【作者】 王磊；

【导师】 徐坤；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2015， 博士

【摘要】 生姜(Zingiber officinale roscoe)为姜科(Zingiberaceae)多年生草本植物,以地下肥大的肉质根茎为食用器官。据联合国粮农组织(FAO)统计,我国是世界上生姜栽培面积最大,生产总量最多的国家。近年来,随着生姜国际贸易量的增加,对生姜品种的经济性状提出了更高的要求,加快生姜新品种选育至关重要。生姜是无性繁殖作物,通过传统有性杂交手段进行新品种选育较难,主要采用对自然变异选择及利用诱变技术和生物技术等手段开展新品种选育。由于种质资源染色体倍性数据是新品种选育必要的基础数据和遗传背景,但关于生姜种质资源倍性分析方面研究较少,因此本研究在对生姜种质资源的染色体倍性和染色体基数进行系统地分析基础上,构建不同倍性生姜芽尖快繁技术体系,开展生姜染色体诱变技术研究,建立生姜细胞悬浮系等生物育种技术体系,可为创新生姜种质材料和新品种的选育提供有力的保证。主要结果如下:1采用流式细胞仪对来自中国各地63份生姜种质资源进行倍性分析,结果表明,16个生姜品种(占总种质资源25.4%)为二倍-四倍混倍体生姜,其中四倍体细胞比率均大于40%,47个生姜品种(占总种质资源74.6%)为二倍体,没有四倍体或更高倍性生姜自然发生,但生姜种质资源中二倍-四倍混倍体的自然发生为首次发现。同时采用根尖压片法对所有生姜种质资源染色体数目进行分析,结果表明,中国地区生姜基本染色体数为x=11。生姜的表型性状随着倍性不同而发生改变,二倍体生姜和混倍体生姜植株之间存在显著形态学差异,混倍体生姜茎粗较粗,分枝数和姜球数较少,单球重和单株根茎鲜重较大,为“大姜类型”生姜;二倍体生姜茎粗较细,分枝数和姜球数较多,单球重和单株根茎鲜重较小,为“小姜类型”生姜。同时分别通过对不同倍性之间的差异表型性状进行聚类分析,最终确定生姜单球重为区分二倍体生姜和混倍体生姜最关键的植物学性状。2不同激素配比显著影响不同倍性生姜离体芽尖接种成活率、初代接种芽尖增殖系数以及继代增值系数和试管苗植株株高。当NAA 0.4 mg·L-1与KT 2.5 mg·L-1时,混倍体生姜“山农1号”的茎尖成活率较高,为73.5%,且此激素浓度配比时,继代增殖系数也较大,为5.8,株高为7.2cm;二倍体生姜“广西小黄姜”适宜初代培养基激素配比与继代培养基也较为一致,为NAA 0.2 mg·L-1、KT 1.5 mg·L-1。采用ISSR分子标记方法检测不同倍性生姜离体快繁中不同继代次数的试管苗、驯化苗以及大田苗(母本)的遗传稳定性,结果表明,“山农1号”和“广西小黄姜”扩增片段长度均在200~2000bp,其中“山农1号”共获得清晰可见带60条,平均每个引物代数大约6条;“广西小黄姜”共获得清晰可见的带43条,平均每个引物代数大约为4条(表6)。通过比较和分析不同引物的电泳图得出,“山农1号”和“广西小黄姜”的不同继代次数试管苗、驯化苗与大田苗相比,issr引物扩增谱带无异,表明在所检测范围内不同倍性生姜离体快繁时dna水平上未发生变异,遗传稳定性好。同时也通过流式细胞仪确定,无论混倍体还是二倍体生姜,通过茎尖离体培养长期保存后,其dna含量比较稳定,倍性一致,且不同倍性生姜的驯化移栽苗与大田苗的植株形态性状也无明显变异,如“山农1号”大田苗的根茎鲜重为1452.0g,驯化苗为1435.3g,两者之间无显著差异。3以混倍体生姜“山农1号”为试材,通过秋水仙素诱导可产生二倍体和四倍体生姜,且秋水仙素处理浓度和诱导时间可显著影响诱导效果,在附加秋水仙素浓度为0.15%时,处理时间为12h~16h时,生姜茎段的成活率高于80%,植株变异率高于25%。与混倍体植株(对照)相比,二倍体生姜株高较矮,茎粗较细,叶片较薄,如二倍体生姜的株高、茎粗和叶厚分别比对照低30.3%,10.5%和27.3%。秋水仙素诱变产生两个四倍体表型,表型Ⅰ株高、茎粗、叶长、叶宽和叶厚分别较对照低47.0%,42.1%,28.6%,26.7%和40.5%;而四倍体表型Ⅱ各指标均较对照高于7.6%、13.2%、34.3%、60.0%和35.7%。4混倍体生姜(对照)和诱导产生的二倍体生姜继代时植株成活率较高,倍性稳定。但四倍体生姜随着继代次数增加成活率显著降低,第10次继代时,表型Ⅰ的植株成活率已低至为0,而表型Ⅱ仅为1.5%,且表型Ⅱ倍性易改变,第7代继代时,就会有17.9%的二倍体植株出现。不同倍性生姜试管苗驯化移栽时,成活率不同,移栽后植株长势也不同。对照和二倍体生姜移栽成活率可达100%,生长较快,而四倍体试管苗的移栽成活率较低,生长缓慢,长势较弱,且随着生长的进行,移栽植株陆续发生死亡,移栽后120d时,四倍体Ⅰ型和Ⅱ型植株成活率为44.0%和28.0%,但移栽后180d时,四倍体两种表型成活率均为0。可见,四倍体可能是生姜不能稳定存在的倍性类型。5以“莱芜大姜”试管苗叶片为外植体,接种到ms+1.0mg·l-12,4-d+0.5mg·l-16-ba+30g·l-1蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到ms+0.15mg·l-12,4-d+6.0mg·l-16-ba+30g·l-1蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0g-1.5g,继代培养的适宜间隔期为15d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基ms+0.2mg·l-1NAA+10.0 mg·L-1 6-BA+30g·L-1蔗糖上可获得再生植株。更多还原

【Abstract】 Ginger(Zingiber officinale roscoe) belongs to family of Zingiberaceae and perennial herb, and its edible organ is fat rhizome underground. According to statistics by FAO(Food and Agriculture Organization of the United Nations), C hina is the largest ginger producers to the world market. Currently, with increase of ginger quantum of international trade, higher demand to commercial character of ginger variety is proposed, therefore it is important for breeding new ginger variety.Due to poor flowering and seed set, conventional crossbreeding is inapplicable, nature variation selection, ploidy breeding and biology breeding provide some promising