Establishment And Application Of DH Lines, And Studies On Shoot Regeneration And Genetic Transformation From Hypocotyl And Cotyledon In Brassica Oleracea Var. Acephala

Ornamental kale(Brassica oleracea var.acephala) is a variation cabbage in Brassica.It is tolerant to frost and chilling,thereby growing vigorously even in regions that experience low temperatures.It is widely used in China as an ornamental plant in winter and spring during the last decade.However,most of ornamental kale cultivars depend on importation with high cost.So it is necessary to develop our own breeding system of ornamental kale for expoilted novel cultivars adapted to native climate.In this study,we have developed an efficient and reliable protocol for microspore culture and doubled-haploid plant regeneration in ornamental kale,and estimated the genetic diversity of DH₀ and DH₁ population derived from microspore culture.Diallel analysis was used to study the heredity of some ornamental traits.And melocluar markers were developed to indentify the genetic diversity of DH and inbred lines and to predict the heterosis of some crosses.Finally,a high-frequency shoot regeneration system for ornamental kale was established from seedling cotyledon and hypocotyl explants,and Agrobacterium tumefaciens-mediated transformation using these explants was preliminarily studied.The results were presented as follows:1.We have developed an efficient and reliable protocol for microspore culture in ornamental kale by evaluating the effects of microspore developmental stage,sampling time,genotype and culture condition on microspore embryogenesis.Of all the 37 genotypes,20 genotypes(54.1%) produced embryos.The genotype 'Peachy Dancing' showed the highest embryogenesis frequency with yield of 123.6 embryos per dish.The embryogenesis frequency of inbred lines was lower than that of commercial cultivar and F₁ hybrid,and the embryo yield decreased with increased generation of inbreding.The microspores at the medium uninucleate to early binucleate stage when the bud size was 3.0-4.0 mm,and sampling after 3d of florescence were suitable for microspore culture. Cold pre-treatment of flower buds for 48 h at 4℃before microspore isolation enhanced microspore embryogenesis in some genotypes,but was negative in other genotypes,due to genotype dependence.The optimal concentration of sucrose for microspore embryogenesis was 16%(w/v).Medium replacement decreased embryo yield significantly compared with continuous culture without medium replacement,but medium addition enhanced embryo yield.Additions of PGR,active charcoal or colchicine into NLN medium had no effect on microspore embryogenesis.2.DH populations were obtained by investigating the effects of genotype,basal medium,agar concentration,cold treatment,embryoid age and PGR on plant regeneration of embryoid from microspore culture.The frequencies of plant regeneration from embryoids of different genotypes varied from 20%to 90%;MS medium solided with 1.0%agar was suitable for plant regeneration;cold treatment of embryoid could promote the regeneration,but had no significant effect;embyoid at 20-30 d age showed highest regeneration capacity;The frequency of regeneration was enhanced when embryoids were cultured on medium MS+1.5 mg/L BA+0.2 mg/L NAA;The rates of spontaneous doubling of microspore plants from three genotypes varied from 38%to 50%,and more than 95%rooting ability was showed in regenerated plants,among which 75%survived when transplanted;Artificial doubling through culturing haploid plantlets in media with different concentrations of colchicine showed low efficiency,with the highest doubling frequency 16%.3.Genetic diversity was evaluated in DH₀ and DH₁ populations of ornamental kale using ISSR markers.The results showed that there was extensive genetic diversity in the two DH₀ populations evaluated.The genotype of DH₀ populations derived from P3 was more abundant than that derived from 'Red Nagoya'.The range of similar coefficient among 46 plants of DH₀ population derived from 'Red Nagoya' was 0.61-0.97,and the range of similar coefficient among 41 plants of DH₀ population derived from P3 was 0.56-0.91.Almost no genetic difference was detected in three DH₁ populations,but several genetic differences in 2 inbred