and inoculated with A. tumefaciens cellsuspension, carrying binary vector with an insert, at thewound sites.

• After 3 successive inoculations at weekly intervals, treatedplants are grown to maturity and seeds are harvested.

• Progeny raised from these seeds is screened fortransformants on a selective medium.

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