Study On Agrobacterium-Mediated Transformation Of Zyziphus Jujube With S6PDH Gene

Zyziphus jujube Mill. is a native fruit crop with an important economical and ecological value in China. The fruit was widely favorited to Chinese, eastern Asian, southeastern Asian. Presently , Chinese date was mostly cultured in the Huanghe River valley, most of the cultural area was attributed to semi-aridity and aridity, salted soil , low temperature and drought environment, which contributed to low yield, coarse quality and low-degree of horticulturalization. All these bad conditions could lead to non-biological stress. The stress was caused by the osmotic unbalance between intracellular and extracellular. Plant cell can accumulate some low weight substance to alleviate the stress, such as sorbitol. Sorbitol-6-phosphate dehydrogenase (S6PDH) is confirmed as a key role in the biosynthesis of sorbitol in Rosaceae. In our research, initially, we prepare to construct a regeneration system from vitro leaves of jujube. And then, Agrobacterium-mediated genetic transformation system was studied and obtain the transformant of jujube containing S6PDH gene in the end. Therefore , jujube tree would be given the ability of synthesizing sorbitol increasing the resistant ability of jujube tree for osmotic stress. The main results of the experiments are as follows.1. Regeneration system of in vitro leaves of Muzao jujube was construced.1.1 Through a experiment with random blocks design, the adventious bud directly regenerated from in vitro leaves of three cultivares was initially studied. The research results showed that TDZ can high efficient induce the regeneration of adventitious bud from leaves, by contrast, BA didn't have so effect. During the process of regeneration, The brown phenomenon of callus was one of the key factors to decrease the adventitious bud regeneration ability, meanwhile, the adventitious bud haredly normally develop into shoot. The research results also showed that the highest regeneration pencentage of Muzao, on the regeneration medium(MS+1.0mg/L TDZ+0.8mg/L IBA) , reached 73.3% , and 76.7% for Goutouzao and Tuanzao on the medium (MS+2.0mg/L TDZ+0.4mg/L IBA) ,the fomer was significantly lower than latter two cultivares on the highest regeneration frequency.We then studed the regeneration system on Muzao in detail.1.2 AgNO₃ supplemented to the MS medium , containing 1.0mg/L TDZ and 0.8mg/L IBA , has a significant effect on lightening brown, the regeneration efficiency was also promoted to 94.4%. By contrast, PVP and Vc added to the above medium didn't help to differentiation of the adventitious bud, AC was harmful to callus differentiation.1.3 In the initial culture stage, dark period was critical for the differentiation of adventitious bud, the research results showed 21 days long is good for it. The regeneration efficiency of leaf explants originated from rooted shoots was higher than multiplication shoots. Based on the gradually optimization, the frequency of adventitious bud regeneration of Muzao reached 98.3% .1.4 The subculture for elongation was very important for adventitious shoot deveplopment. The adventitious bud appearance in 20^th day after inoculation, then, the leaf explants should be immediately transferred to elongation medium.If the PGR combination was 0.1mg/L TDZ and 0.2mg/L IBA, the adventitious bud will developed into cluster short shoots. Compared with it, the combination adopted 0.6mg/L BA and 0.2mg/L IBA, normal shoot would be frequently obtained.1.5 The shoot was propagated on the MS medium containing 1.2mg/L BA and 0.2mg/L IBA . The root could be induced on the rooting medium(1/2MS+0.4mg/L IBA+0.25%AC), the rooting frequency reached 100%.2 The Agrobacterium-mediated transformation system of jujube was constructed, the transformed clones with S6PDH gene was obtained.2.1 The critical concentration of Km for adventitious bud regeneration from leaf explants was 20 mg/L, 25 mg/L was the critical concentration of Km for shoot multiplication. Root induction was sensitive to the Km, 15mg/L Km was suitable.2.2 Cb was a ideal antibiotic for killing Agrobacterium, the comfortable concentration was 400 mg/L, it can also encouraged the calluse inducing from jujube leaf explants, on the contrary, Cef had not such effect on callus development.2.3 The preculture and co-culture period had an important influence on the regeneration of Km-resistant clone. The long time of the period would decrease the regeneration frequency, whereas it could also enhance the proportion of chimerism of adventitious buds. The GUS gene could be transient expressed in the explants through 1～3d co-culture, especially at 3d.2.4 In these study, 128 Km-resistant clone was firstly obtained and 24 clones of them existed after several times selection. Three of the 24 clones were confirmed by PCR the S6PDH gene in the end, the transformation efficiency in general was 2.3%.