Optimization Of Eggplant Anther Culture System And Identification Of Regeneration Plant Ploidy

Eggplant(Solanum melongena L.,2n=2x=24)is an annual herb of solanum with high nutritional value.Anther culture is one of the important means for new variety breeding and germplasm innovation,which can obtain a large number of homozygous haploid and diploid plants in a short time.Previous studies revealed that eggplant anther culture can obtain regeneration plants which come from the original pollen cells.In addition,eggplant anther culture is much easier than eggplant pollen culture,studies on haploid breeding of eggplant in China are mainly based on anther culture.But the eggplant anther culture system is incompleted,there are still many problems in practical applications such as high rate of anther contamination and low rate of embryogenesis.Eggplants were used as materials in this study.Three aspects of the optimum culture period,the best disinfection method and the best culture medium selection were conducted to establish an efficient and stable culture system of eggplant anther culture in vitro.The aims of this study were to obtain plants from origin of pollen and lay the foundation for the creation of new germplasm of eggplant.Indirect identification methods of plant ploidy by comparison of plant morphology,stomatal size and chloroplast counts in guard cells were combined with direct identification by chromosome counting to determine the ploidy of regenerated plants.Convenient and reliable methods were selected to identify the ploidy of eggplant regeneration plants,and improve the efficiency of haploid breeding of eggplant.The main results obtained in this study were as follows:1.The relationship between external morphology of flower buds and development period of pollen was established by making glass observation section.The pollen at the uninucleate stage when sepal split base difference is 1-2 mm and the anthers showed yellow green,which was the most suitable timepoint for anther culture.The best method for the disinfection of anther was established.The buds without flower sepals was treated with 75%alcohol for 2 min,and 6.5%sodium hypochlorite added a drop of polysorbate was used for disinfection for 12 min,and then the buds were washed 4-5 times with distilled water for inoculation.2.A stable and reliable regeneration system of eggplant anther culture was established.Pretreatment medium:MS+2,4-D 0.2 mg/L+Vc 8.0 mg/L+KT 1.0 mg/L+sucrose 30 g/L+agar powder 8 g/L.Inducing medium:MS+2-BA 1.0 mg/L+ NAA 1.0 mg/L+KT2.0 mg/L+sucrose 30 g/L+ agar powder 8 g/L.Differentiation medium:MS+NAA 0.1 mg/L+ ZT 1.0 mg/L +Glu 800 mg/L+GsH 30 mg/L+L-Ser 100 mg/L+ sucrose 30 g/L+ agar powder 8 g/L.Rooting medium:1/2MS+2-BA 0.2 mg/L+NAA 0.1 mg/L+ sucrose 20 g/L+ agar powder 8 g/L.3.Identified ploidy of regeneration plants by observing the chromosome number in root tip and anther cell of regeneration plant,combined with observing morphological characters and fertility of regeneration plants,used the methods of observation of stomatal size and chloroplast count of the lower epidermis of the leaves.Anther source primitive diploid plant was used as control.Among the six regenerated plants,four were haploid,one was diploid and one was tetraploid.The best method for identified haploid was chromosome counting in root tip cell.The multiploid can be identified by meiosis.The indirect methods of stomatal size,chloroplast number in guard cells,and the adult-plant stage leaf index can be used as ploidy identification.Three kinds of indirect identification methods combined with flowering and fecundity of regeneration plants could be used for plant ploidy identification accurately.