利用卡那霉素抗性基因作为筛选标记对麻疯树进行基因转 Kanamycin resistance gene as a marker gene transfer of Jatropha

化的方法 Of the method

技术领域 Technical Field

[0001] 本发明涉及麻疯树的组织培养技术领域及基因工程领域，尤其是指利用卡那霉素作为筛选标记对麻疯树进行基因转化的方法。 [0001] The field of tissue culture techniques and genetic engineering of the present invention relates to Jatropha, especially refers to the use of kanamycin as a marker of Jatropha genetic transformation methods were.

背景技术 Background

[0002] 能源危机是当今世界面临的巨大挑战。 [0002] The energy crisis is a huge challenge facing the world today. 中国的石油储量仅占全世界的2%，消费量却居世界第二，因此，迫切地需要寻找一种新型的能源来替代目前广泛使用的化石能源。 China's oil reserves account for only 2% of the world, consumption is second in the world, therefore, an urgent need to find a new energy sources to replace fossil fuels is currently widely used.

[0003] 与化石资源相比，种植能源植物获取生物柴油具有可再生的优势，是解决能源危机的根本途径。 [0003] Compared with fossil resources, planting energy plants to obtain biodiesel is renewable advantages, is the fundamental way to solve the energy crisis. 目前已发现的能源植物，主要集中在夹竹桃科、大戟科、萝摩科、菊科、桃金娘科以及豆科等。 It has been found in energy plants, mainly in Apocynaceae, Euphorbiaceae, Asclepiadaceae, Asteraceae, Myrtaceae and legumes like.

[0004] 麻疯树OaioMa curcas L.)，又名小桐子、柴油树，为大戟科，麻疯树属，多年生落叶灌木或小乔木。 [0004] Jatropha OaioMa curcas L.), also known as Jatropha, diesel tree, Euphorbiaceae, Jatropha, a perennial deciduous shrub or small tree. 小桐子种仁含油量高达61. 5%，可作为生产生物柴油的原料，是最有可能成为未来替代化石能源的树种，具有巨大的开发潜力。 Jatropha seeds oil content of up to 61.5%, as the production of biodiesel, is most likely to be the future alternative to fossil energy species, has great potential for development. 小桐子耐干旱贫瘠，分布于热带、 亚热带和雨量稀少、条件恶劣的干热河谷地区，主要分布于中国云南、贵州、四川、广东、广西、海南等地。 Jatropha resistant to arid, distributed in tropical, subtropical and low rainfall, poor conditions of dry and hot valley region, mainly in China, Yunnan, Guizhou, Sichuan, Guangdong, Guangxi, Hainan and other places.

[0005] 植物转基因技术可将外源基因导入植物，定向改造植物性状而日益受到人们的重视。 [0005] plant transgenic technology may be a foreign gene into a plant, the transformation of plant traits and increasingly directed people's attention. 根癌农杆菌介导法是最常用的植物基因转化方法之一。 Agrobacterium-mediated transformation method is one of the most common plant genes. 根癌农杆菌含有Ti质粒，其上有一段T-DNA区，农杆菌通过侵染植物伤口进入细胞后，可将T-DNA插入到植物基因组中， 并且可通过减数分裂稳定的遗传给后代，这一特性成为农杆菌介导法植物转基因的理论基础。 Agrobacterium tumefaciens containing a Ti plasmid, on which there is a T-DNA region, after the attack on the plant by Agrobacterium wound into the cells, T-DNA can be inserted into a plant genome, and can be stabilized by meiosis to their offspring This feature has become Agrobacterium-mediated transgenic plants theoretical basis. Ti质粒上含有另一个重要的区域，是Vir区，它编码实现质粒转移所需的蛋白质。 Ti plasmid contains another important area, is Vir region, which encodes a protein to achieve the desired plasmid transfer. 我们将目的基因插入到经过改造的T-DNA区，借助农杆菌的感染实现外源基因向植物细胞的转移与整合，然后通过细胞和组织培养技术，再生出转基因植株。 We will target gene is inserted into the engineered T-DNA region, with Agrobacterium infection realization and integration of exogenous gene transfer to plant cells, and then by cell and tissue culture techniques, regenerating transgenic plants.

