科研之友
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摘要
A protocol is developed for Agrobacterium-mediated genetic transformation of Amaranthus tricolor via explain co-cultivation with Agrobacterium rhizogenes. Bacteria-plant specific factors which influenced transformation were optimized. Of the two Agrobacterium strains employed, LBA9402 was more infectious compared to A4. Bacterial suspensions grown overnight with 100 mu M acetosyringone and experiencing O.D(.660) = 0.6 followed by dilution to a density of 10(9) cells ml(-1) were the most effective. Explants from garden-grown plants were more responsive than those from in vitro cultures; stem internodes being better than leaves. Immersion of the pre-pricked explants in bacterial suspension resulted in a markedly higher transformation frequency compared to the direct injection method. The infection of internode explants with the LBA9402 strain followed by co-cultivation on growth regulator-free MS medium (MSO) for 5 days resulted in emergence of hairy roots up to a maximum frequency of 97.22%. Roots were individually cultured in MSO, but fortified with bactericidal antibiotic (500 mu g ml(-1) cefotaxime). Rhizoclones showing prolific growth were renewed through successive subcultures in MSO. Opine gene expression was revealed by positive agropine and mannopine synthesis in all selected transformed rhizoclones. Shoot regeneration from root clones, capable of auxin-independent growth and opine proficiency, was stimulated in MS augmented with 2.0 mg l(-1) zeatin. pRi TL-DNA rolB and pRi TR-DNA man2 ORF were detected in leaf tissues of regenerated plants from selected hairy root clones through PCR amplification. The implication of such findings is discussed on the possibility of conferring protection to crop amaranths against biotic stress challenges, particularly due to insects, viruses or fungal pathogens.
- 出版日期2010-6-28
