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39.768 Plant Molecular Genetics Lecture 2, part 4 of 4

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e) Selection and regeneration of transformants.

i) transfer upside down to same medium, supplemented with 500 µg/ml carbenicillin (to kill A.t) and 300µg/ml kanamycin sulfate to select for transformed calli.

ii) In about 4 weeks shoots should appear from transformed calli. To promote shoot formation, you want a high cytokinin:auxin ratio, as was used above.

Brassica napus plantlets transformed with pBI1121-based construct regenerating in the presence of kanamycin, using the method of Moloney M. M., Walker M.J. and Sharma K.K. 1989. High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports 8:238-242.[P. Zhang (1999) Ph.D. thesis, University of Manitoba]

Typically, ~ 1/3 of regenerated shoots produce mature plants which express foreign genes, the remainder being escapes or transformants in which the genes have shut off.  For example, you often get transient expression of non-integrated DNA in the nucleus. This will still confer kanamycin resistance to the callus cells, but the gene will not be propogated to adult plants.
 

iii) Transfer plants to soil as soon as first roots are observed. This reduces shock to new root system.

Regenerated B. napus plant in soil [P. Zhang (1999) Ph.D. thesis, University of Manitoba]

f) Analysis and verification of gene expression in transformed plants.
 

-PCR: Quick; no copy number information

-Southern blot: Provides information on numbers of copies of gene inserted. To a first approximation, the number of bands in a digest indicate the number of T-DNA insertion sites.

-Northern blot; tells whether gene is expressed

-reporter gene assay (eg. assay for production of GUS). If reporter gene is separate from gene of interest, this assay is not always reliable. In some cases, the gene of interest is not expressed at the same level as the reporter gene. Additionally, in many cases, only part of the T-DNA inserted, excluding the gene of interest.

- antibiotic assay [Peijun Zhang, Ph.D. thesis, University of Manitoba, 1998].
It is often possible to screen plantlets for presence or absence of a transgene using the kanamycin resistance gene for selection. In transgenic B. napus, transformed plants expressing Kan^R show full root growth when plantlets are geminated and grown on media containing Kanamycin (left). Root growth of non-transformed plants is almost completely inhibited (right).

e) Selection of homozygous transgenic lines

For many practical purposes, lines homozygous for the transgene are needed. The only way to determine homozygosity is through Mendellian genetics. The progeny of parents from T1 or later generations can be screened for segregation of the transgene. If the plant has a single-copy insertion, homozygous parental lines can be identified by lack of segregation for the transgene. It is usually safest to assay directly for the gene of interest (eg. by PCR) rather than relying on the selective marker (eg. Kan^R). This is because one can often get partial T-DNA insertions, missing the gene of interest.
 
 

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39.768 Plant Molecular Genetics Lecture 2, part 4 of 4

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