[0006] 然而，麻疯树的研究工作起步较晚，关于麻疯树组织培养和遗传转化技术的报道还很少。 [0006] However, research work began late jatropha, jatropha reports on tissue culture and genetic transformation technique is also very little. 2010年，Pan等在African Journal of Biotechnology上发表文章，以卡那霉素作为筛选剂，利用农杆菌介导法对麻疯树进行转化。 2010, Pan et of the Biotechnology article published in the African Journal to kanamycin as a selection agent, by Agrobacterium-mediated transformation of Jatropha. 该方法是在筛选阶段不添加卡那霉素而获得转基因植株，然而120株再生植株只有1株生根，成功率太低而不具有实用性。 This method is not added during the screening phase kanamycin to obtain transgenic plants, but 120 regenerated plants only one rooting success rate is too low and not practical.

发明内容 DISCLOSURE

[0007] 本发明所要解决的技术问题是，提供一种利用卡那霉素抗性基因作为筛选标记对麻疯树进行基因转化的方法，以便高效地获得转基因植株。 Technical Problem [0007] The present invention solves is to provide a use of kanamycin resistance gene as a selectable marker gene transformation method of Jatropha tree to efficiently obtain transgenic plants.

[0008] 为解决上述技术问题，本发明采用如下技术方案：一种利用卡那霉素抗性基因作为筛选标记对麻疯树进行基因转化的方法，包括以下步骤： [0008] In order to solve the above problems, the present invention adopts the following technical solution: a kanamycin resistance gene as a selection marker gene transformation method of Jatropha tree, comprising the steps of:

步骤（1)，分别制备麻疯树外植体以及农杆菌菌液，再将外植体经农杆菌液浸染后进行 Step (1), were prepared Jatropha explants and Agrobacterium suspension, and then the explants were disseminated after Agrobacterium

共培养； Co-culture;

步骤（2)，将共培养后的外植体脱菌后接入筛选培养基C13KC进行培养，从而获得抗性愈伤组织，所述筛选培养基C13KC是含有1. 0-3. Omg/L的6-苄基嘌呤、0. 25-1. Omg/L的3-吲哚丁酸、1-50 mg/L的卡那霉素以及50-200mg/L的特美汀的MS培养基； Explants step (2), after the co-cultured bacteria - access screening C13KC cultured medium to obtain resistant callus, the screening medium C13KC containing 1. 0-3. Omg / L .. 6-benzyladenine, 0 25-1 Omg / L indole-3-butyric acid, 1-50 mg / L of kanamycin and timentin of MS 50-200mg / L of culture medium;

步骤（3)，将抗性愈伤组织转入分化培养基SR13KC进行培养，分化出抗性不定芽，所述分化培养基SR13KC是含有1. 0-3. Omg/L的6-苄基嘌呤、0. 25-1. Omg/L的3-吲哚丁酸、 l-50mg/L的卡那霉素以及50-200mg/L的特美汀的MS培养基；以及 Step (3), the resistant callus were transferred to regeneration medium SR13KC culture, differentiation of adventitious buds resistance, the differentiation medium SR13KC containing 1. 0-3. Omg / L 6-benzyladenine .., 0 25-1 Omg / L indole-3-butyric acid, l-50mg / L of kanamycin and 50-200mg / L of timentin MS medium; and

步骤（4)，将抗性不定芽转入生根培养基R2KC中进行培养，从而获得转基因植株，所述生根培养基R2KC是含有0. 1-1. Omg/L的3-吲哚丁酸，l_50mg/L的卡那霉素，50_200mg/L 的特美汀的1/2MS培养基。 Step (4), the resistance into the rooting medium R2KC buds were cultured to obtain transgenic plants containing the rooting medium R2KC 0. 1-1. Omg / L indole-3-butyric acid, l_50mg / L of kanamycin, 50_200mg / L of timentin of 1 / 2MS media.

[0009] 进一步地，步骤（1)中，共培养时采用C13A固体培养基，该C13A固体培养基是指含1. 0-3. 0mg/L 6-苄基嘌呤，0. 25-1. 0mg/L 3-吲哚丁酸，50-200 μ M 乙酰丁香酮，20-30 g/L 蔗糖，7 g/L琼脂的MS培养基，pH 5. 8-6. 0。 [0009] Further, the step (1), when the co-culture using a solid medium C13A, C13A the solid medium containing means 1. 0-3. 0mg / L 6- benzyladenine, 0. 25-1. 0mg / L 3- IBA, 50-200 μ M acetosyringone, 20-30 g / L sucrose, 7 g / L agar MS medium, pH 5. 8-6. 0.

