| Chinese cabbage vegetable belongs to genus Brassica?B.campestris,syn.B.rapa?of Cruciferae,most of their young leaf or flower were edible organs,there are many kinds of varieties,and more than ten species of vegetables including pakchois,savoy,greens komatsuna,Chinese flowering cabbage and purple flowering stalk,they were adaptable and easy to cultivate.Vegetables of annnual supply in China?s vegetable market ocuppies an important position.This article mainly using pakchois,savoy,greens komatsuna,Chinese flowering cabbage and purple Chinese flowering cabbage,and the hybrids offspring between the above vegetable as the test materials.On the basis of optimizing the protocol of isolated microspore culture,homozygous double haploids were rapidly obtained and identified.The main results of research are as follows:1.Optimization of microspore culture protocol of Chinese cabbage vegetablesIn the optimization protocol of isolated microspore culture,different types of embryoids were successfully obtained in the optimal protocol of isolated microspore culture.Among three genotypes XM2,QW3 and QW4,the addition of TDZ or FDT promoted the induction of embryoids.When the concentration of TDZ was between 0.10.5 g·L-1,the embryo induction rate was significantly higher than that of the control,and when the concentration of TDZ was 0.5 g·L-1,the embryo induction rate of XM2 was the highest,reaching 37.67 embryos per bud.When the concentration of FDT was between 0.10.3mg·L-1,the embryo induction rate of the three genotypes was significantly higher than that of the control.When the concentration of FDT was at 0.3 mg·L-1,the embryo induction rate of QW4 was the highest,which was 25.33 embryos per bud.The addition of ZT or BR promoted the embryogenics of SC1,SC2 and SC3.When the concentration of ZT at 0.02 mg·L-1,embryo induction rates of the three vegetables reached the highest,which were 6.60,15.00 and 16.40,respectively;When the concentration of BR was at 0.02 mg·L-1,the embryo induction rate of SC3 was the highest,21.67 embryos per bud.When the concentration of sorbitol was within the range of 510 g·L-1,the embryo induction rate improved,and the genotype QW4 had the highest induction rate at the concentration of 10 g·L-1,reaching 20.75 embryos per bud.When the concentration of MT,IAA or NAA was 0.025 mg·L-1,the embryo induction rate was the highest?17.67 embryos per bud?.When the concentration of the three surfactants was 0.0001% mg·L-1,the induction rate was the highest,with 21.33 embryos per bud.When the optimal speed of shock culturing was 50 rpm·min-1,not only improved the aeration conditions of the culture medium,promoted the formation of cotyledon embryos,but also promoted the synchronization of embryoid development.The time of heat shock treatments for one day not only improved the temperature condition of the medium,but also improved the synchronization of embryoid development.2.Creation of double haploids of homozygosis pakchoi that have more leaves and bolting resistance.Microspore culture technology was used to guide microspore culture on the F2 generation material of Pakchois,A total of 235 new DH lines of more leaves and bolting resistance vegetables were developed.Addition MT,NAA and IAA promote the embryogenesis induction of QW1 and QW2,and the embryogenesis induction rate was the highest when the concentration was 0.025 m g·L-1.When the embryoids were inoculated into MS culture medium,the plant regeneration rate of QW4 was the highest,reaching 68.67%.The seedling rate was the highest when the embryoids were cultured for 1525 days,and the plantlet regeneration rate was 63.3%76.7%.When NAA concentration is between 0.050.1 mg·L-1,it is conducive to the formation of regenerated seedling root.The spontaneous doubling rate of the regenerated plants of the four genotypes was above 58%.A total of 163 plants with persistant green traits were obtained by using QW1 and QW2 genotypes.All the DH lines of the five excellent Pakchois had an affinity index of less than 0.3 in the flowering period and greater than 5 at bud period,which could be used as self-incompatibility lines in hybrid breeding.3.Creation of double haploids of homozygosis of multi-branch Chinese flowering cabbageIn this study,203 homozygous DH lines with multiple branches,brilliant green color leaves of Chinese flowering cabbage were obtained,and the isolated microspore culture was carried out using the offspring of hybrids between pakchoi and Chinese flowering cabbage,which greatly improved the embryo induction rate and plant regeneration rate.Microspore embryo induction rate was significantly improved when the concentration of TDZ between 0.10.5 mg·L-1,and the average direct plantlet regeration rate was above 60%,especially genotype 17sy06,reaching 81.67% with 0.5 mg·L-1TDZ.The spontaneous doubling rate of microspore regenerated plants was over 70%.The highest total plot yield was SC09,up to 5775.7g.Comprehensive flavor for CC01,CC11,CC02 and CC03 were better.The DH line from genotype 17sy07 had floral affinity index less than 0.3 and bud affinity index greater than 3.9,with self-incompatibility.DH line SC04 has the highest general combining ability and can be used as the parent materials to obtain the required stable varieties through recombination of sexual hybridization genes.The combination with the highest special combining ability is SC07 × Qi 2 sterile lines,followed by SC16 × 019 sterile lines,which can be used as parents to select and breed the first-generation hybrid.4.Creation of double haploids homozygosis of more leaves Greens KomatsunaThe hybrids from Greens Komatsuna with maer were used as test materials,after isolated microspore culture has received more than 197 DH lines of more-leaves Greens Komatsuna,their leaves number increased significantly,bright green,leaf blade rushed;they could ber used as the parent material for breeding hybrid with strong tillering ability,disease resistance and high yield.Brassinolide can effectively improve the frequency of induction and plant regeneration of microspore culture.The optimal concentration of BR required by ?XM1?,?XM2?and ?XM3? are 0.02 mg·L-1,0.01 mg·L-1 and 0.02 mg·L-1,respectively.The average induction rate was 25.00,20.05 and 8.5 embryos per bud,and the direct plant regeneration rate was 87.00%,83.67% and 74.00%,respectively.When BR at 0.08 mg·L-1,both embryo induction rate and direct plant regeneration rate decreased.The spontaneous doubling rate of microspore embryos in the three genotypes of Greens Komatsuna was more than 68.5%,genotype XM1 has the highest spontaneous doubling rate,70.63%.The morphological characteristics of 14 DH lines received from XM2 were identified,and the characters of DH lines were different and showed stable characteristics.5.Creation of double haploids of homozygosis in purple Chinese flowering cabbageIn isolated microspore culture,the embryo induction rate of purple Chinese flowering cabbage was low.After hybridization with purple pakchoi,the embryo induction rate was increased when the offsprings were cultured with microspore,a total of 122 new purple Chinese flowering cabbage DH lines were obtained,and the results were quite different.Only genotype ZC1 and ZC2 in the three hybrid combinations induced embryoids,and ZC3 did not produce embryos even with the addition of surfactant in microspore culture.0.0001% g·L-1surfactant played an important role in promoting the induction of microspore embryoids and plant regeneration.The embryoid induction rate was more than 12 embryos per bud,and the direct plant regeneration rate was more than 50%.The embryoid induction rate of microspores gradually decreased when the surfactant concentration beyond a certain range,and the phenomenon of inhibition or even no embryo appeared in high concentration.The average double haploid rate of the two genotypes was over 70%,and ZC1 was the highest,up to 81.82%.Morphological characteristic of 9 excellent DH lines were identified and there is a big difference.We found that the height and thickness of the main and side stem on yield were significant in Chinese flowering cabbage. |
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