lines.This indicated that DH₁ lines derived from microspore culture have been pure on genotype while F6 inbred lines have been far from pure.4.In order to select the right lines for crossing of ornamental kale,genetic diversity of DH and inbred lines was assessed by morphological traits,ISSR,SRAP and ISSR+ SRAP markers.Analysis of 11 morphological traits showed that appearance(plant shape and leaf color) was not the important index for morphological classification,while plant height,No.of exterior leaf and inner leaf,and breadth of exterior leafs and inner leafs can identify 18 lines of ornamental kale well.80 ISSR and 84 SRAP marker fragments generated severally from 9 primers were employed to discriminate polymorphism between 18 lines.The number of polymorphic loci,the percentage of polymorphic loci, Shannon's Information index(I) and effective number of alleles(Ne) was 68,85.0%, 0.4055,1.4397 for ISSR markers,and 70,83.3%,0.4123 and 1.4687 for SRAP markers, respectively.The Mantel-test indicated that correlation between ISSR and SRAP markers (R=0.1645) was not significant.The clustering analysis showed that the clustering result of ISSR+SRAP marker was most consistent with pedigree relations of lines.The morphological matrix had a low correlation with molecular marker matrices.The correlation between the result of SRAP marker and that of morphological maker was the highest(R=0.4058),the relation of ISSR marker data and morphological maker data was farthest(R=0.0467) and the relation of ISSR+SRAP marker and morphological maker data was medium(R=0.2952).5.The 7 lines(DH or inbred lines) of ornamental kale were selected to predict the heterosis of some crosses by diallel analysis.The results showed that there was high heterosis in all 5 traits.The correlations between genetic distances based SRAP and ISSR +SRAP markers and F₁ morphological values and heterosis were not significant. However,correlation between the SCA and heterosis of inner leaf number was significant. These suggested that the genetic distances based on molecular marker could not be used to predict the heterosis of ornamental kale.6.A high-frequency shoot regeneration system from seedling cotyledon and hypocotyl explants for ornamental kale was established.The effects of genotype,seedling age and medium on shoot regeneration were evaluated.The maximum shoot regeneration frequency(65.0%for cotyledons,76.1%for hypocotyls) and the average number of shoots per explant(4.3 for cotyledons,8.2 for hypocotyls) was obtained when the explants of cultivar 'Red Nagoya' from 4-day-old seedlings were cultured on MS medium supplemented with 3 mg/L BA and 0.1 mg/L NAA.Among the four varieties tested,'Red Nagoya' showed the best shoot regeneration response.Hypocotyl explants were found to be more responsive to regeneration as compared with cotyledons.The addition of 3.0 mg/L AgNO₃ was beneficial to shoot regeneration.Morphological lower end of hypocotyl segments showed better regeneration capability than the morphological upper end of the hypocotyl segments.The optimal shoot multiplication medium was determined as MS medium containing 1.0 mg/L BA and 0.05 mg/L NAA.In addition,we investigated the genetic stability of regenerated plants randomly,by using ISSR markers.The absence of molecular polymorphism demonstrated that regenerated plants from the same variety were genetically stable.7.Conditions suitable for Agrobacterium-mediated transformation of ornamental kale of seedling cotyledon and hypocotyl explants were studied.Effects of selection agent, kanamycin(Km),on shoot regeneration were investigated.The results indicated that ornamental kale was comparatively sensitive to Km.Selection pressures were determined as 10 mg/L Km.The concentration of cefotaxime(Cef) which restrained shoot regeneration of explants was decided as 300 mg/L.3 d of pre-culture promoted invasion of Agrobacterium evidently,and prevented the browning of explants.The most green shoots were achieved when pre-cultured explants infected with Agrobacterium tumefaciens which has been diluted as twofold for 5 min.And hypocotyls produced more green shoots than cotyledons.The results of detection of GUS expression and PCR analysis showed that the GUS gene in the T-DNA had been integrated into the genome of plants.