[0010] 进一步地，步骤（1)中，麻疯树外植体的制备经由以下步骤：挑选麻疯树种子经消毒后，取出完整的胚和子叶，接种于MS培养基培养萌发，获得子叶，切制成外植体，本步骤中采用的MS培养基含有20-30g/L蔗糖和7g/L琼脂，pH5. 8-6. 0。 [0010] Further, the step (1) to prepare Jatropha explant through the following steps: selection of Jatropha seeds after disinfection, remove the complete embryo and cotyledon inoculated in MS medium for germination, get cotyledons , cut into explants, MS medium employed in this step contained 20-30g / L sucrose and 7g / L agar, pH5. 8-6. 0.

[0011 ] 进一步地，步骤（1)中，制备麻疯树外植体时，将完整的胚和子叶接种于MS培养基后先暗培养2-4天，再光照培养10-12天，种子即萌发，光照培养条件是：温度23-26°C，光照12-16小时/天，光照强度1800-2000 Ix0 [0011] Further, the step (1), the preparation of Jatropha explant, the intact embryo and cotyledon inoculated in MS medium to dark culture after 2-4 days, 10-12 days and then light culture, seed That germination, light culture conditions: temperature 23-26 ° C, light 12-16 hours / day, light intensity 1800-2000 Ix0

[0012] 进一步地，步骤（1)中，准备农杆菌菌液、浸染及共培养的具体步骤为： [0012] Further, the step (1) Agrobacterium suspension prepared, disseminated and co-culture specific steps are as follows:

(a)挑取农杆菌的单克隆于含有抗生素的YEP液体培养基中J8°C，200rpm震荡培养20-M小时，然后按照1 ：100的体积比转接，震荡培养至对数生长期，OD600=O. 8-1.0，将菌液转入50ml离心管，常温，4000rpm离心20分钟收集菌体，采用C13A液体培养基重新悬浮离心收集的农杆菌，调至0D_=0. 3-0. 震荡培养1-2个小时，获得农杆菌菌液备用， 采用的C13A液体培养基是指含有1. 0-3. 0mg/L 6-苄基嘌呤，0. 25-1. 0mg/L 3-吲哚丁酸， 50-200 μ M 乙酰丁香酮，20-30g/L 蔗糖的MS 培养基，ρΗ5· 8-6. 0 ； (A) picked Agrobacterium monoclonal in YEP liquid medium containing antibiotics J8 ° C, 200rpm shaking culture 20-M hours, then a 1: 100 volume ratio transfer, culture shock to the logarithmic growth phase, OD600 = O. 8-1.0, the broth into 50ml centrifuge tubes, at room temperature, 4000rpm centrifuged for 20 minutes to collect the cells, using C13A liquid culture medium was collected by centrifugation and resuspended Agrobacterium, adjusted 0D_ = 0. 3-0. shaking culture for 1-2 hours, to obtain spare Agrobacterium suspension, C13A liquid medium used means a 1. 0-3. 0mg / L 6- benzyladenine, 0. 25-1. 0mg / L 3- indole butyric acid, 50-200 μ M acetosyringone, 20-30g / L sucrose MS medium, ρΗ5 · 8-6 0.;

(b)将切好的外植体放入容器中，加入准备好的菌液进行，置于摇床以不高于100转/ 分的速度震荡5-15分钟进行浸染；以及 (B) The explants were cut into the container, add broth were prepared and placed in a shaker with not more than 100 rev / min for 5-15 minutes concussion disseminated; and

(c)取出浸染过的材料，将其置于无菌滤纸上吸除多余的菌液后再接入C13A固体培养基，置于23-26°C黑暗共培养2-4天。 (C) removing the material disseminated over, placing it sucked excess bacteria on sterile filter paper and then access C13A solid medium, placed in co-culture dark 23-26 ° C for 2-4 days.

