【作者】 张雷；

【导师】 梁国鲁； 杨静慧；

【作者基本信息】 西南大学 ， 果树学， 2007， 硕士

【摘要】 在天津市20万hm~2果园中，有1/4果园属于盐碱地，该果园中果实品质差、产量低，果树易早衰，经济效益低。所以，改良盐碱果园土壤理化性质，改善其土壤结构，是盐碱果园迫切需解决的问题。许多果园绿肥作物具有较强的耐盐能力，种植绿肥改变可以改良土壤，如田菁(Sesbania rostrata)、草木樨(Melilotus suavena)、柽麻(Critalaria jumcea L．)等，它们一方面可以提高土壤肥力，另一方面可以降低土壤含盐量和土壤pH。草木樨(M.suavena)作为一种优良的果园绿肥，有着较为广泛的用途，属于盐生植物，有较强的抗逆性和适应性。它具有固氮作用，还是优良的牧草。但是草木樨也只能耐0.6％的土壤含盐量，只能在中度盐碱地上生长，所以进行耐盐胁迫试验的盐胁迫浓度为0.8％。另外草木樨的出苗率和苗木的耐盐性较差。因此，提高草木樨的耐盐性、选育优良的耐盐碱草木樨品种是盐碱果园土壤改良，特别是中、重度盐碱果园的关键。本研究首先收集了7个不同地域的耐盐草木樨：宁夏草木樨，赤峰草木樨，黑龙江草木樨，西宁草木樨1，西宁草木樨2，西宁草木樨3，天津草木樨。分别进行无菌培养和耐盐胁迫试验和耐盐筛选，通过盐胁迫下的生理反应——生理指标的测定(细胞膜透性、脯氨酸、丙二醛、株高、鲜重、干重)和耐盐性综合分析，得出天津草木樨、西宁草木樨1、赤峰草木樨为耐盐最强的品种或类型。由于赤峰草木樨种子萌芽率高、变异小，被选择为基因转化的外植体。通过不同激素、除草剂、不同外植体器官的筛选建立了再生体系和遗传转化体系，获得了耐盐转基因草木樨植株。主要内容如下：1．在0.8％盐胁迫下，对所试草木樨叶片细胞质膜透性分析，表明宁夏草木樨叶片细胞质膜透性最高，其余依次是宁夏草木樨＞宁草木樨3＞宁草木樨2＞龙江草木樨＞宁草木樨1＞峰草木樨＞天津草木樨；在0.8％盐胁迫下，对所试草木樨游离脯氨酸含量分析，表明西宁草木樨1叶片游离脯氨酸含量最高，其余依次是赤峰草木樨＞天津草木樨＞西宁草木樨3＞宁夏草木樨＞西宁草木樨2＞黑龙江草木樨；在0.8％盐胁迫下，对所试草木樨叶片丙二醛含量分析，表明赤峰草木樨叶片丙二醛含量最高，其余依次是天津草木樨＞西宁草木樨2＞西宁草木樨3＞宁夏草木樨＞黑龙江草木樨＞宁夏草木樨；在0.8％盐胁迫下，对所试草木樨株高分析，表明天津草木樨株高最高，其余依次是西宁草木樨1＞西宁草木樨3＞赤峰草木樨＞宁夏草木樨＞西宁草木樨2＞黑龙江草木樨；在0.8％盐胁迫下，对所试草木樨鲜重分析表明，西宁草木樨1鲜重最重，其余依次是赤峰草木樨＞天津草木樨＞西宁草木樨3＞宁夏草木樨＞西宁草木樨2＞黑龙江草木樨；0.8％盐胁迫下，对所试草木樨干重分析表明，天津草木樨干重最重，其余依次是西宁草木樨1＞赤峰草木樨＞西宁草木樨3＞宁夏草木樨＞西宁草木樨2＞黑龙江草木樨。经过对不同草木樨品种的细胞膜透性、脯氨酸含量、丙二醛含量、株高、鲜重和干重的六个生理指标的测定和筛选，及指标的综合评价分析，得出天津草木樨、西宁草木樨1、赤峰草木樨为1级，耐盐性最强；其次是西宁草木樨3为2级，耐盐性较强；宁夏草木樨、西宁草木樨2的耐盐性为3级，耐盐性较差；黑龙江草木樨耐盐性为4级，耐盐性最差。所有指标表明天津草木樨、赤峰草木樨、西宁草木樨1的抗盐碱能力最强，可以作为耐盐基因的转化受体的首选材料。2．赤峰草木樨的最优再生体系为：外植体为下胚轴，诱导再生芽培养基配方为：MS+4mg/LBA+0.2 mg/LIAA上；伸长培养基配方为：MS+1mg/LBA+0.2 mg/LIAA；生根培养基配方为：1/2MS+0.2mR/LNAA的上。当除草剂的浓度为2mg/L时，没有任何芽再生，是理想的草木樨转基因芽的筛选浓度。以草木樨下胚轴(破坏生长点的)为外植体的基因转化率为6.35％。3．经过对赤峰草木樨耐盐抗逆HS转录因子的遗传转化，通常选择压除草剂的筛选得到了转基因抗盐草木樨植株。更多还原