strategies for the crop improvement of ginger. Ploidy level of genetic resources is fundamental and essential to develop an efficient breeding program. However, information on ploidy of the ginger germplasm is scarce. The object of this study was to determine DNA ploidy levels and chromosome numbers of the C hinese ginger germplasm resources, on this basis, to set up shoot propagation system in vitro of different ploidy ginger, to study ginger ploidy breeding technique, and to develop ginger cell suspension cultures method, and these knowledge will offer further insights into ginger germplasm creation and breeding new variety. The main results are as follows:1 Sixty-three ginger varieties representing diverse geographic locations in C hina were analyzed for ploidy by flow cytometry. O ur results indicated that 16 ginger varieties(25.4% of the accessions) were mixploids with diploid and tetraploid cells, which tetraploid cell percentage was higher than 40%, and 47 ginger varieties(74.6% of the accessions) were diploid. No tetraploids or other polyploidy occurred in nature and it is the first time to report natural occurrence of mixploid ginger. C hromosome numbers were counted in actively growing root tips using conventional rapid squash techniques. The results showed he basic chromosome number of ginger in C hina is x = 11. Phenotypic alterations occurred in response to changes in ginger ploidy level. Mixploid ginger was typed as a ?giant ginger? with stronger stems, fatter fingers, bigger rhizomes, fewer shoots, and the diploid was typed as ?small ginger? with thinner stems, more shoots, and a smaller single finger and rhizomes. Based on clustering analysis of each discrepant trait, it was found that the single finger mass of ginger was the most important and key morphological character distinguishing the mixploid from the diploid.2 Plant growth regulator combinations had significant effect on in vitro shoot survival rate, shoot initiation, shoot multiplication and plant height of plantles. With regulator combinations of 0.4 mg·L-1 NAA and 2.5 mg·L-1 KT, higher shoot survival rate of mixploid ginger “No.1 shannong” was 73.5%, and bigger shoot multiplication in subculture is 5.8. For diploid ginger “Guangxi small ginger”, the combination of plant growth regulator suitable for shoot initial was same as shoot multiplication, which NAA was 0.2 mg·L-1and KT was 1.5 mg·L-1. The genetic stability of micropropagated clones of different ploidy ginger was evaluated at different subculture times using nter-simple sequence repeat(ISSR). The results showed primers utilized in this study gave rise to a total of 60 scorable bands in “No.1 shanong” and 43 scorable bands in “Guangxi small ginger”, ranging from 200 bp to 2000 bp in size. ISSR analysis of micpropagated plants of different ploid ginger showed a profile smiliar to that of the field plants indicating that no genetic variation had occurred in vitro. FCM analysis showed no significant variation in nuclear DN A contents and ploidy levels in long-term in vitro maintained thorough different ploidy ginger