摘要
为探索竹类植物的遗传改良技术,选择丛生竹中分布较广、适应性较强的孝顺竹(Bambusa multiplex)为材料,开展了地理居群表型性状变异的调查分析,基于AFLP标记的遗传多样性分析,开花生物学特性研究和杂交试验,以及不同外植体类型的愈伤组织再生技术研究。主要获得以下研究结果:
1.对孝顺竹自然分布区内5个天然地理居群开展胸径、地径、全长等19个表型性状调查和统计分析,发现居群间和居群内存在丰富的表型变异。19个表型性状在5个居群间的差异均达极显著或显著差异,泸州居群有14个表型性状的均值具5个居群之首,而仁化居群有11个性状的表型均值具5个居群之末。相关分析表明,胸径、地径、胸径/地径等14个表型性状间相关性均达到极显著或显著水平;仅秆节数、秆节数/全节数、叶长、叶宽、叶长/宽等5个性状与其它性状间的相关性较弱。表型性状与地理生态因子间的相关系数显示,秆重/全重与纬度、叶长/宽与海拔呈显著性正相关,叶长与经度、叶长/宽与经度、秆节数/全节数与纬度等分别呈显著性负相关。表型性状主成分分析的结果可获得3个主成分,累计贡献率达97.435%。第一主成分以胸径、地径、全长等8个性状的权重较大;第二主成分以叶长/宽的作用最大;第三主成分以秆节数、秆节数/全节数权重较大。将主成分分析的结果代入群体平均值开展系统聚类,可以从表型上将5个居群划分为3类,即温岭居群首先与长汀居群聚为一类,龙山居群与仁化居群聚为一类,泸州居群单独一类。2.从56对AFLP引物中筛选出10对较好的引物用于5个孝顺竹居群150个样品的PCR扩增,统计分析结果表明:10对引物在5个居群中共检测到939个位点,多态性位点比率71.99%。5个孝顺竹居群内检测到的多态位点数A在106-464之间,平均为255.8个;多态位点百分率P在11.29%-49.41%之间,平均为27.24%;观测等位基因数na在1.1129-1.4941之间;有效等位基因数ne在1.0713-1.3147之间;Nei’s基因多样性指数h在0.0406-0.1794之间;Shannon信息指数I在0.0601-0.2656之间;多态位点数A、多态位点百分率P、等位基因数na、有效等位基因数n、Nei’s基因多样性指数h、Shannon信息指数I的等指标在5个居群间的排序均表现为长汀>仁化>温岭>泸州>龙山,表明长汀居群存在丰富的遗传变异,而龙山居群的遗传多样性相对较低。孝顺竹物种水平遗传分化系数Gst=0.4393,即孝顺竹群体间的遗传变异占总变异的43.93%,群体内的遗传变异占总变异的56.07%;居群间基因流Nm =0.6383。5个孝顺竹居群间的遗传一致度在0.8165-0.9675之间,遗传距离在0.0330-0.2027之间,利用遗传距离采用UPGMA法对5个孝顺竹地理居群进行聚类的结果显示,泸州居群内和龙山居群最先聚在一起,然后与温岭居群相聚,长汀居群最后聚在一起。
3.孝顺竹开花生物学和杂交试验表明,孝顺竹常于4月份形成花序,小花在早晨至上午开放,下午14:00-15:00陆续闭合;花丝伸长生长结束时花粉生活力最高,萌发率可达50%以上;稃片完全张开时柱头可授性较强,上午9:00-12:00柱头可授性较好。孝顺竹种内自交授粉、及其与麻竹的杂交试验表明,孝顺竹自交种实与杂交种实未表现出明显区别,均为长椭圆形,有不甚明显的腹沟,具喙,成熟时黄褐色。成熟的孝顺竹种子纯净种子长0.82-1.10 cm,宽0.26-0.28 cm,百粒鲜重3.32 g。孝顺竹×麻竹杂交苗在四个月龄时即表现出与母本自交苗明显的差异,叶片明显大于后者,此时表型杂种的叶长、叶宽、叶长/宽、地径、发笋数、叶脉数、叶片形状、叶鞘高度、叶鞘繸毛、叶耳、叶舌等性状均介于母本和父本自交个体之间。对表型杂种开展AFLP分子标记鉴定,10株表型杂种中父本特异位点占21.97%-28.10%,非亲本位点占1.88%-4.05%,表明杂种引入了父本的遗传基础且进行了遗传重组。杂种秆箨在15个月龄时表现出刺毛性状的分离,一种为父本的多刺毛表型,一种为母本的光滑秆箨。孝顺竹开花生物学的研究和种间杂种的获得为实现丛生竹重要性状的遗传学分析和新品种选育奠定了基础。
4.利用孝顺竹小穗和种胚外植体诱导出胚性愈伤组织并实现植株再生。适宜愈伤组织诱导的培养基组成为NB+500 mg·L-1脯氨酸+500 mg·L-1谷氨酰胺+300 mg·L-1水解酪蛋白+30 g·L-1蔗糖+8 g·L-1卡拉胶+4 mg·L-1 2,4-D的培养基上,小穗和种胚愈伤组织诱导率分别可达87.30%、76.27%。对愈伤组织增殖的培养基进行了优化,较好的培养基组成为KNO3475 mg.L-1+ NH4NO3 825 mg.L-1+ MgSO4·7H2O 185 mg.L-1 + KH2PO4340 mg.L-1 + CaCl2·7H2O 440 mg.L-1+MS basal micronutrients + MS Iron + MS vitamins +1.0 mg.L-1 proline+30 g.L-1 maltose+8 g.L-1carrageenan+4 mg.L-1 2,4-D,部分愈伤组织可耐10.0 g.L-1 NaCl胁迫,75 mg.L-1的潮霉素可用于愈伤组织的抗性筛选。悬浮培养时,接种细胞密度以4.0%为宜,转速设120 r/min较好,2,4-D浓度4~6 mg·L-1,7-10d为转接周期较佳。植株分化过程经历预分化转绿和发芽两个阶段,经MS+30 g·L-1蔗糖+10 g·L-1卡拉胶+4 mg·L-1 KT培养基预分化7 d后,转入无激素的MS培养基上分化14 d,小穗愈伤组织仅个别长出绿色芽头;但种胚愈伤组织芽分化率可达80.0%。分化芽中有8%左右的白化苗,并伴有花叶苗出现。优化后的种胚愈伤组织预分化最佳培养基是MS+3 mg·L-1 6-BA+3 mg·L-1 KT。孝顺竹再生植株生根较容易,分化芽苗在添加2 mg·L-1 NAA的MS培养基上生根良好,移栽成活率可达70%以上。孝顺竹高效愈伤组织再生体系的构建为实现基于农杆菌介导的遗传转化提供了条件。
