摘要
本研究建立了‘大五星’枇杷花药离体培养及成年植株叶片高频再生体系,采用农杆菌介导法以及花粉管通道法,将单性结实iaaM基因导入枇杷,研究结果如下：
1.以‘大五星’枇杷花药为外植体,通过间接胚胎发生建立了枇杷花药培养及植株再生技术体系,结果表明：枇杷小孢子发育时期与花蕾直径存在相关性,横径为4.68mm左右、纵径为4.52mm左右的花蕾(此时枇杷花蕾处于单核靠边期)适宜用于枇杷花药胚状体的诱导；4℃低温处理2d愈伤组织诱导率最高,达69.89%；花药愈伤组织诱导的最适培养基是MS+2.4-D0.5mg/L+6-BA2.0mg/L,愈伤组织诱导率为78.33%；枇杷花药愈伤组织诱导胚状体的最适培养基为MS+ZT0.05mg/L+NAA0.01mg/L+IBA0.05mg/L,胚状体诱导率为25.69%；枇杷花药胚状体最适的增殖培养基为MS+ZT0.05mg/L+NAA0.02mg/L+IBA0.02mg/L,增殖系数达5.8；枇杷花药胚状体经4℃低温+饱和CaCl2脱水处理7天后接种于萌发培养基1/2MS+蔗糖30g/L上,胚状体成苗率最高,为78.2%；将再生植株移栽入配比为腐熟有机肥：园土：锯末=1：2：1的基质中时,组培苗移栽成活率达85.25%。
2.以12年生‘大五星’枇杷幼叶为外植体,成功再生出了完整植株。重点研究了外植体表面灭菌方法、取材时间、基本培养基及植物生长调节剂对叶片愈伤组织诱导、增殖、芽苗分化及生根的影响,结果表明：枇杷叶片的灭菌处理以先用75%酒精处理12s,后用0.1%的升汞处理8min为宜；取材时间以春季最佳,愈伤组织启动萌发时间最快,平均为20.3天,诱导率为81.6%；适宜愈伤组织诱导的培养基是MS+BA0.5mg/L+2,4D0.5mg/L,在此培养基上,愈伤组织诱导率为89.2%；适宜愈伤组织增殖的培养基是MS+BA0.5mg/L+2,4D0.25mg/L,愈伤组织在此培养基上增殖最快,增殖系数达4.11；叶片基部的再生能力强于叶中部及叶尖；芽诱导的最适培养基配方是MS+TDZ0.8mg/L+NAA0.3mg/L+AgN030.2mg/L,芽诱导率为30.6%；芽苗增殖适宜培养基是MS+6-BA1.0mg/L+NAA0.3mg/L+Tryptone750mg/L,增殖系数最高,为3.7；芽苗在1/2MS+NAA0.5mg/L培养基上,可获得86.92%的生根率。生根试管苗炼苗后,移栽到无菌锯末、泥炭和腐殖土(1：3：1)中,经过精心的温度和水分管理,可获得92.1%的成活率。
3.以‘大五星’枇杷花药胚状体为受体材料,采用农杆菌介导法转化单性结实iaaM基因,探讨卡那霉素浓度、预培养时间、农杆菌侵染时间、共培养时间以及乙酰丁香酮浓度等因素对遗传转化的影响。结果表明：卡那霉素最佳浓度为100mg/L,花药胚状体经预培养2-3d,农杆菌侵染15min,共培养3d,共培养基中加入AS10mg/L,最适合进行遗传转化。抗菌素选用250mg/L的头孢霉或安比西林750mg/L脱菌效果较好。采用此转化体系进行遗传转化,最终获得了16个卡那霉素抗性芽,对抗性芽次生胚进行iaaM和NPTII基因的特异性扩增检测,结果显示有9个胚系扩增出了预期目的条带,PCR阳性率为56.25%,进一步对抗性芽进行Southern杂交,结果显示其中有8个胚系出现了明显的杂交信号,表明iaaM基因已导入枇杷胚状体中,转化率约为0.5%。
4.利用枇杷叶片愈伤组织为受体建立了根癌农杆菌介导的遗传转化体系。研究了预培养时间、农杆菌侵染时间、共培养时间以及乙酰丁香酮浓度等因素对遗传转化的影响。结果表明：卡那霉素的最佳浓度为90mg/L,叶片愈伤组织经预培养6d,农杆菌侵染20min,在含有AS5mg/L的共培养基中共培养3d,最适合进行遗传转化；抗菌素选用300mg/L的头孢霉脱菌效果较好；采用此体系进行遗传转化,从1500块愈伤组织中最终获得了卡那霉素抗性愈伤组织124块,随机抽取8块进行iaaM和NPTII基因的PCR扩增鉴定,结果显示其中6块扩增出了预期条带,PCR阳性率为75%,进一步对抗性愈伤组织进行Southern杂交分析,结果显示其中有6块出现了明显的杂交信号,证实iaaM基因已导入枇杷愈伤组织基因组中,表明根癌农杆菌介导外源基因转化枇杷叶片的愈伤组织是完全可行的,为以后通过基因工程手段进行枇杷的遗传改良奠定了基础。
5.以‘大五星’枇杷为材料,对枇杷花粉管通道法转基因技术进行探索性研究,将携带iaaM和GUS基因的Pbi121质粒DNA导入枇杷,结果表明：最佳注射时间为授粉后60h,此时花粉管已到达胚囊；采用“切除柱头+整个花柱”的方法注射外源DNA,转化率最高,为2.6%；随着注入外源DNA浓度的增加,坐果率逐渐降低,DNA浓度为500μg/ml是适宜的DNA注射浓度,坐果率为11.8%,而转化率可达3.2%。
In this study, high frequency regeneration systems from anther and leaf explants of adult trees in 'Dawuxing' loquat (Eriobotrya japonica Lindl.) were established. Agrobacterium-mediated transformation and pollen tube-pathway were adopted to introduce iaaM gene to obtain possible seedless lines. In this research, factors influencing the efficiency of transformation were investigated systematically, and the major technique parameters of agrobacterium-mediated transformation were optimized. The main results are as follows.