shoot. There was also no phetotypic significance between accamalited plantl and field plant, for example, fresh weight of rhizome with filed plant of “No.1 shannong” was 1452.0g, similar to that of accamalited plant, 1453.3g, which showed no significant difference.3 Taking mixploid ginger “No.1 shannong” as material, different colchicines concentrations and time duration of treatment were combined to induce diploid and tetraploid ginger. Survival rate of stem cutting in vitro is higher than 80%, and mutation rate is more than 25%, when colchicine concentration increased to 0.15% for 12h~16h. Compared to mixploid ginger(control), diploid ginger was a plant with shorter plant height, thinner stem and leaf. Plant height, stem diameter and leaf width of diploid ginger was lower than these of CK by 30.3%, 10.5% and 27.3%. Two tetraploid phenotypes was induced from mixploid by colchine, which type Ⅰ?s plant height, stem diameter, leaf length, leaf width and leaf thickness were lower than those of CK by 47.0%, 42.1%, 28.6%, 26.7% and 40.5%, these indexes of type Ⅱwere higher than those of CK by 7.6%, 13.2%, 34.3%, 60.0% and 35.7%.4 Mixploid giner(CK) and induced diploid had higher plant survival rates, more stable ploidy in subculture. With increase of subculture times, survival rate of tetraploid reduced. At 10 th subculture, plant survival rate of type Ⅰhad reduced to 0, and typeⅡis only 1.5%. ploidy level of typeⅡwas inconstantly, which 17.9% diploid ginger was generated from typeⅡat 7th subculture. When different ploidy ginger plantlet in vitro was acclimated and transplanted, their survival rate and growth vigor were different. Transplanting survival rate of CK and diploid ginger was 100%, which growed quickly, and tetraploid ginger had lower transplanting survival rate, weaker growth vigor. With plant growing, transplanted tetraploid plant died successively. After 120 d transplanted, plant surviva l rates of type Ⅰ and Ⅱwere 44.0% and 28.0%, but their survival rates all were 0 after 180 d transplanted. Therefore, tetraploid may not be stable ploidy level ginger.5 “Laiwu” giant ginger was taken the material to study induction of leaf-derived callus, establishment of cell suspension and plant generation in vitro ginger. The results showed that young leaves of in vitro ginger as explant were cultured on revised Murashige and Skoog medium supplemented with 2,4-D 1.0 mg·L-1, 6-BA 0.5 mg·L-1 and sucrose 30 g·L-1, and it resulted in callus with loose structure rapid growth. Obtained callus were cultured in modified liquid MS medium supplemented with 2,4-D 0.15 mg·L-1, 6-BA 6.0 mg·L-1 and sucrose 30 g·L-1, and shook at 25 ℃ under dark condition for 25-30 d, then good-diversity and rapid-growing cell suspension cultures were established. The optimal parameter of cell-suspension cultures were: 1.0g-1.5g initial inoculum, a sub-culture every 15 d, and 3/4 volume of updating liquid medium during subculture. Plantlet formation occurred when suspension cell were transferred to solid MS medium containing 2,4-D 1.0 mg·L-1, 6-BA 0.5 mg·L-1 and sucrose 30 g·L-1.更多还原