【作者】 陈凌；

【导师】 李凌；

【作者基本信息】 西南大学 ， 园林植物与观赏园艺， 2010， 硕士

【摘要】 越橘为杜鹃花科(Ericaceae)越橘属(Vaccinium. SPP)多年生落叶或常绿灌木,在欧洲、俄罗斯、北美、阿尔卑斯山以及中国的部分地区有分布。全世界有400多个栽培品种。我国越橘属植物分布在华东、华中、华南至西南及东北地区,多见于丘陵及海拔400-1400米的地区。越橘果实蓝紫色,果肉细腻,种子极小,甜酸适度,具宜人的香气。可鲜食,也可加工成果汁饮料、果酒饮品。果实内所特有的蓝浆果花青素等物质具有提高视力、抗衰老和防癌等功效,被誉为“21世纪功能性保健浆果”和“水果中的皇后”,并被国际粮农组织列为人类五大健康食品之一,近年来风靡欧美及日本。然而,由于重庆夏季多伏旱,并且越橘的根系浅以及抗旱性差,导致越橘在重庆的种植区域狭窄。要改变这种现状的有效途径之一,就是应用诱变育种等常规育种手段和现代分子生物学技术相结合的方法,即人工诱导产生遗传变异,经多代筛选和鉴定,培育高产优质和抗性强的越橘新品种。目前,对越橘体细胞无性系变异方面的研究多集中在组织培养条件和药品配方的筛选上,在组织培养过程中筛选变异植株。但是生物体在诱变剂的作用下,细胞内的遗传物质在结构和功能上也常常会发生改变,并能够改变植物的遗传性状,使DNA分子受到损伤,从而使其在复制过程中发生改变,引起基因的突变。采用化学诱变剂,能诱导无性系的高频率变异,通过一定的选择压力,可以筛选到一些有益的突变性状和性状优良的突变体。本试验采用化学诱变和体细胞无性系变异相结合的方法,诱导和筛选越橘抗旱突变体,并对处理后的植株进行了初步研究,总体上可分为三部分：1.在已建立的越橘再生体系上,对越橘进行化学诱变剂处理,并对再生植株的株高、叶长、叶宽、变异率、生根率等进行统计。结果表明：越橘茎尖在经0.1%---0.6%EMS后试管苗的死亡率为零；当EMS浓度为0.3%时为最佳浓度,材料V3和3#得到的试管苗的平均株高达到了4.74cm和4.52cm,平均叶长与平均叶宽的乘积为1.173cm2和1.073cm2,当EMS浓度为0.6%时,其生长势弱,中上部狭小并为黄绿色,部分叶片脱落。2.对处理后获得的再生植株进行初步研究,通过测定干旱处理前后与耐旱性相关的一些生理指标,并筛选出可能出现有抗旱突变体的EMS浓度。结果表明：(1)在干旱胁迫下,随胁迫时间的延长CK中MDA的含量增加的幅度最大；相形之下,0.1%处理和0.5%处理中MDA的含量比处理前只有少量的增加,差异不显著。(2)经EMS处理后的再生植株其可溶性糖含量均高于对照,材料V3和3#经0.3%EMS处理后其可溶性糖含量分别比对照增加了107.3%和83.7%,差异显著。(3)材料V3和3#经0.3%EMS处理后的M0代植株SOD、POD活性依然明显高于对照,分别增加了2.09%和35.97%；188.73%和142.8%,差异显著。(4)对于其CAT活性,材料V3经0.6%EMS处理后其活性比对照增加了84.6%,经0.2%和0.5%EMS处理后其活性低于对照；材料3#经0.1%和0.6%EMS处理后其活性分别比对照增加了48.72%和62.15%。M0代植株经干旱胁迫后进行初步筛选后,过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性增强,丙二醛(MDA)含量减少,对干旱环境表现出一定的抗性。这两组材料经浓度为0.3%EMS诱变处理后可能存在抗旱性较强的突变植株。3.越橘表型变异植株的筛选。在诱变剂处理得到的460株M0代植株中,共筛选出了9株具有明显表型变异的单株,主要表现为整株叶片形态和叶片颜色的变异。诱变产生的表型变异频率非常低,仅有1.96%。变异植株通过对叶片厚度、主脉直径、栅栏组织厚度和叶片组织结构紧密度等9项与抗旱相关的叶片解剖结构指标的单因素多重比较,选定了4项灵敏度最高的指标进行隶属函数分析,分别为：气孔密度、叶片厚度、上表皮厚度、栅栏组织厚度。应用隶属函数法以高灵敏度的抗性指标对所筛选出的变异植株进行抗旱能力的综合评价。结果表明：9株变异越橘植株与对照材料的抗旱能力依次为：V-5>V-9>V-4>V-7>V-3>V-6>CK>V-1>V-2>V-8。更多还原

