Histological Studies Of Leaf Regeneration And Fact Ors Influencing The Efficiency Agrobacterium Mediated Transformation In Zizyphus Jujube. Mill.

Jujube (Zizyphus jujuba Mill.) is the first major dry fruit tree in China. However,jujube has smaller buds, lower natural fruit setting rate and higher embryo abortion rate. Conventional breeding was resulted in slow progress owing to long growth and reproductive cycle. The effects of explant maturity,basal medium, plant growth regulators with different concentration combinations, explant orientation, and sucrose concentrations on adventitious bud regeneration were compared, and the pattern of bud regeneration was observed through histological study. Modern biotechnology creates a new path for jujube breeding.The genetic transformation through Agrobacterium tumefaciens-mediate. The main conclusions are as follows:1. Establishment of in vitro leaf regeneration system in Zizyphus jujuba Mill.The results showed that WPM supplemented with 0.5mg/L TDZ and 0.2mg/L NAA was effective for in vitro shoot regeneration of'Huizao'. The optimal explant was young, and thick expanded leaves from the intermediate shoot. The regeneration frequency was higher in those from petiolar end than those farther. The regeneration frequency remained as high as 69.61% when leaf explants were placed firmly with the abaxial surface contacting the woody plant medium. When sucrose concentration was 40g/L, the hyperhydric degree of adventitious buds was reduced. A 10-day dark culture was necessary for the shoot regeneration.Addition of 0.5 mg/L AgNO3 and 0.1mg/L STS to the medium could pomote leaf regeneration.2. Anatomical observations of adventitious bud regeneration from leaf explants of Ziziphus jujube Mill.'Huizao'.The histological process of adventitious shoot regeneration from the leaf explants of Zizyphus jujuba Mill.'Huizao'was reported in this study. Shoot regeneration was obtained via culture of the leaf explants on woody plant medium (WPM) supplemented with 0.5mg/L thidiazuron (TDZ), 0.2mg/Lα-naphthalene-acetic acid (NAA) and 0.5mg/L silver nitrate (AgNO3) for 10 days in the dark followed by 3 weeks at a 14-h photoperiod. Histological studies revealed that the adventitious buds directly originated from the parenchymatous cells around the vascular bundles without an intermediate callus phase. The parenchymatous cells in the leaf explants started dividing, which resulted in the formation of meristemoids. Then the meristematic cells continued division and subsequently gave rise to adventitious bud. 3. Agrobacterium-mediated transformation of Ziziphus jujube Mill.'Huizao'Effects of kanamycin on shoot regeneration of leaves, and days of co-cultivation on transformation frequency were investigated. Our experiments showed the percentage of shoot regeneration was reduced to zero,therefore 40mg/LKan was used in the subsequent selection process. The transformation frequency reached the highest rate(21.1%)when the length of the co-cultivation was 2d. A histochemical staining analysis confirmed the integration of the target gene into the genome of regenerated plants.