Study On Technique Optimization Of Constructing DH-Line Population In Brassica Napus L.

Rapeseed is one of the four main oil crops worldwide as well as in China,and is of strong adaptability,extensive use,high economic value and great developing potential.At present,the application of modern biotechnology,such as the haploid culture technology,in crop breeding has greatly promoted the combination of good quality,high-yielding and resistance,a lot of new germplasm resources have been developed.Based on previous studies,the optimization on the microspore culture technology of Brassica napus L.under field condition was studied systematically in the paper.1.The nine sowings,each sowing spacing 10 days from Aug.28th to Nov.16^th,was conducted group by group.The results showed that the third,fourth,fifth and the sixth sowing were all good for embryogenesis of microspores,and the forth sowing could get the greatest number of embryogenesis,indicating that the sowing period from Sep.27th to Oct.7th in Guiyan is much more suitable for microspore culture.Among the four different genotypes,the embryogenesis produced from the fourth and the fifth sowing accounted for 71.1%in all embryoids.2.The data from the study on the sampling period of the microspores showed that the microspores during the late bud stage and the beginning flowerage were most suitable to induce embryogenesis.The production of embryogenesis was influenced by genotypes of plants.The period from the mono-nucleolus early stage to twi-nucleolus,the buds were mostly 3.0mm～3.5mm in length,could produce a lot of embryogenesis,but it was hard to produce embryogenesis when the buds were smaller than 2.5mm or bigger than 4.0mm.3.Data of present study indicated that the buds pretreated under 4℃from 4h to 12h were all suitable for the inducing of embryogenesis,the most efficiency of which was under the treatment for 4h,then followed the treatment for 8h.Little embryogenesis was induced when buds was pretreated more than 48h.Concerning the inflorescence,the pretreatment under 4℃from 4h to 24h were all suitable for inducing embryogenesis.No significant difference of embryogenesis yield was detected between the two treatments of plants with different genotypes.However,the production of embryogenesis was higher in inflorescence pretreatment than in bud pretreatment in the ame genotype. 4.Good effects of surface sterilization and culture of buds were detected under the treatment of NaClO with concentration of 2.0%for 15 to 20 minutes,and HgCl₂ of 0.1%for 10 minutes.5.The concentration of sucrose in B₅ medium could be modified into 2%to dissociate and centrifuge microspores.The medium containing 1.2%of agar,0.02%of NAA,2.0mg/L of 6-BA,3.4 mg/L of AgNO₃ and 3%of sucrose was more suitable for inducing embryogenesis into young plant than others.6.It was about 30 days for the embryogenesis of cotyledon-type or fish torpedo-type to differentiate into plant in 1/2 MS solid medium.Small part of the abnormal embryogenesis with certain genotype could differentiate into plant after 30 days culture in medium with GA₃,while most of the abnormal embryogenesis only shift into callus without GA₃ and then dead.7.The plants could be conserved in bottles about 3 months after treated with paclobutrazol in concentration of 2.0～3.0mg/L,with healthy bodies and strong roots and no affection on the transplantation.8.It was good for the rooting of plants when added 0.5mg/L IBA into the rooting medium, whereas it was harmful to the growth of plants with high concentration of IBA.9.The transplantation could be conducted after rooting cultured for 25 days and the adaptation treatment about 10～15 days.It was good for the survival of plants when transplanted at cloudy day, associated with the disinfection of soil and careful management of the field.10.Totally,240 plants were gained after the microspore inducement,embryogenesis differentiation and plants rooting in the ZB05 medium,and 191 plants were gained after transplantation,112 of which were certified to be fertility normal plants by the fertility test in the field.11.The purity test of the recessive genetic male sterile(GMS) NAB-2 and its plants of embryoid were conducted using 20 SSR primers,18 of which were detected polymorphism among accessions and 32 polymorphic sites were amplified.6 primers could obviously differentiate the plants of embryoid from the first-cousin cross offspring of NAB-2.One real plants of embryoid was detected with 11 of 18 primer combinations.Among DH plants,6 individuals showed polymorphism at the genome level,while no polymorphism be found in the 24 first-cousin cross offspring,which showed that plants of embryoid were more suitable to investigate the genetic diversity of donor population.