1. The plant regeneration system was established through indirect embryogenesis from anther culture of loquat(Eriobotrya japonica Lindl. cv.'Dawuxing'). The results showed that there was correlation between diameters of flower bud external morphological characteristics and the microspore developmental stages, and when the transverse and vertical diameters of flower buds were4.68mm and4.52mm respectively, the microspores were at the late-uninucleate developmental stage which were suitable for inducing embryoids.2days of4℃cold treatment in darkness to anthers were more favorable, resulting in69.89%of callus formation. The most suitable medium for anther callus induction was MS medium supplemented with2,4-D0.5mg/L and6-BA2.0mg/L, in which anther calluses were induced at a frequency of78.33%. The optimal induction of anther-derived embryos was achieved on MS medium supplemented with ZT0.05mg/L, NAA0.01mg/L and IBA0.05mg/L, in which embryos were induced at a rate of25.69%. The most suitable medium for anther-derived embryo proliferation was MS supplemented with ZT0.05mg/L, NAA0.02mg/L and IBA0.02mg/L, in which the multiplication coefficient was5.8.78.2%of complete plantlets were succesfully obtained on1/2MS medium supplemented with3.0%sucrose after the embryos were pretreated for7days in4℃cold treatment and dehydration. The rooted plantlets survived at a rate of85.25%in the transplantation matrix of decomposed organic fertilizers, garden soil and sawdust(1:2:1).2. A plant regeneration technique was successfully developed using in vitro culture of leaf explants harvested from12year-old mother plants of loquat. The effects of sterilization method, time of sampling, basal medium and plant growth regulators on the callus induction, proliferation, shoot differentiation and rooting were studied. A satisfactory sterilization was achieved by treating the explants with75%alcohol for15s, then0.1%mercuric chloride for8min before culture. Sampling in spring was better, and calluses occurred within20.3days and at a high frequency of81.6%. The optimal induction of calluses was achieved on MS medium supplemented with6-BA0.5mg/L and2,4-D0.5mg/L, in which calluses were induced at a frequency of89.2%. The most suitable medium for callus proliferation was MS medium supplemented with6-BA0.5mg/L and2,4-D0.25mg/L, in which calluses were proliferated fast with a high multiplication coefficient of4.11. The regeneration capacity of leaf blade base was better than leaf blade centre and tip. The most suitable medium for shoot differentiation was MS medium supplemented with thidiazuron (TDZ)0.8mg/L, NAA0.3mg/L and AgNO30.2mg/L, in which shoots were regenerated at a rate of30.6%. The most suitable medium for shoot proliferation was MS medium supplemented with6-BA1.0mg/L, NAA0.3mg/L and Tryptone750mg/L, in which a high multiplication coefficient of3.7was obtained.86.92%of the shoots rooted and grew into complete plantlets on1/2MS medium supplemented with NAA0.5mg/L. The rooted plantlets, after hardening, survived at a rate of92.1%in the transplantation matrix of autoclaved sawdust, peat and humus (1:3:1) under careful temperature and water management. The plant regeneration technique developed in this study could be useful for Agrobacterium-mediated genetic transformation in loquat.
