Establishment Of High Frequency Regeneration System And Genetic Transformation With IaaM Gene In Loquat

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%.