【Abstract】 One third of 2,000,000 acres orchards in Tianjin belong to the salt alkaloid area. Soil Stalinizations, soil fertility decline sand and low productivity of soil result in low fruit output and bad quality. As the fruit tree is easily in the progress of premature senility, the economic efficiency fell lower and lower. So the improvement of the grow condition for soil physics and chemistry nature have be an urgent problem.Many orchards green manure crop has the ability of bearing the salty, and planting green manures could improve the soil characters, such as Agati sesbania, Melilotus suavena and so on. They could enhance the soil fertility; on the other hand, they could also reduce the soil salt content and soil pH.As one kind of fine orchard green manure, M. suavena has a more widespread use. Comparing with clover, M. suavena has higher salt-resistant. Because it belongs to the halophyte, M. suavena which is used to be a kind of fine forage has very strong resistance adversity and the ability of nitrogen fixation.But M. suavena could only bear no more than 0.6% soil salt content and inhabit in the moderate salt alkaloid soil. Moreover M. suavena’s rate of seedlings emergence and the rate of nursery stock survival is low. Therefore, enhancing M. suavena salt-resistance to achieve the aim of selective fine breeding is the key for salt alkaloid orchard soil amelioration, especially for moderate and heavy salt alkaloid soil.This research firstly collected 7 different regions M. suavena: Ningxia M. suavena, Chifeng M. suavena, Heilongjiang M. suavena, Xining M. suavena 1, Xining M. suavena 2, Xining M. suavena 3, Tianjin M. suavena. Separately through aseptic culture, salt force disposal and salty tolerance screens, the physiological target after the disposal (cell membrane penetrability, leaf blade drifts away praline content, leaf MDA content, height, fresh weight, dry weight) demonstrate that Tianjin M. suavena, Xining M. suavena 1, Chifeng M. suavena bear salt most strongly. Because Chifeng M. suavena seed germinating rate is high and the variation is small, it is chosen as explants of the gene transfer. Through the selection of different hormones, herbicide and explants, regeneration system gene transferred system was set up and the genetransferrd salt-resistant sweet clove is obtained. Main content as follows: 1. Under the 0.8% salt forces, the cell membrane penetrability of Ningxia M. suavena is the highest. The rest in turn are Xining M. suavena 3＞Xining M. suavena 2＞Heilongjiang M. suavena＞Xining M. suavena l＞Chifeng M. suavena＞Tianjin M. suavena. Under the 0.8% salt forces, the leaf blade drifts away praline content of Xining M. suavena 1 is the highest. The rest in turn are Chifeng M. suavena＞Tianjin M. suavena＞Xining M. suavena 3＞Ningxia M. suavena＞Xining M. suavena 2＞Heilongjiang M. suavena. Under the 0.8% salt forces, the height of Chifeng M. suavena is the highest. The rest in turn are Tianjin M. suavena＞Xining M. suavena l＞Xining M. suavena 2＞Xining M. suavena 3＞Heilongjiang M. suavena＞Ningxia M. suavena. Under the 0.8% salt forces, the height of Tianjin M. suavena is the highest. The rest in turn are Xining M. suavena 1＞Xining M. suavena 3＞Chifeng M. suavena＞Xining M. suavena 2＞Heilongjiang M. suavena. Under the 0.8% salt forces, the fresh weight of Xining M. suavena 1 is the highest. The rest in turn are Chifeng M. suavena＞Tianjin M. suavena＞Xining M. suavena 3＞Ningxia M. suavena＞Xining M. suavena 2＞Heilongjiang M. suavena.Under the 0.8% salt forces, the dry weight of Tianjin M. suavena is the highest. The rest in turn are Xining M. suavena l＞Chifeng M. suavena＞Xining M. suavena 3＞Ningxia M. suavena＞Xining M. suavena 2＞Heilongjiang M. suavena. Under the 0.8% salt forces, the dry weight of Tianjin M. suavena is the highest. The rest in turn are Xining M. suavena l＞Chifeng M. suavena＞Xining M. suavena 3＞Ningxia M. suavena＞Xining M. suavena 2＞Heilongjiang M. suavena.After the measure and screen for the cell membrane penetrability, leaf blade drifts away praline content, leaf MDA content, height, fresh weight, dry weight of the different M. suavena variety, it demonstrates that the rank of salt-resistant is that Tianjin M. suavena, Xining Melilotus Suavenal and Chifeng M. suavena is the 1st level; Xining M. suavena 3 is the 2nd level; Ningxia M. suavena and Xining M. suavena 2 is the 3rd; the Heilongjiang M. suavena is the 4th.All the indexes demonstrate that the salt-resistant of Tianjin M. suavena, Chifeng M. suavena, Xining M. suavena 1 are best of all and could be taken as the primary materials for the gene-transfer.2. The best regeneration system of Chifeng M. suavena: the culture direction for hypocotyls of M. suavena used as explants regeneration was on the medium : MS+4mg / LBA+0.2 mg / LIAA ;The buds of regeneration can be elongated by the medium : MS+1mg / LBA+0.2 mg / LIAA; The seeding growth 2-3cm high was put on the MS medium containing of different concentration. The experimental results suggested that the optimal medium for rooting of adventitious buds is : 1/2MS+0.2 mg / LNAA.As no sprout regenerating the concentration of the herbicide is 2 mg/L, so that concentration is the most ideal screen one.The rate for gene-transfering of M. suavena hypocotyls used as explants (growth point damaged) is 6.35%.3. After the gene-transferring of salt-resistant HS for the Chifeng M. suavena, the aim M. suavena was obtained after the screen of herbicide concentration experiment.更多还原