Study On The Technology Of Isolate Microspore Culture In Radish (Raphanus Sativus L.)

Raphanus sativus L. belongs to raphanus of Cruciferae. It is an annual herbaceous plant and also an important vegetable in China. In recent years, with the development of vegetable production and the raise of people's living standard, the requirement on radish quality, resistance to disease and pest, tolerance to environmental stress and yield is more and more. It is needed to raise efficiency of radish breeding and accelerate the advancement of radish breeding. Isolated microspore culture is of great importance in crop genetics and breeding, through which homozygote could be obtained rapidly and it is easy for us to screen out breeding material with resistance to disease and pest or tolerance to environmental stress in a shorter time. On the other hand, the genotype and phenotype of double haploid (DH) obtained from microspore culture are identical, which is suitable for genetic analysis, genetic map construction, gene discovery, etc.In the process of microspore culture, determination of the condition suitable for embryoid induction and plant regeneration is very important. In this experiment, directed towards the difficulties in radish microspore culture, radish materials of different genotypes were chosen. The suitable size of flower bud, pretreatment method, and culture condition of the microspore culturewere studied. Screen of the mediums of the plant regeneration were done for embryoid regeneration culture. The main results are as follows:The relationship between the cytological characteristics of microspore at different development stages and the corresponding morphological characters of flower organ.Through the observation on the morphological characters of flower organ of four radish materials, the result showed that the lengths of the flower bud ranged 2.99 to 3.51mm, the ratios of the length to the transverse diameter were 1.41to 1.48, the petal length covered 1.89 to 2.56 mm, and the ratios of flower length and anther length were 0.92 to 1.06, when the microspore was at the uninucleate stage. At the same time, the external features of the flower bud were: plump flower bud, the anther with light yellow to yellow color, the anther closely covered by sepal, and petal and anther having the same length or roughly the same. Based on the observation resoult, it is possible to rapidly and conveniently choose the flower bud at suitable developmental stages.The effect of low temperature, high temperature, starvation and mannite treatments upon survival rate of microspore from four factors: The result indicated that low temperature pretreatment in four materials could raise the survival rate of microspore in certain time. In the process of culture, high temperature pretreatment for 2d also raised the survival rate. In the four materials, microspore of radish could not stand for starvation of carbon. Mannite pretreatment brought about different results in different materials. An orthogonal design experiment based on six factors including 4℃low temperature treatment, 32.5℃heat shock treatment, NAA concentration, medium type, 6-BA concentration, and sucrose concentration in the culture process was conducted.The effects of 16 treatment combinations on embryo production rate of radish microspore were researched in 22 radish materials.Visual analysis and variance analysis indicated that the important factors ranked in the front places in 9 materials which produced embryos were 4℃treatment, 32.5℃treatment, and sucrose concentration in order, among them 2 materials obtained regeneration plants.On this basis, the conditions suitable for microspore culture of radish were optimized: 32.5℃treatment 2d, 6-BA 0.1mg/L,NAA 1mg/L,sucrose concentration 130g/L, NLN medium.The plant regeneration of radish is difficult. Regeneration culture medium that is most using embryo from seed germination as explant in tissue culture, the regeneration culture medium suitable for differentiation of embryoid was screened out. In six materials, B5 culture medium is the most suitable for differentiation; phenylurea cytokinins TDZ had no effect upon gemmulation of the six materials; B5+6-BA and B5+6-BA+NAA had the best effect for gemmulation.The rooting medium: MS+NAA.The plants regenerated from embryoid were gotten on the suitable medium.