Study On Microspore Culture Technology Of Brassica Napus And Common Wheat

Microspore culture is the main way to obtain haploid,a common method in crop breeding.Through microspore culture technology,many haploids can be obtained in a short time,and the homozygous diploid plants can be obtained by doubling the haploid,thus speeding up the breeding process.However,it is easy to limit microspore culture technology by many influencing factors,such as material genotype,pretreatment method,separation and purification method,medium type,etc.,which seriously hinders the application of microspore culture technology in practical breeding.Brassica napus,the main oil crop,belongs to dicotyledon plant of Cruciferae.Common wheat（Triticum aestivum）,the main food crop,belongs to the gramineous monocotyledon plant.In this study,microspore separation,culture,and regeneration system of Brassica napus and common wheat were established.By exploring the key influencing factors in microspore culture,including pretreatment method,disinfection method,separation and purification method,activated carbon,agar concentration,the procedure has been optimized.The application of microspore culture technology in crop genetic population construction and rapid haploid breeding was explored.The main results of this study are as follows:1.The effects of disinfection methods of materials,size of raw buds,activated carbon,and agar concentration on microspore culture of Brassica napus L.and the microspore culture and differentiation system of Brassica napus L.were further optimized.The disinfection method of 75%alcohol 30 s+3%sodium hypochlorite solution could significantly improve the disinfection effect of microspore source buds and reduce the contamination rate of medium as well.The size of flower bud can be used as the basis to determine the optimal harvesting time of different genotypes.When the bud sizes of DH20FB2759/HY62,DH19YB437/FC111 and DH19YB437/18JHB161 genotypes were 3.7±0.3 mm,3.0±0.3 mm and 3.7±0.3 mm,respectively,the corresponding microspore development period ranged from late uninucleate stage to early binuclear stage.This was the best sampling period for microspore culture,and the induction rate of microspore embryo was the highest,embryoid induction rate were 17.47 embryo/bud,14.55 embryo/bud and 14.91embryo/bud,respectively.Totally,0.1%activated carbon could improve the quality of the subsequent embryoid and promote the development of embryoid into seedlings.The agar concentration of 0.9%-1%can remarkably increase the green seedling rate,and the maximum green seedling rate can reach 86.67%,which is beneficial to obtain robust regenerated plants.2.The microspore separation,culture,and differentiation system of Brassica napus was established,including:（1）Microspore separation system:about 40 buds with the size of 3.0-3.7 mm were taken→75%alcohol 30 s+3%sodium hypochlorite solution+2drops of surfactant were disinfected→microspores were isolated and purified in B₅ liquid medium by glass rod grinding and centrifugal methods;（2）Microspore culture system:1×10⁶ cells/ml microspore→N13 medium containing 50mg/L colchicine（NLN medium+13%sucrose）→heat shock at 32℃for 2 days→elute colchicine→static culture in N13 medium for 2-3 weeks→low light culture in 50 rpm for about 2 weeks.Embry form was seen and embryoid induction rate was up to 17.47 embryo/bud;（3）embryoid differentiation system:embryoid→inoculation into B₅ solid medium for culture（40-50 days）→transplanting into vernalization.Green seedling rate was up to 86.67%.3.The influences of disinfection method,pretreatment method,sampling period and separation and purification method on wheat microspore culture were studied.The results proved that the disinfection method of 75%alcohol wiping wheat spikes+2%sodium hypochlorite solution for 10 min disinfection could achieve good disinfection effect and reduce the contamination rate of medium.Pretreatment with0.5 g/L copper sulfate solution for 14 days could significantly improve the microspore induction rate,which reached 3.5 embryos per spike.High temperature heat shock pretreatment could improve the ratio of cell expansion.When the outer morphology of the spikes of Xinong 979 and Lunxuan 987 were stage 2（the top of the wheat spike was 1 cm away from the sheath of flag leaf,and the wheat was just exposed）and Kenong 199 was stage 3（the wheat was completely exposed,but the wheat spikes were not exposed from the bracts）,the ratio of microspore in the late uninucleate stage was the highest,reaching 63.11%,56.78%and 58.71%,respectively.Microspore was effectively separated from anther by using glass rod grinding method.The microspore with cell integrity of 91.99%and cell viability of 79.02%was obtained by grinding medium of microspore extract（0.3 mol/L mannitol+1 g/L MES+1.1 g/L Ca Cl₂·2H₂O）.The best purification method of microspore was the gradient centrifugation method of microspore extract+21%maltose solution,aiming to obtain a high percentage of late uninucleate stage microspore,the ratio of which could reach 80.71%.4.The isolation,purification,and culture system of wheat microspore was established,including:（1）microspore separation and purification system.Wheat spikes of external from stage 2 to stage 3 were taken and placed in 0.5g/L copper sulfate solution at 4℃for 14 days→75%alcohol was used to wipe the bracts.The wheat spikes were disinfected for 10 min with 2%sodium hypochlorite solution→anthems were stripped and placed in microspore extraction solution→microspore was isolated and purified by glass rod grinding method and 21%maltose-density gradient centrifugation method;（2）Microspore culture system:1×10⁴ cells/ml microspore→embryoids appeared after 40 days of static culture in NPB99 liquid medium.This study established and optimized microspore isolation,culture,and regeneration system of Brassica napus and wheat,which laid a methodological foundation for exploring microspore culture technology in crop genetic population construction and haploid breeding.