[0013] 进一步地，步骤（2)进一步包括以下工艺步骤： [0013] Further, the step (2) further comprises the following process steps:

(a)取出共培养后的外植体，置于灭菌后的无菌吸水纸上，吸去菌液； (A) Remove the explants co-cultured on sterile absorbent paper after sterilization, absorb bacteria;

(b)将吸去菌液的外植体接入筛选培养基CUKC进行培养，所述筛选培养基C13KC是指MS培养基，含有1. 6-3. 0mg/L6-苄基嘌呤、0. 25-1. 0mg/L的3-吲哚丁酸、l_50mg/L卡那霉素、50-200mg/L特美汀、2-3%蔗糖及0. 7%琼脂，且ρΗ5· 8-6. 0 ；以及 (B) the bacteria absorb the explants were cultured CUKC access filter medium, the filter medium C13KC refers to MS medium containing 1. 6-3. 0mg / L6- benzyladenine, 0. 25-1. 0mg / L indole-3-butyric acid, l_50mg / L kanamycin, 50-200mg / L timentin, 2-3% sucrose and 0.7% agar, and ρΗ5 · 8-6 0; and

(c)置于23-26°C暗培养14-21天，获得抗性愈伤组织。 (C) placed 23-26 ° C for 14-21 days dark culture, acquired resistance callus.

[0014] 进一步地，步骤（3)采用的分化培养基SR13KC是指MS培养基，其含有0. 25-1. ang/L6-苄基嘌呤、0. 1-1. 0mg/L3_ 吲哚丁酸、0. 01-0. ang/L 赤霉素、l_50mg/L 卡那霉素、50-200mg/L特美汀、20_30g/L蔗糖以及7g/L琼脂，且pH5. 8-6. 0 ；接好的抗性愈伤组织在23-26°C，光照培养30-60天即可得到抗性不定芽，培养条件为，光照12-16小时/天， 光照强度1800-2000 Ix。 [0014] Further, the step (3) refers to the differentiation medium SR13KC MS medium containing 0. 25-1. Ang / L6- benzyladenine, 0. 1-1. 0mg / L3_ indol-butoxy acid, 0. 01-0. ang / L GA, l_50mg / L kanamycin, 50-200mg / L timentin, 20_30g / L sucrose and 7g / L agar, and pH5. 8-6. 0 ; then good resistance callus 23-26 ° C, light cultivation 30-60 days to get resistance adventitious buds culture conditions, light 12-16 hours / day, light intensity 1800-2000 Ix. [0015] 进一步地，步骤（4)中，首先将抗性不定芽在0. 1-1. Omg/L的3_吲哚丁酸溶液中浸泡2-10分钟后，再接种到R2KC培养基进行生根培养，培养30-60天即可生根获得转基因植株，生根培养条件为：2316°C，光照12-16小时/天，光照强度1800-2000 Ix0 After the [0015] Further, the step (4), the first resistance at 0. 1-1 buds. 3_ indole butyric acid solution Omg / L soak 2-10 minutes, then inoculated into medium R2KC rooting culture conditions, cultivation 30-60 days to obtain transgenic plants take root, rooting for the: 2316 ° C, light 12-16 hours / day, light intensity 1800-2000 Ix0

[0016] 进一步地，所述生根培养基R2KC是含有0. 1-1. 0mg/L3-昭丨哚丁酸、l_50mg/L卡那霉素以及50-200mg/L特美汀的1/2MS培养基。 [0016] Further, the rooting medium R2KC containing 0. 1-1. 0mg / L3- Zhao Shu IBA, l_50mg / L kanamycin and 50-200mg / L timentin of 1 / 2MS media.

[0017] 进一步地，所述农杆菌携带有目的基因。 [0017] Further, Agrobacterium carrying the desired gene.

[0018] 本发明的有益效果如下：本发明利用卡那霉素抗性基因作为筛选标记，对麻疯树进行基因转化，以获得转基因植株，转化率可达1-5%左右(转基因植株数占外植体总数的比例)。 [0018] The beneficial effects of the present invention are as follows: Using the card of the present invention kanamycin resistance gene as a marker of Jatropha genetic transformation to obtain transgenic plants, up to about 1-5% conversion rate (number of transgenic plants accounting for the total number of explants body proportions). 因农杆菌介导的转化方法具转化的基因多为单拷贝，且有明确的边界序列，遗传稳定，多数符合孟德尔遗传规律，价格低廉等，为利用植物基因工程技术对麻疯树进行遗传改良，获得高产，高油，抗寒等新品种奠定了良好的基础。 Because transformation methods with Agrobacterium-mediated transformation more than a single copy of the gene, and there is a clear boundary, genetic stability, most consistent with Mendelian inheritance, low cost, etc., for the use of plant genetic engineering techniques Jatropha genetic improved access to new varieties of high yield, high oil, cold and so laid a good foundation.