Bambusa multiplex (Gramineae, Bambusoideae), a sympodial bamboo species with pachymorph rhizome was chosen as material for genetic improvement research, considering its wide distribution and strong stress-resistance. In this study, phenotypic traits of five wild populations of B. multiplex in China were investigated and analyzed, genetic diversity was measured by AFLP markers, flowering habit and crossing test were conducted, and callus induction and plantlet regeneration of different explant types was experimented, the results are as follows:
1. Phenotypic variation of B. multiplex were conducted by using five geographical populations of Wen Ling, Long Shan, Lu Zhou, Ren Hua and Chang Ting, China, 20 culms of each were cut down for samples. Total 19 phenotypic traits were measured and analyzed, including diameter of breast height (dbh), diameter of culm base (dcb), dbh/dcb, culm length (cl), full length (fl), cl/fl, culm fresh weight (cfw), full fresh weight (ffw), cfw/ffw, number of culm internode (nci), mumber of full internode (nfi), nci/nfi, maximum internode length (mil), number of internodes under branching (nib), height under branching (hb), angle of branching (ab), leaf length (ll), leaf width (lw) and ratio of ll/lw. The results showed that abundant variation existed within and among populations, 14 traits of Lu Zhou population toped the five and 11 traits of Ren Hua population bottomed. 7, 5, 4, 2 and 1 traits in Long Shan, Chang Ting, Ren Hua, Lu Zhou and Wen Ling had the largest variance coefficient, respectively. Correlation analysis demonstrated that difference at 1% to 5% significant level existed between 14 phenotypic traits including dbh, dcb, etc. While five phenotypic traits of number of nci, ratio of nci/nfci ,ll, lw and ratio of ll/lw. Some traits exhibited correlation to geographical and ecological factors, for example, significant positive correlation of cfw/ffw to latitude, ll/lw to altitude, and negative correlation of ll to longitude, ll/lw to longitude, etc. 3 principal components were obtained after analysis by using 19 phenotypic traits with a cumulative contribution of 97.44%. Index of principal components were introduce into the original values of each population and a cluster analysis was developed, five populations were divided into 3 groups by phenotypic traits, Wen Ling and Chang Ting joined together, Long Shan and Ren Hua joined, Chang Ting was the third group alone.
