Primary Establishment Of Plant Agrobacterium Tumefaciens-mediated Transformation System Of Euhalophyte Suaeda Salsa

Halophytes are a class of the natural plant taxa which can complete their life cycle in the saline with salt content equivalent of 200 mM NaCl or higher. They developed a series of survival strategy in particular to adapt to the high salinity in the process of co-evolution with the saline soil. Therefore, studies on genetic and molecular mechanism of salt tolerance of halophytes will be very important for cultivating salt-tolerant crops through biothechnology, such as genetic engineering, which could have important ramification in exploitating saline-alkali soil. However, transformation systems of euhalophytes have not yet been established. Suaeda salsa L. is a kind of succulent euhalophyte, which can completed the life cycle in the high saline soil, and even intertidal zone. Agrobacterium-mediated transformation is the most widely used methods for plant molecular research. In the present paper, stem tips of S. salsa L. were used as explant to examine the factors affecting agrobacterium-mediated transformation efficiency.The main results are as follows:1. Stem tips of S. salsa L. were used as explant to study effects of antibiotics on in-vitro callus induction. With the increase of hygromycin concentration, the meristem differentiation rate markedly decreased. Meristem differentiation from shoot explants was completely inhibited in presence of 10 mg.L-1 hygromycin, about 100% shoot died. It was considered that piperalillin sodium (500 mg.L-1) was suitable for Suaeda salsa to elimination of Agrobacterium tumefaciens during transgenic callus induction; hygromycin 10 mg.L-1 was suitable in the selection of transgenic versus false-positive shoot from shoot explants.2. Stem tips of S. salsa L. were used as explant to study agrobacterium tumefaciens-mediated transformation efficiency. Hygromycin-resistant shoot were induced after co-cultivation of shoot with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI1301) with the genes for GUS and HPT. Using stable expression frenqueny of GUS gene, the optimal parameters for Agrobacterium tumefaciens-mediated transformation of Suaeda salsa were examined, such as bacterium concentration, preculture time, infection time, co-culture time and recovery time. Histochemical GUS assay showed that the optimal parameters for Agrobacterium tumefaciens-mediated transformation of Suaeda salsa are as follows: one to two millimeters of stem tip of the 10 day-old sterile seedlings, preculture for 2 days, infection for l0 min, co-culture for 2 days, bacterium concentration OD600=0.5.Then the shoot tip to restore in the differentiation medium 2 to 3 days and eventually transferred to selection medium containing 3 or 4 consecutive screening generations, each generation for 12 days, leading to a tansformation frequency of about 3%.3. The described above successful transformation systems was further confirmed by histochemical analysis of GUS activity and PCR in hygromycin-resistant shoot. The results showed that the extra gene Hyg was integrated into Suaeda salsa shoot genomes and GUS gene had stable expression. An efficient plant regeneration protocol from Agrobacterium tumefaciens-mediated transformed shoot is being studied.