Abstract
Abstract To establish a mass micropropagation procedure for Cephalotus follicularis, the effects of varying the strengths of solid Murashige Skoog (MS) medium were investigated using subcultured shoot explants. After a 60 day primary culture from root mass, the regenerated shoot explants were subcultured every 60 day in solid MS medium. To facilitate shoot proliferation, liquid MS (LMS) medium was applied with or without exogenous auxin and cytokinin. Our results demonstrate that shoot proliferation and survival of C. follicularis is most effective in modified MS (MMS) medium containing 1/5- or 1/10-strength macronutrients and full strength micronutrients. Successful shoot proliferation and development of C. follicularis explants were obtained in 1/5 or 1/10 modified liquid MS medium (MLMS) without auxin and cytokinin, or with addition of 5 □M IAA /1 □M BA for 45 day. The liquid medium consistently produced more explants than the solid medium and shortened the culturing time. Plantlets cultured in hormone free 1/5 MMS medium developed greater root systems. Using the liquid culture we established, vigorous plants with extensive roots were obtained within four months. Plant survival in the greenhouse reached 100%. The Arabidopsis thaliana gene At1g68290 was expressed as a C-terminal hexahistidine fusion protein, affinity purified from transgenic plants and its identity confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. Purified AtBFN2 digested RNA, single-stranded DNA, double-stranded homoduplex DNA and circular plasmids, with a substrate preference for single-stranded DNA and RNA. The AtBFN2 activity towards ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH, and it cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases, as effectively as it cleaves ssDNA, and more effectively than homoduplex DNA without mismatches. We conclude that AtBFN2 is a mismatch-specific endonuclease. The AtBFN2 expression was detected in expanding cotyledons, root tips and lateral roots, differentiating xylems, senescent leaves and floral organs, using GUS gene driven by a 1.2 kb AtBFN2 promoter. Strong GUS activity was observed in the abscission zone of siliques and in the ruptured testa, suggesting that AtBFN2 may be involved in plant programmed cell death. A decrease in AtBFN2 activity was accompanied with the delayed cotyledon expansion and reduced anthocyanin accumulation in the BFN2-RNAi transgenic plants.