Primary Construction Of Plant Regeneration System And Agrobacterium Tumefaciens-mediated Transformation System Of Halophyte Suaeda Salsa

Suaeda salsa is a kind of euhalophytes with significant succulence of the leaves. Itpossesses some specific mechanisms in salinity tolerance during the evolution. Suaedasalsa is native to saline soils and finishes its life cycle in the high salinity region in thenorth of China. Calli of Suaeda salsa also possess strong salt tolerance. Some aspects ofenvironmental stresses appear to be perceived similarly by cells and by whole plants,leading to similar biochemical and molecular reactions. Therefore, Suaeda salsa hasattracted much attention not only at whole plant but also at cell level because of its uniquemechanism of salt-tolerance and potential significance in genetic engineering in plantsalt-tolerance, which is regarded as the best strategy for breeding salt-tolerant crops onsaline soils. So far, physiological and molecular responses of Suaeda salsa to salinitystress have been extensively studied in our laboratory, which encourages us to set up thesystem of genetic transformation of Suaeda salsa.To our knowledge, this is the first study on an efficient plant regeneration protocol invitro and Agrobacterium tumefaciens-mediated genetic transformation for Suaeda salsa.Agrobacterium tumefaciens-mediated genetic transformation of Suaeda salsa should be apowerful technique to understand the mechanism of salt tolerance in halophytes such asSuaeda salsa by using antisense RNA and RNA interference. The main results of thisstudy are as follows: 1. An efficient plant regeneration protocol in vitro for Suaeda salsa was achieved viaorganogenesis from immature inflorescence explants. In this paper, effect of differentconcentration combinations of growth regulators on the organogenesis is examined.Adventitious shoots were induced from immature inflorescence cultured on MS medium(Murashige and Skoog, 1962) supplemented with 1.0 mg.L-1 6-benzylaminopurine (6-BA)and 0.4 mg.L-1 indole-3-acetic acid (IAA) for 25 days. Induction frequency ofadventitious shoots was about 82.1%. Adventitious shoots were further multipledvigorously and maintained for a long time on the same medium for adventitious shootinduction. Rooting was achieved on MS medium without any plant growth regulatorsafter adventitious shoot was transplanted for 2-3 weeks. We proposed that highconcentration of 6-benzyladenine (1.5 mg.L-1) inhibited shoot formation, and also resultedin vitrifiation.2. Effects of antibiotics on in-vitro callus induction of Suaeda salsa from hypocotylexplants were examined. With the increase of kanamycin concentration, the callusformation rate markedly decreased. Callus induction from hypocotyl explants wascompletely inhibited in presence of 30 mg.L-1 kanamycin. When 50 mg.L-1 kanamycinwas present in the callus induction medium, about 95% calli died. Results also showedthat 500 mg.L-1 piperalillin sodium had no significant influence on callus induction. 600mg.L-1 piperalillin sodium significantly inhibited callus induction. It was considered thatpiperalillin sodium (500 mg.L-1) was suitable for Suaeda salsa to elimination ofAgrobacterium tumefaciens during transgenic callus induction;kanamycin 30 mg.L-1 wassuitable in the selection of transgenic versus false-positive callus from hypocotyl explants.3. Agrobacterium tumefaciens-mediated transformation of hypocotyl explants fromSuaeda salsa was studied. Kanamycin-resistant calli were induced after co-cultivation ofhypocotyl segments with Agrobacterium tumefaciens strain LBA4404 that harbored abinary vector (pBI121) with the genes for GUS and NPTII. Using stable expressionfrenqueny of GUS gene, the optimal parameters for Agrobacterium tumefaciens-mediatedtransformation of Suaeda salsa were examined, such as bacterium concentration,preculture time, infection time, co-culture time and the adding way of acetosyringone.Histochemical GUS assay showed that preculture for 2d, infection for 5-l0min, co-culturefor 4d, bacterium concentration OD600 0.5 with 100 μmol.L-1 acetosyringone andco-culture medium with 100 μmol.L-1 acetosyringone are the optimal parameters for thehighest level of GUS gene expression. GUS gene expression rate reached about 63%.4. Successful transformation was confirmed by histochemical analysis of GUSactivity and PCR in kanamycin-resistant calli. The results showed that the extra geneNPTII was integrated into Suaeda salsa calli genomes and GUS gene had stableexpression. An efficient plant regeneration protocol from Agrobacteriumtumefaciens-mediated transformed calli needs to be studied.