Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Establishment on Isolated Microspore Culture Optimized System for Restorer of New Cytoplasmic Male Sterile (NER) of Brassica napus L.

[Objective]The aim was to establish optimized system for NER isolated microspore culture of Brassica napus L.. [Method]Twenty varieties of NER of Brassica napus were grown under uncontrolled temperature and light conditions,and their isolated microspore from anthers were used as explants in vitro culture. The influencing factors of microspore culture were preliminarily studied. [Result]The difference of genotypes was important influencing factors to embryoid yield. The embryoid yield increased by supplementing with 6-BA and NAA,culturing in solid-liquid double layer medium with activated charcoal; The difference was not significant of embryoid yield between culturing in medium supplemented with colchicines and the CK. The rates of cotyledonous embryoids directly developed into normal plantlets increased through enriching with 0.1-0.2 mg/L NAA and being treated on slim illumination two days before being inducted into normal plantlets. [Conclusion]The technical system of microspore culture of restorer of new cytoplasmic male sterile （NER） was established,which and lays a foundation for accelerating genotype purification of NER introgressive line.

Isolated Microspore Culture and Plant Regeneration of Leaf Mustard(Brassica juncea(L.)Czern.et Coss.)

Isolated micmspore culture was carried out with Brassica juncea to study the effects of sterilization methods, culture period of embryos, hormone formula and the concentration of aetivatod carbon on embryogenesis, and the concentration of NAA on induction of roots. The results showed that the embryogenesis on medi- um subjected to filter sterilization is better than medium subjected to autoclaved sterilization. The embryos cultured for 15 d had the highest plantlat regenexafion rate （52%）. NLN-13 liquid medium including 0. 1mg/L 6-BA and 0.2 g/L activated carbon significantly improved the planflet regeneration rate. 1/2 MS inclu- ding 0.3 mg/L NAA had the highest plantlet regeneration rate （ 100% ）. Key words Brassica juncea ; Isolated micmspore culture ; Embryogenesis ; Planflet regeneration

Extra-Large Seed Germplasms of Brassica Napus Created through Microspore Culture

Thousand Seed Weight （TSW） is one of the major yield components of rapeseed （Brassica napus L.）. Here reports an extra-large seed germplasm GM01 which was obtained through isolated microspore culture. Three-way cross was made： H8--a Yunnan spring early-maturing rapeseed variety, ＂Legeney＂--a Canadian canola variety and ＂020010＂--a semi-winter late-maturing rapeseed variety. One hundred and forty eight doubled hyploid lines were obtained from the F~ plants of three-way cross through isolated mierospore culture. Among them, the TSW of GM01 amounted to 8.68 g and the TSW of 53 lines were above 5.0 g. The TSWs of GM01 were relatively stable among the multi-location field trials from 2007 to 2014 with variation being only 10%-15% among the locations and years. Compared with H8, GM01 had larger flowers, stigmas, siliques and seed diameters, but less branches, siliques per plant and seeds per silique.

Phytohormones Accumulation and Distribution in Shoots and Roots of Haploid,Diploid and Tetraploid Barley Seedlings Derived from Microspore Culture

Phytohormones play important roles in plant growth and development,and polyploids are thought to be an important method for plant breeding.However,the relationship between ploidy and phytohormone is still unclear.In this study,barley at three ploidy levels were produced by microspore culture.Therefore,we further analyzed the phytohormone content in the shoots and roots of the three kinds of barley materials to study the effect of ploidy on phytohormones accumulation and distribution.The results showed that Abscisic acid(ABA),gibberellin(GA),jasmonic acid(JA),auxin(IAA),salicylic acid(SA)and cytokinin(CTK)were successfully determined in shoots and roots using LC-MS(liquid chromatography mass spectrometry).By comparing the shoots of the haploid and diploid plants,it was found that the distribution trend of the six phytohormones was consistent,and another consistent trend was found in the roots of the diploid and tetraploid plants.In addition,we further analyzed the shoot/root ratio of the different phytohormones to identify the potential differences for haploid,diploid and tetraploid.Here,the relationship between ploidy and phytohormone we provided would provide new insights into understanding the new phenotypes that occur in polyploid species.

Embryogenesis and Development of Isolated Microspore in Chinese Cabbage

[Objective] The aim was to observe embryogenesis and development of isolated microspores in Chinese cabbage. [Method] Chinese cabbage F 1 hybrids were used as the experimental materials, and optical microspore was employed to observe the embryogenesis and development of isolated microspores. [Result] Cells swelled after heat shock treatment, which was the critical factor of embryoid induction. Three pathways equal division, unequal division and germination of microspores were discovered to lead to the embryogenesis from isolated microspores after swelling. Microspore could grow directly to embryoid through germination path way. Equally divided microspores formed the original embryos after successive multiple equal divisions. Original embryos could develop into cotyledon-shaped embryos via globular, heart-shaped and torpedo-shaped embryos. The large one of the two cells from unequally divided microspores continued to divide and finally formed a polar embryoid. [Conclusion] The study will provide cytological basis for high induction frequency and embryoid of Chinese cabbage.

Embryogenesis and Development of Isolated Microspores in Raphanus sativus

[Objective] The aim was to observe embryogenesis and development of isolated microspores of Raphanus sativus.[Method] By using Chinese radish variety Shandong Huaye Xinlimei as the experimental materials,the caryocinesia features and development of isolated microspores in radish were studied.[Result] Two types of cell division appeared in the isolated microspore：unequal division and equational division.The unequal divided microspores had two different size nucleuses,while the equational divided microspores had two same size nucleuses.The equational divided microspores developed to 4-cell structure,multi-cell structure,ball-shaped embryos,heart-shaped embryos,torpedo-stage embryos and cotyledon-stage embryos.[Conclusion] This result will provide cytological basis for the isolated microspore culture of radish.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).