2. 10 primer combinations of AFLP were screened out for genetic analysis of the above five populations of B.multiplex, each with 30 samples. Total 939 loci were detected with a polymorphic percentage of 71.99%. In each population, polymorphic loci (A) varied from 106-464 with an average of 255.8; polymorphic percentage (P) changed from 11.29%-49.41% with an average of 27.24%; Observed alleles number (na) was 1.1129-1.4941; Effective alleles number (ne) was 1.0713-1.3147; Nei's gene diversity (h) was 0.0406-0.1794; Shannon's Information index (I) 0.0601-0.2656. All of these index showed a tendency of Chang Ting>Ren Huap> Wen Ling>Lu Zhou>Long Shan in five B.multiplex populations, indicates that a rich variations existed in Chang Ting while a low genetic diversity in Wen Ling. Coefficient of genetic differentiation of B.multiplex (Gst)was 0.4393, which means a 43.93% genetic variation occurred between populations while 56.07% genetic variation in population; gene flow (Nm) among populations was 0.6383. Nei’s genetic identities in five B.multiplex populations were between 0.8165-0.9675, and genetic distances were between 0.0330-0.2027. Five populations showed a molecular phylogeny of Luzhou and Long Shan were clustered together firstly, then Wen Ling joined, and the Chang Ting was the last.
3. Studies on flowering biology and crossing of B.multiplex showed that it’s inflorescences usually formed in April, spikelets opens in the morning and closes at 14:00-15:00 p.m.. Pollen germinated percentage can be over 50% at the time that the elongation growth finishes of filament. Stigma receptivity is the best when glumes open during 9:00-12:00 a.m.. Seeds from selfing of B.multiplex and B.multiplex×Dendroclamus latiflorus doesn’t show apparent difference, they were long oval, inconspicuous ventral furrow, browning yellow when mature with 0.82-1.10 cm long, wideth0.26-0.28 cm wide ,weight 3.32 g per 100 seeds. Progenies showed significant difference from selfing and crossing at four month’s age, putative hybrid showed a more larger leaf than that of selfed ones. At this age, the leaf length, leaf width, leaf length-width ratio, ground diameter, shoot numbers, leaf vein numbers, leaf shapes, leaf sheath height, leaf sheath hair, ligules, auricle of putative hybrids shows a middle value between the paternal and maternal selfed progenies. AFLP identification of hybrids were conducted, paternal particular loci changed from 21.97%-28.10% in 10 hybrids, and non-parental loci were 1.88%-4.05%, gave a molecular confirmation of true hybridity. Sheath of hybrids shows segregation in cilia at fifteen month’s age, one is the paternal ciliate and the other shows the maternal glabrous. Such a study make a base for elite hybrids selection and genetic analysis of important taxonomic taits of sheath ligule, auricle, blade and spot, etc..
4. Embryogenic callus was initiated and plant regeneration has been achieved by culturing of spikelets and seed embryo explants of B. multiplex. 87.30% and 76.27% callus were induced 475 mg.L-1+ NH4NO3 825 mg.L-1+ MgSO4·7H2O 185 mg.L-1 + KH2PO4340 mg.L-1 + CaCl2·7H2O 440 mg.L-1+MS basal micronutrients + MS Iron + MS vitamins +1.0 mg.L-1 proline+30 g.L-1 maltose+8 g.L-1carrageenan+4 mg.L-1 2,4-D, callus can proliferate under stress of 0.0 g.L-1NaCl, 75 mg.L-1 Hygromycin was useful in callus resistance selection. A good suspension culture system of callus was an inoculation density of 4.0%, rotation speed was 120 r/min, 2,4-D concentration was 4~6 mg/L, and subculture every 7 days.
Plantlet regenerated by two stage of pre-differentiation and germination, after a 7 d pre-differentiation on MS supplemented with 30 g·L-1 sucrose, 10 g·L-1 Carrageenan and 4 mg·L-1 kinetin (KT), and a 14 d auxin-free germination culture on MS, only a few spikelet calli germinated; whereas embryo calli regenerated by up to 80.0%; about 8% plantlets were albinal, and mosaic plantlets were occasionally found. Optimal pre-differentiation medium for seeds embryoids was MS supplemented with 3 mg·L-1 6-benzylaminopurine (6-BA) and 3 mg·L-1 KT; plantlets rooted in MS supplemented with 2 mg·L-1 naphthaleneacetic acid (NAA). Rooted plantlets transferred to soil with over 70% success. Such an effective regeneration system of B.multiplex made it possible for genetic transformation mediated by agrobacterium tumefaciens.
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