【作者】 戴希刚；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 园林植物与观赏园艺， 2009， 博士

【摘要】 羽衣甘蓝（Brassica oleracea var.acephala）是十字花科芸薹属甘蓝种的一个变种,由于其耐寒性极强,可忍受低温霜冻,近年来,已成为我国冬春季的主栽观赏植物。目前国内市场上羽衣甘蓝品种大部分为进口,且价格较高。因此选育出性状优良的自交亲本,建立一套完善的制种技术,培育出具有自主知识产权的羽衣甘蓝品种具有重要的意义。本文主要以羽衣甘蓝杂交育种为目的,利用小孢子培养、分子标记、双列杂交和遗传转化等方法对羽衣甘蓝DH系的获得、DH₀和DH₁代的遗传多样性、DH系与自交系的遗传关系的评价、杂种优势的表现、分子标记对杂种优势的预测、遗传转化受体系统的建立等进行了系统的研究。主要研究结果如下:1.建立了羽衣甘蓝稳定、高效的游离小孢子培养体系。详细地研究了小孢子发育时期、取样时期、基因型、培养条件等因素对小孢子胚胎发生的影响。结果表明,供试的37个基因型中,有20个获得了胚状体,占供试材料的54.1%,其中基因型‘桃舞’获得了最高的出胚率,为123.6个/皿;自交系的出胚率比商业品种和F₁代杂种的出胚率要低得多,且自交代数越高,小孢子的胚胎发生能力就越弱;在供试植株开花后3天取样,选择花蕾大小为3.0-4.0 mm的处于单核中期到双核早期的的小孢子进行培养时胚胎发生能力最强;小孢子分离前,4℃低温预处理48 h有利于部分基因型的小孢子胚胎发生,而对某些基因型有副作用;16%的蔗糖浓度最适合羽衣甘蓝小孢子的胚胎发生,在热激培养48 h后更换培养基对小孢子的发育有副作用,而加液培养能起到积极作用;向培养基中添加激素、活性炭和秋水仙素对小孢子的胚胎发生无促进作用。2.对小孢子胚状体再生过程中基因型、基本培养基、琼脂浓度、低温处理、胚龄、激素等影响因素进行研究,从而建立遗传稳定的DH系。各不同基因型的小孢子植株再生率水平在20%-90%之间;附加1.0%的琼脂的MS培养基最适合植株再生;低温处理对胚状体的再生有一定作用,但不明显;25-30 d胚龄的胚状体再生能力较强。MS+1.5 mg/L BA和0.2 mg/LNAA培养基有利于胚状体再生成苗;被鉴定的3种基因型的小孢子植株的自然加倍率在38%-50%之间,95%以上的植株能够很好的生根,移栽成活率在75%以上。无菌苗在培养基中进行秋水仙素加倍处理效果不佳,加倍率最高仅为16%。3.利用ISSR标记评价了羽衣甘蓝DH₀代及DH₁代群体的遗传多样性。结果发现被评价的两个DH₀代群体均存在较大的遗传多样性,其中由P3小孢子培养得到的DH₀群体比‘名古屋-红’所得到的DH₀群体的基因型更丰富。‘名古屋-红’的DH₀群体46个株系间相似系数范围为0.61-0.97;P3的DH₀群体的41个株系间相似系数范围为0.56-0.91。3个DH₁代群体各株系间基本无遗传差异,而作为对比的两个自交系存在一定的遗传差异,这表明由小孢子培养所得的植株经过自交后的DH₁代各株系基因型是完全纯合的,而自交6代的自交系在遗传上远未达到纯合。4.为了正确地选择羽衣甘蓝杂交育种的优良亲本,利用表型性状、ISSR、SRAP以及ISSR+RAP标记对羽衣甘蓝DH系和自交系间进行了遗传多样性的评估。通过对11个表型性状的分析,结果表明外观特征（株型和叶色）不是羽衣甘蓝表型性状分类的重要指标,而株高、外叶数及开展度、心叶数及开展度能很好地鉴别羽衣甘蓝纯系聚类群。从38条ISSR引物和170对SRAP引物中筛选到具有多态性的ISSR和SRAP引物各9条。ISSR引物共扩增出80条带,多态性位点数为68,多态性位点比率为85.0%,平均多样性指数为0.4055和有效等位基因数为1.4397;SRAP引物共扩增出84条带,多态性位点数为70,多态性位点比率为83.3%,平均多样性指数为0.4123,有效等位基因数为1.4687。ISSR与SRAP标记的聚类结果微弱相关（R=0.1645）;ISSR+SRAP的聚类结果与品系的系谱来源最为一致;形态标记和分子标记之间存在着一定的相关性,但是一致性并不高。其中SRAP标记与形态标记的结果相关性最高,相关系数为0.4058,其次为ISSR+SRAP,ISSR标记与形态标记结果相差最远,其相关系数仅为0.0467。5.选取羽衣甘蓝7个纯系作为亲本进行双列杂交,结果表明,各个性状上都表现出较强的杂种优势。利用SRAP和ISSR+SPAP标记的羽衣甘蓝品系间遗传距离与各性状的F₁表现和杂种优势相关性均不显著,而特殊配合力与心叶数的杂种优势呈显著相关。基于分子标记的亲本间遗传距离难以有效地预测羽衣甘蓝的杂种优势。6.通过羽衣甘蓝无菌苗子叶和下胚轴建立了高效的再生体系。子叶和下胚轴的再生能力主要受基因型、苗龄和培养基的影响。4 d苗龄的外植体培养在附加3 mg/LBA和0.1 mg/L NAA的MS培养基上获得了最高的再生率（子叶65.0%,下胚轴76.1%）和平均出芽数（子叶4.3,下胚轴8.2）,其中下胚轴的再生能力比子叶强;4个被试基因型中以‘名古屋-红’的再生能力最高;培养基中添加3.0 mg/L AgNO₃对子叶和下胚轴的再生有促进作用;下胚轴切段的形态学下端比上端的芽分化能力要强。MS+1.0 mg/L BA+0.05 mg/L NAA用于再生芽的增殖效果最好;经过ISSR分子标记检测,4个基因型的再生植株均表现出较强的遗传稳定性。7.以羽衣甘蓝子叶和下胚轴为受体,对根癌农杆菌介导的遗传转化条件进行了摸索。羽衣甘蓝对卡那霉素（Km）反应较为敏感,最终确定转化过程中的选择压力为10 mg/L;抑菌剂头孢霉素（Cef）浓度定为300 mg/L,但其对外植体再生有抑制作用。侵染之前对外植体预培养3 d可以防止外植体褐化死亡,提高农杆菌的转化效率。菌液稀释2倍,侵染时间为5 min时分化出的绿芽数量最多,其中下胚轴获得的绿芽数比子叶最多。获得的绿芽经过筛选继代培养后经过GUS检测及PCR检测,证明GUS基因已经转入羽衣甘蓝基因组中。更多还原

【Abstract】 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.更多还原