葡萄原胚团的诱导、保存、扩繁及其细胞学观察  

Induction,preservation,propagation and cytological observation of proembryonic masses of grapevine

作　　者：程远[1] 万东艳 张宝林[1] 王跃进[1] 文颖强[1] 

机构地区：[1]西北农林科技大学园艺学院.农业部西北地区园艺作物生物学与种质创新重点实验室.旱区作物逆境生物学国家重点实验室,陕西杨凌712100

出　　处：《果树学报》2017年第8期968-987,共20页Journal of Fruit Science

基　　金：国家自然科学基金(31372022);陕西省科技研究发展(攻关)计划(2014K02-02-03)

摘　　要：【目的】通过诱导、保存和扩繁葡萄原胚团,为葡萄外源基因转化及基因编辑研究提供受体材料。【方法】以4个欧亚种葡萄品种和1个野生种葡萄的花器官为材料,比较不同外植体在3种培养基上胚性愈伤组织的诱导率,通过二次再生途径诱导、保存和扩繁次生原胚团并利用改良苯酚品红染色法对其进行细胞学观察。【结果】‘无核白’葡萄的雄蕊接种在PIV培养基上胚性愈伤组织诱导率最高(10.8%);初生原胚团细胞核大、染色深且具有旺盛分裂能力,二次再生途径诱导的次生原胚团具有同样的细胞学特征且可以正常成苗。【结论】葡萄体细胞胚诱导受基因型、外植体类型影响显著;二次胚再生途径可以长期诱导、保存和扩繁原胚团,能为葡萄遗传转化持续提供受体材料。【Objective】Grapevine（Vitis vinifera L.） is one of the world＇s most important fruit crops because of its high yielding and value. However,it is susceptible to pathogens. Genetic engineering is a powerful tool for plant improvement and has the potential to allow the integration of desirable disease-resistance genes into existing genomes. Successful application of gene technology to grapevine requires an efficient regeneration system. Somatic embryogenesis is the most commonly adopted method in genetic engineering research in grapes. Flowering in grapes occurs only once a year,which limits the opportunity for induction and transformation. Therefore it is important to produce regenerative embryogenic tissues and maintain them over a long period. In previous reports,proembryonic masses（PEM） were used in genetic transformation and gene editing. Therefore,it is necessary to induce,preserve,propagate and cytologically understand proembryonic masses.【Methods】In this study,Vitis vinifera L.‘Thompson Seedless＇,‘Semillon＇,‘Riesling＇,‘Shiraz＇and Vitis yeshanensis J. X. Chen accession‘Yanshan-1＇were used to induce somatic embryos（SE）. To obtain higher induction efficiency,the optimum sampling period was determined according to microscopic observation. The pistils,stamens and small buds from grapevine were used to induce somatic embryos on three embryogenic culture initiation mediums. A secondary embryogenesis system was utilized to obtain more proembryonic masses. Induced callus from different explants were observed under stereoscopic microscope. Embryonic callus（EC） and non-embryonic callus were also observed under stereoscopic microscope. Cells of embryonic callus and non-embryonic callus werestained with improved carbolfuchsin solution and observed under microscope. In secondary embryogenesis,primary proembryonic masses,primary somatic embryos,secondary embryonic callus and secondary proembryonic masses were observed,which were stained with improved carbolfuchsin solution and ob

关 键 词：葡萄 体细胞胚 二次胚再生 次生原胚团 细胞学观察 

分 类 号：S663.1[农业科学—果树学]