[0019] 本发明选择了麻疯树的成熟种子萌发后的子叶作为外植体，不仅抗性愈伤组织得出率和分化率较高，且不受季节的限制，为大批量进行农杆菌转化奠定了良好的基础。 Cotyledon after germination [0019] The present invention is selected Jatropha mature seeds as explants, derived not only resistant callus differentiation rate and higher rate, and not subject to seasonal restrictions, large quantities of Agrobacterium Transformation has laid a good foundation.

[0020] 本发明在抗性愈伤组织阶段，分化阶段及生根阶段，分别选择了合适的筛选剂浓度，既避免了非转化愈伤的逃逸及后期大量的分子检测工作，又获得了大量抗性愈伤组织， 保证了抗性植株的正常生长和分化。 [0020] In the resistant callus stage, stage of differentiation and rooting stages of the invention were screened to select the appropriate concentration, both to avoid the non-transformed callus escape and later a lot of molecular detection work, but also get a lot of anti- Callus, to ensure the normal growth and differentiation of resistant plants.

具体实施方式 DETAILED DESCRIPTION

[0021] 本发明利用卡那霉素抗性基因作为筛选标记对麻疯树进行基因转化的方法，主要涉及以下步骤： [0021] The present invention utilize card kanamycin resistance gene as a marker for genetic transformation method Jatropha trees, mainly involves the following steps:

步骤（1)：又包括如下子步骤：种子的消毒及萌发、农杆菌准备、农杆菌转化和共培养； 步骤（2)：抗性愈伤组织的诱导； 步骤（3):分化出不定芽； 步骤（4):生根培养。 Step (1): also includes the sub-steps: disinfection and seed germination, Agrobacterium preparation, Agrobacterium and co-culture; Step (2): induced resistance callus; Step (3): adventitious buds ; Step (4): rooting.

[0022] 此外，还可进行移栽及检测步骤。 [0022] In addition, also for transplanting and testing procedures.

[0023] 下面对各步骤作进一步的具体描述。 [0023] In the following each step is further detailed description.

[0024] 步骤（1)的具体工艺步骤如下： 1、种子的消毒及萌发 [0024] Step (1) of the specific process steps are as follows: 1, disinfection and seed germination

首先采集麻疯树种子，挑取饱满的种子，剥去外壳，经消毒处理后剥去胚乳，将完整的胚和子叶接种在MS培养基上萌发，得到无菌苗。 First Jatropha seed collection, picked full seed, stripped shell, stripped of endosperm after disinfection, will complete embryo and cotyledon inoculated on MS medium germinate, get no vaccine.

[0025] MS培养基配方及pH值为：20-30g/L蔗糖及7g/L琼脂，pH 5. 8-6. 0。 [0025] MS culture media and pH value: 20-30g / L sucrose and 7g / L agar, pH 5. 8-6 0..

[0026] 种子的消毒方法包括以下工艺步骤： Disinfection Methods [0026] Seeds of the following process steps comprising:

(1) 75% (体积比）乙醇浸泡30秒； (1) 75% (by volume) ethanol for 30 seconds;

(2)无菌水洗2-3次； (2) sterile water 2-3 times;

(3)0. 1%升汞浸泡3-5分钟； (3) 01% mercuric chloride for 3-5 minutes;

(4)倒出升汞，用无菌水洗4-6次，每次5分钟； (4) poured mercuric chloride, 4-6 times with sterile water, for 5 minutes each;

(5)加入无菌水，浸泡2-4小时； (5) Add sterile water, soaked 2-4 hours;

(6)剥去胚乳，取完整的胚和子叶，接种于MS培养基（20-30g/L蔗糖，7g/L琼脂， PH5. 8-6. 0)中，每瓶培养基接2-3颗；(7)接好的材料先在暗培养2-4天，然后光照培养10-12天。 (6) Remove the endosperm, germ and cotyledon take full inoculated in MS medium (20-30g / L sucrose, 7g / L agar, PH5. 8-6. 0), a bottle of medium access 2-3 stars; (7) then good material culture first 2-4 days in the dark, then light culture for 10-12 days.