【Abstract】 Vaccinium L., Ericaceae, the genus vaccinium spp, is a perennial defoliate or green-like shrub. It abrading, are found in north American, distribute the Northern Hemisphere, Antartic beach of American city and Southeast of Canada are discovered., and have more than 400 planting breeds. Vaccinium is distribute in the eastern, middle, southern, southwest and Northeast areas in China, appearing much on Knap and the areas of elevation 400-1400 meter. The fruit isblue-purple, sarcocarp is slender-greasy, grem is very small, the sapor is moderation and possess amenity fragrance. There peculiarly have blueberry anthocyanin which in their’s fruit possess efficacy of improve eyesight, resist decrepitude and prevent cancer. It in praised "functionality and health protection berry in 21th century" and "queen of fruitage", also ranked the one of five healthy eatable about human being by International Food and Agricultural Organization(FAO). While blueberry is seen as a very promising emerge plant, the main problems of its development are of shallow roots without the larger main ones. These roots are either thin or underdeveloped with few hairs and small seeds and the limited growing regions caused by poor drought resistance. One of the efficient ways to change the situation is to combine the classical method of mutagenic breeding with marker-assisted selection (MAS), i.e., to induce and screen variations artificially so as to obtain the new blueberry germplasm resources with high yield, good quality and strong resistance.Currently most of the research on somaclonal variation of blueberry is still focused on direct regeneration system. The chemistry mutation changes the hereditary properties of the vegetative, because of it makes the DNA molecular construction suffered the harm, resulting duplicate mistake, consequently cause the gene mutation. So in order to increase the variety of somaclonal variatinon, the chemical mutagen is introduced; the useful variation and mutant with excellent characteristic are selected by using chemical mutagen under certain stress. In this study, we employed the means of combining the use of chemical mutagen and somaclonal variation, induced and selected the group of drought-resistant mutant in blueberry and pilot study was conducted on the group of mutant.Firstly, it focused establishment direct regeneration system of blueberry under the treatment of chemical mutagen. And the percentage of germination, the number of adventitious shoot, leaf length, leaf width and normal seedling of blueberry is counted. The results showed that the shoots of blueberry in 0.1%-0.6% EMS mortality rate of zero; concentration for inducing chilling resistant mutant in blueberry is 0.3%, with increased the average height of blueberry to 4.74cm (V3)or 4.52cm(3#); with increased the average area of blueberry to 1.173 cm2 (V3)or 1.073 cm2 (3#);significant difference. When the EMS concentration of 0.6%, its growth potential is weak, and for the upper part of small yellow-green, some leaves fall off.Secondly the paper is focused on the physiological mechanism of obtained chilling resistant mutant in blueberry. With the drought tolerance of some physiological indicators, and have selected may appear drought-resistant mutants EMS concentration.(1) Under drought stress, with stress time in the content of MDA was CK increase was most dramatic; contrast, the content of MDA was 0.1% and 0.5% increased slightly. The difference was not significant.(2) After EMS treatment, the soluble sugar content of regenerated plants were higher than control. The content of Soluble sugar was increased by 107.3% and 83.7%, when the treatment of 0.3% EMS for V3 and 3#. Difference was significant also.(3) After EMS treatment, the activity of SOD, POD of regenerated plants were higher than control. They were increased by 107.3% and 83.7%,188.73% and 142.8%, when the treatment of 0.3% EMS for V3 and 3#. Difference was significant also.(4) For V3, the activities of CAT of regenerated plants were higher than control, it was increased by 84.6%, when the treatment of 0.3% EMS. The activities of CAT of regenerated plants were less than control, when the treatment of 0.2% and 0.5% EMS. For 3#, the activities of CAT of regenerated plants were higher than control, they were increased by 48.72% and 62.15%, when the treatment of 0.13% and 0.6% EMS.Drought tolerant variants were selected from the MO generation plants, then the activity of SOD, CAT, POD enhanced and the content of MDA decreased which means that the regenerated plants’ ability of drought resistant was improved. The strongest drought resistant mutant plants were obtained at the treatment of 0.3% EMS.Thirldly, the screening of phenotypic variant plants.From the 460 MO generations mutagenic plants, there were a total of 9 phenotypic variant plants to be screened out. The 9 variant plants were mainly varied in the morphological characteristics of whole leaves. The rate of phenotypic variation was very low, only 1.96%.In addition, we measured and analyzed 9 leaf anatomical structure indexes related to drought resistance of the 10 seedings, including stoma density, leaf thickness, top epidermis thickness, thickness of palisade tissue and so on. According to the result of multiple comparisons, we screened out 4 indexes with a high sense.According to the 4 indexes, an order of drought resistance of the 10 plants was given by using memberhip function values analysis:V-5>V-9>V-4>V-7>V-3>V-6>CK>V-1>V-2>V-4.更多还原