梨花药愈伤组织诱导和悬浮培养体系的建立及应用  

Establishment and application of callus induction and suspension culture system of pear anther

作　　者：庄梦弟 李涛 辛骞祎 周梦瑶 张海霞 马辉 钱稷[1] 张玉星[1,2] 亓宝秀 许建锋 ZHUANG Mengdi;LI Tao;XIN Qianyi;ZHOU Mengyao;ZHANG Haixia;MA Hui;QIAN Ji;ZHANG Yuxing;QI Baoxiu;XU Jianfeng(College of Horticulture,Hebei Agricultural University,Baoding 071001,Hebei,China;Hebei Pear Technology and Innovation Center,Baoding 071001,Hebei,China;School of Pharmacy and Biomolecular Sciences,Liverpool John Moores University,James Parsons Building,Byrom Street,Liverpool L33AF,UK)

机构地区：[1]河北农业大学园艺学院,河北保定071001 [2]河北省梨技术创新中心,河北保定071001 [3]英国利物浦约翰摩尔大学药学和分子生物学学院,英国利物浦L33AF

出　　处：《果树学报》2023年第3期577-587,共11页Journal of Fruit Science

基　　金：国家重点研发计划(2019YFD1001404-5);河北省重点研发计划项目(21326308D-1-4);河北省现代农业产业技术体系梨创新团队项目(HBCT2018100202);河北省现代农业赵县梨创新驿站项目。

摘　　要：【目的】建立梨悬浮细胞体系,并利用悬浮细胞体系获得高质量悬浮细胞,进行遗传转化和原生质体的分离。【方法】以新梨7号花药为试材,诱导获得梨花药愈伤组织,研究了细胞悬浮系建立和影响悬浮细胞增殖的主要因子。【结果】花药在1/2 MS+1.0 mg·L^(-1)2,4-D+0.4 mg·L^(-1)NAA+30 g·L^(-1)蔗糖+7 g·L^(-1)琼脂(pH值为5.8~6.0)培养基上诱导的愈伤组织,经4~5次继代得到了黄白色、颗粒状,状态相对较好的愈伤组织;将愈伤组织接种于液体培养基中,于28℃,200 r·min-1的黑暗条件下悬浮振荡培养,经4~5次悬浮继代培养建立了悬浮细胞系,适宜的启动和增殖培养基为:MS+1.5 mg·L^(-1)2,4-D+1.0 mg·L^(-1)6-BA+30 g·L^(-1)蔗糖,细胞生长曲线呈“S”型,活细胞率达81.98%,近圆细胞率达83.66%,可作为遗传转化的受体,转化率为20.00%。也可直接用于原生质体的分离,原生质体产量达3.67×10^(6)个·mL^(-1),活力可达92.00%。【结论】建立了梨悬浮细胞培养体系,利用所建体系获得了高质量的悬浮细胞,可直接用于遗传转化和原生质体的分离。【Objective】Pear is one of the important fruit species in the world,and pear growing has become an important pillar industry in many places for its huge economic value,which plays a significant role in farmers’income and social stability.Pear’s genome is highly heterozygous,which is resulted from cross-pollination,bringing serious difficulties to the genetic transformation.Cell suspension culture is an important culture method for cells.It can not only provide raw materials for protoplasm preparation,regeneration,genetic transformation and somatic cell hybridization,but also be used for the production of secondary metabolites.In this paper,we firstly obtained calluses by inducing the anthers of Xinli No.7 pear and then studied effects of culture medium types and hormone combinations on the growth of pear callus under suspension culture conditions by selecting the loose calluses from the vigorous growths after induction as materials,in order to establish a suitable suspension culture system for pear anther calluses,obtain pear calluses of high quality and provide good materials for pear protoplast preparation and pear genetic transformation.【Methods】Pear anthers were taken as the callus induction materials.Flower buds at the“small-balloon stage”(transverse diameter=2.7-3.0 mm,longitudinal diameter=8.0-8.5 mm)of Xinli No.7 pear trees were selected,cleaned and soaked in 75%alcohol for 25 s and 0.1%HgCl_(2) for 8min successively,and then the petals were removed and the anthers were taken out.The anthers were cultured on the induction medium(1/2 MS+1.0 mg·L^(-1)2,4-D+0.4 mg·L^(-1) NAA+30 g·L^(-1) sucrose+7 g·L^(-1) agar powder,pH 5.8-6.0)for 30 days.After several subcultures on the medium(MS+1.5 mg·L^(-1)2,4-D+0.5 mg·L^(-1)6-BA+30 g·L^(-1) sucrose+7 g·L^(-1) agar powder),loose calluses were selected for the suspension culture experiments,in which the basal medium was MS+30 g·L^(-1) sucrose(pH 5.8-6.0),and the suspension medium was prepared by adjusting the concentration of 2,4-D(1.0,1.5,2.0 mg�

关 键 词：梨 愈伤组织 细胞悬浮培养 生长曲线 

分 类 号：S661.2[农业科学—果树学]