Cultivation of the‘Zhongnonghong’pomegranate tissue and genetic transformation of the leaf receptor material were obtained

Abstract：【Objective】In order to obtain a stable and sterile pomegranate genetic transformation system re⁃ceptor material. This is the basis for conducting genetic transformation experiments，and it is also a power⁃ful guarantee for improving reproducibility and reliability，when the proliferation buds reached 3 to 5 cm，they were cut from the basal part putted into MS medium and subcultured. Add 1 mg·L^-1 PVP and 200 mg·L^-1 coconut and different concentrations of 6-BA，NAA or IBA. After two subcultures，the shoots necrosis，shoots and stem segments were observed and recorded. The rate of shoot formation was the ratio offresh shoots to original plants on the original plants. The rate of stem formation was the ratio of stem seg⁃ments grown to these shoots. A stable and sterile pomegranate proliferation medium was screened to ob⁃tain a stable and robust leaf receptor material.【Methods】The shoots of adult plants were collected from 2to 5 knots as explants from February to March. The leaves were harvested and cutted into 1-2 stem segments，dilute the solution soak the oscillation wash 30 min，tap water rinse，placed in the clean bench ster⁃ilization, 75% alcohol surface sterilization 30 s，0.1% HgCl2 solution soaking at different times，sterile wa⁃ter rinse 5 times; remove the stem section inoculated with different concentrations of 6-BA，NAA or IBA MS，B5 or WPM on the primary medium. The culture conditions were as follows: temperature（24±1）℃，light intensity 2 500-3 000 lx，light time 16 h·d-1. Each treatment of 20 bottles，each bottle inoculatedwith 3 to 4 explants，repeated 3 times. Counting the fungi，bacteria and browning from 10 d to 30 d afterinoculated. When the induced axillary bud grew from 2 to 3 cm，it was cut from the basal part and trans⁃ferred into the MS multiplication medium，adding 6-BA，KT，NAA and IBA，and with different concentra⁃tions. Adding 1 g·L^-1 PVP anti browning，adding anti coconut juice stem tip necrosis and yellow leavesfall off. The proliferation coefficient and growth status were recorded after 30 d. Comparing the effects ofPVP and coconut on the pomegranate material and screening the different medium and concentration ofthe first generation medium.【Results】The medium with the first generation of pomegranate stem was MS+1.0 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+1 mg·L^-1 PVP. After two subcultures，the appropriate medium wasscreened: 1.0 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+1 mg·L^-1 PVP+200 mg·L^-1 coconut juice induced into budsfor the best，0.3 mg·L^-1 6-BA+0.1 mg·L^-1 IBA+1 mg·L^-1 PVP+200 mg·L^-1 coconut juice for induction ofstem elongation. In the growth of pomegranate plants，can get green，stretched the robust red peony leaves.【Conclusion】The stable and robust pomegranate experimental material was obtained，which laid the foun⁃dation for the establishment of stable pomegranate genetic transformation system.