3. Anther-derived embryos were used as experimental materials, and the parthenocarpic gene iaaM was transferred into anther embryos, which would lay the basis for further research of haploid breeding and development of seedless cultivars in loquat. The factors affecting the transformation, such as kanamycin concentration, the pre-culture, infection time, co-cultivation time and inclusion of acetosyringone, were examined. The results showed that2-3day preculture,15min infection and3d co-cultivation with10mg/L AS concentration in the co-culture medium would lead to an increase in transformation efficiency. Cefotaxime at250mg/L or Ampicillin at750mg/L were optimal. Using Agrobacterium-mediated transformation method, the parthenocarpy gene iaaM was introduced into loquat anther embryoids and16transgenic lines were obtained. The iaaM and NPTII gene-specific amplification results showed that eventually9strains were amplified the expected target bands, the PCR positive rate was56.25%. The PCR-Southern analysis indicated that8strains showed hybridization signal, which further proved that iaaM gene was integrated into genome of the embryos of "Dawuxing" loquat, and the transformation rate was about0.5%.
4.Calluses from leaves were used as receptors to establish the Agrobacterium-mediated transformation system. The effects of f precuhure time, infection time, co-cultivation time and concentration of acetosyringone on transformation frequency were studied. The optimal concentration of kanamycin was90mg/L.6day preculture,20min infection and3d co-cultivation with5mg/L AS concentration in the co-culture medium would lead to an increase in transformation efficiency. Cefotaxime at300mg/L was optimal. Using Agrobacterium-mediated transformation method, the iaaM gene was introduced into loquat calluses and124resistant lines were obtained from1500calluses.8resistant calluses of124lines were chosen randomly for PCR amplification, and eventually6strains were amplified the expected target bands, the PCR positive rate being75%. The PCR-Southern analysis indicated that6strains showed hybridization signal, which further revealed that iaaM gene was integrated into genome of the calluses of "Dawuxing" loquat. This experiment proved that Agrobacterium-mediated transformation of loquat calluses was feasible, which would lay the basis for further research of haploid breeding and seedless cultivars in loquat.
5. Experiment was conducted for the application of pollen-tube path way in loquat. Plasmid Pbi121containing gene iaaM and reporter gene GUS was introduced into 'Dawuxing' loquat via pollen tube pathway. The best time of inoculation was60hours after pollination when the pollen tubes had reached the embryo sac. The optimal transformation method was 'removal of stigma and the whole style' with a high transformation rate of2.6%. With the increase of the concentration of DNA, the rate of fruit set decreased gradually, and the concentration of500μg/ml DNA was suitable, resulting in the rate of fruit set of11.8%and the transforation rate of3.2%.
引文
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