Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianBrassica juncea lines |
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Authors: | Anand Pandian Clive Hurlstone Qing Liu Surinder Singh Phil Salisbury Allan Green |
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Institution: | (1) CSIRO Plant Industry, PO Box 1600, 2601 Canberra, ACT, Australia;(2) The University of Melbourne, 3010 Victoria, Australia |
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Abstract: | In recent years,Brassica species have acquired an important position in the oilseed industry. Even thoughBrassica transformation protocols are well established,there is still a need for the development of new transformation protocols for
elite AustralianB. juncea lines,because regeneration inB. juncea is highly genotype-dependent and in addition, their hypocotyl explants are susceptible to necrosis.Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianB. juncea lines is described here. To overcome necrosis, we have adopted 2 strategies: extension of precultivation time of hypocotyl
explants, and use of a 2-stage hygromycin selection process.The frequency of recovery of transformants from AustralianB. juncea andBrassica napus lines was 1.7% and 0.9%, respectively. Polymerase chain reaction tests confirmed that allBrassica plants that survived through stringent screening procedures were positive for the inserted hygromycin resistance gene,hph. Progeny from 6Brassica lines tested segregated for thehph gene, and χ2 analysis suggested a 3:1 segregation ratio.This is in line with a tDNA integration into a single locus, which is an important
feature of a transformation protocol for subsequent breeding purposes.
Although the scientific content of this article has been reviewed,the full-text Web publication has not been edited in detail. |
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Keywords: | Brassica transformation Agrobacterium necrosis PCR |
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