| Anther culture has special significance for the genetic improvement of plants and has been proved as one of the best effective ways of obtaining haploid and homozygotic plants.In the present study,investigations were carried out of the anther culture and plant regeneration system,the embryogenesis and embryo development process,several primary physiological and chemical changes during the development of anther-derived embryos,RAPD analysis of regenerated plants and EMS in vitro mutation of anther-derived embryos in loquat(Eriobotrya japonica Lindl.cv.'Dawuxing').The main results are as follows.(1) A stable and repeatable anther culture and plant regeneration system of loquat (Eriobotrya japonica Lindl.cv.'Dawuxing') was improved in this study.(2) The callus induction rate was 68.14%on MS medium supplemented with 0.05mg·L-1ZT,0.02mg·L-1NAA,0.02mg·L-1IBA,70 g·L-1 sucrose and 6.5g·L-1 agar.The rate of embryogenesis of anther callus was 58.33%on MS medium supplemented with 0.05mg·L-1 ZT,0.02mg·L-1 NAA,0.02mg·L-1 IBA,30 g·L-1sucrose and 6.5g·L-1agar.(3) The multiplication ability of anther-derived embryos was strong.When cultured on MS solid medium supplemented with 0.05mg·L-1ZT,0.02mg·L-1NAA,0.02mg·L-1IBA, 30 g·L-1 sucrose and 6.5g·L-1agar,each primary embryo could produce 5.39 new embryos on the average.When cultured in MS liquid medium with the same supplements,the primary embryos averaged 26.10 new embryo formation.Compared with solid culture, the liquid culture helped produce abundant anther-derived embryos with comparatively identical development stages in short time and with few abnormal embryos.(4) Though the germination rate and conversion rate of anther-derived embryos were low,the desiccation of anther-derived embryos was effective for improving the germination rate and conversion rate:the anther-derived embryos hardly germinated without desiccation,and after desiccation,the germination rate of anther-derived embryos was 23.38%,in which 29.23%of the embryos germinated normally and 68.42% of normally germinated embryos at last formed plantlets.(5) Chromosome observation showed that most of plantlets were haploids,the others were diploids.The acquisition of haploids indicated that some of these embryos originated from microspores.(6) Both microscope and histological observations showed that the anther-derived embryos of loquat developed through the typical globular,heart,torpedo and cotyledon stages.But some abnormal embryos were also observed.Histological observations showed that the origin of secondary embryogenesis of loquat anther had two ways:one was from epidermal cells and under epidermal cells and the other was from embryonic calluses.(7) Culture methods had some effects on secondary embryogenesis.When adopting solid culture,the origin of secondary embryogenesis was from both epidermal cells and under epidermal cells of embryonic calluses.When adopting liquid shake culture,the origin of secondary embryogenesis was mostly from epidermal cells and under epidermal cells.The rate of abnormal embryos was quite high in solid culture medium,whereas in liquid culture medium abnormal embryos were seldom observed.(8) In the whole developmental stages of anther-derived embryos,the water contents were high,and the dry mass contents were low.As a whole,the dry mass contents increased gradually with the anther-derived embryos development.Soluble sugars, soluble protein and nucleic acid were closely related to the development and maturation of loquat anther-derived embryos.In the important turning point of the development of loquat anther-derived embryos,soluble sugars,soluble protein and nucleic acid changed significantly in metabolism.During development of loquat anthers embryos,the dynamics of protein synthesis were roughly in "S" shape.The anther-derived embryos contained high soluble proteins from embryogenic calluses to globular embryos.The two accumulation peaks appeared at the embryogenic callus stage and differentiation stage. Alkaline protein contents were higher than acidic protein contents,and alkaline proteins contents and total soluble protein contents had the same change trend during the development and maturation of anther-derived embryo.Soluble sugar contents and fructose contents had the same change trend.The two accumulation peaks appeared at the embryogenic callus stage and differentiation stage.At early stage,soluble protein accumulation was due to the sugar transformation.After cotyledon stage,proteins were transformed to sugars in order to prepare energy for the anther-derived embryos' further development,maturation,germination and plant regeneration.RNA contents of anther-derived embryos were low at embryogenic callus stage and cotyledon stage. However,DNA were actively synthesized at the two stages.The DNA synthesis peak appeared earlier than that of RNA during the development of'anther-derived embryos.At the early development stage,the ratio of RNA to DNA ascended,which showed that the transcription and translation function were boosted up,and protein and ribosome accumulated gradually.But the ratio of RNA to DNA diminished gradually with the maturation of anther-derived embryos.(9) The RAPD analysis with four primers showed that the bands of some regenerated plants were coincident,but of a few regenerated plants the bands were inconsistent obviously.RAPD analysis primarily showed that the anther-derived embryo regenerated plants had comparative hereditary stability.At the same time,mutation variations occurred in some of the regenerated plants.(10) When EMS concentration ranged 0.1%-0.7%and the exposure time of treatment was 0.5h,1h and 2h,respectively,loquat anther-derived embryos could retain different survival rates.But 0.9%was approximately the deadly concentration of EMS to anther-derived embryos.Maximal survival rate(91.56%) was obtained in the treatment of 0.3%EMS for 0.5h.After EMS treatments,the percentage of primary embryos producing new embryos was remarkably lower than CK.The new embryos produced on all treatments could maintain good multiplication ability like CK.The anther-derived embryos needed more time to produce new embryos when EMS concentration was 0.7%. And these new embryos had stronger multiplication ability than others.(11) EMS induced abnormal mitosis in anther-derived embryo cells and embryonic cells.The rate of abnormal mitosis of these cells increased with the EMS concentration. The abnormal mitosis included chromosome multipolar,dikaryon,chromosome bridge, chromosome multi-bridge,chromosome laggards,and chromosome unequal division.In some cells the chromosome number changed.(12) The secondary embryos after EMS treatments had the approximately same germination rate and conversion rate as CK.Chromosome observation revealed that the chromosome number changed in some of the radicels germinated from anther-derived embryos with EMS treatment.After EMS treatment the haploid(n=17) proportion decreased,diploid proportion increased,and there were also certain frequencies of polyploids(2n=3x=51,2n=4x=68) and aneuploids(2n+1,2n-1).The maximal proportion (16.67%) of polyploids and aneuploids were obtained in the treatment of 0.3%and 0.5% EMS for 2h. |
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