| Zizyphus jujuba Mill.,as one of native fruit crop with an important economical and ecological value,distributed widely in China. Jujube witches'broom (JWB) caused by phytoplasma,is becoming the most destructive systematic disease of Chinese jujube and has brought much economic damage to our country. A highly efficient regeneration system and genetic transformation system of Taishan sour jujube (Zizyphus spinosus Hu) and Xilv jujube (Zizyphus jujuba cv. Xilv) were established. In vitro leaves of Taishan sour jujube (Zizyphus spinosus Hu) were selected as explants, GUS (β-glucuronidase) reporter gene ( uidA) and AG (antimicrobial peptide gene gene) gene under the control of the Arabidopsis sucrose-H+ symporter gene (At-SUC2) promoter which is phloem-specific was introduced into Taishan sour jujube by Agrobacterium tumefaciens. Optimal conditions were established for efficient transformation from leaf explants of Taishan sour jujube. The transplants of jujube tree should be given the resistant ability to phytoplasma. The main results were as follows:1,The new shoots from dormancy branches cultured in water were treated by a series of sterilizations. Rapid micropropagation of in vitro shoots of Taishan sour jujube and Xilv jujube were established.2,A highly efficient regeneration system of Taishan sour jujube (Zizyphus spinosus Hu) was established. The optimal hormone combinations for adventitious bud initiation are TDZ 1.0mg/L and IAA 0.5mg/L; The optimal hormone combinations for adventitious bud elongation are IAA 0.1 mg/L and GA3 0.5mg/L.The leaf explants were first cultured 3 weeks under dark, then, transferred to light, highest regeneration rate was obtained, reached 100%. And no shoot regeneration was obtained when leaf explants cultured under direct light.The optimal rooting medium of sour jujube is 1/4 MS medium plus IBA 0.5 mg/L,rooting percentage was 98.5% and root number per plantlet was 4.1; Dark culture was unnecessary for rooting, highest rooting rate was obtained under direct light.3,A highly shoot regeneration system from leaf explants of Xilv jujube (Zizyphus jujuba cv. Xilv) was established. The optimal hormone combinations for adventitious bud initiation are TDZ 1.0mg/L and IAA 0.5mg/L; The optimal hormone combinations for adventitious bud elongation are BA 0.1 mg/L and IBA 0.5mg/L.The basal medium WPM is more effective to induce adventitious bud initiation than that MS medium.The optimal rooting medium for Xilv jujube is 1/4 MS medium plus IBA 0.5mg/L same as sour jujube, rooting percentage was 98.5% and root number per plantlet was 4.1. Dark culture was necessary for the shoots to root, highest regeneration rate was obtained when shoots were first cultured 10d in the dark.4,A highly efficient genetic transformation system of Taishan sour jujube (Zizyphus spinosus Hu) was estabished. The critical concentration of Km for adventitious bud regeneration from leaf explants was 20 mg/L, the critical concentration of Km for shoot multiplication was 25 mg/L .25mg/L km was employed for transformation selection.5,Km-resistant shoots were propagated. The histochemical analysis of GUS activity was performed in some of GUS transgenic plants, results showed that no GUS activity was observed. The integration of AG gene was checked by PCR and no expected amplification bands were detected. These results showed that maybe the transgene is transient expression, or maybe km-resistant bud is false positive, all these need to be further study. But these results showed that sour jujube as transformation receptor is very difficult to receive foreign gene. Key words:Sour jujube;Xilv jujube;in vitro leaves;dark-culture;adventitious buds;plantlet regeneration... |
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