[0027] 种子萌发的培养条件是，温度23_26°C，光照12_16小时/天，光照强度1800-2000 Ix0 Culture Conditions [0027] seed germination, the temperature 23_26 ° C, light 12_16 hours / day, light intensity 1800-2000 Ix0

[0028] 2、农杆菌菌液的准备 [0028] 2, Agrobacterium suspension preparation

挑取农杆菌的单克隆，如EHA105，LBA4404，GV3101等，分别含有表达载体pBI 121，于含有相应抗生素的YEP液体培养基中，28°C，200rpm震荡培养20-¾小时，然后按照体积比为1 :100转接YEP液体培养基中，震荡培养至对数生长期，农杆菌的浓度为OD6tltl=O. 8-1.0。 Monoclonal picked Agrobacterium, such as EHA105, LBA4404, GV3101, etc., were an expression vector pBI 121, YEP liquid medium containing the appropriate antibiotic, 28 ° C, 200rpm shaking culture 20-¾ hours, and then follow the volume ratio 1: 100 Adapter YEP liquid medium, shake culture to logarithmic phase, concentration of Agrobacterium was OD6tltl = O 8-1.0.. 将菌液转入50mL离心管，常温，4000rpm离心20分钟收集菌体，用C13A液体培养基重新悬浮农杆菌，调至OD6tltl=O. 3-0. 6J8°C培养1-2个小时，即获得农杆菌菌液备用。 The bacteria transferred to 50mL centrifuge tubes, at room temperature, 4000rpm 20 minutes to collect the cells by centrifugation, resuspended with Agrobacterium C13A liquid medium, adjusted to OD6tltl = O. 3-0. 6J8 ° C for 1-2 hours culture, i.e. obtaining spare Agrobacterium suspension.

[0029] 其中，所用YEP液体培养基配方及pH值为：10g/L蛋白胨，5g/L酵母提取物，IOg/ L氯化钠，pH7. 0。 [0029] wherein YEP liquid medium used and the pH of the formulation: 10g / L peptone, 5g / L yeast extract, IOg / L NaCl, pH7 0..

[0030] C13A液体培养基是指含有1. 0-3. Omg/L 6-苄基嘌呤、0. 25-1. Omg/L 3-吲哚丁酸、50-200 μ M乙酰丁香酮及20-30g/L蔗糖的MS培养基，pH5. 8-6. O。 [0030] C13A liquid medium means a 1. 0-3. Omg / L 6- benzyladenine, 0. 25-1. Omg / L 3- indole butyric acid, 50-200 μ M acetosyringone and 20-30g / L sucrose MS medium, pH5. 8-6. O.

[0031] 3、农杆菌转化和共培养具体包括如下工艺步骤： [0031] 3, Agrobacterium and co-culture specifically includes the following process steps:

(1)取灭菌的盘，中间垫上两层滤纸； (1) take a sterilized tray, two intermediate pad paper;

(2)取出无菌苗，用剪刀剪下长势较好的子叶，放入盘中； (2) Remove aseptic, cut with scissors cotyledons growing well into the dish;

(3)然后加入少量水，用刀将其切成0.5X0.5 cm大小； (3) then adding a small amount of water, a knife to cut into the size of 0.5X0.5 cm;

(4)将切好的叶片放入50或IOOmL三角瓶中，加入准备好的菌液，置于摇床以不高于100转/分的速度震荡5-15分钟； (4) The blade cut into 50 or IOOmL flask, add the prepared broth, placed in a shaker with not more than 100 rev / min for 5-15 minutes concussion;

(5)取出浸染过的材料，倒掉菌液，将叶片放在滤纸上，吸去多余的菌液，接入C13A固体培养基，其中C13A固体培养基是指含1. 0-3. Omg/L 6-苄基嘌呤、0. 25-1. Omg/L 3-吲哚丁酸、50-200 μ M乙酰丁香酮、20-30g/L蔗糖及7g/L琼脂的MS培养基，pH 5. 8-6. O ； (5) removing the material disseminated over, drained broth, the leaves on filter paper to absorb excess bacteria, the access C13A solid medium, which means C13A solid medium containing 1. 0-3. Omg / L 6- benzyladenine, 0. 25-1. Omg / L 3- IBA, 50-200 μ M acetosyringone, 20-30g / L sucrose and 7g / L agar MS medium, pH . 5. 8-6 O;

(6)接好的材料在23-26°C暗培养2-4天。 (6) then good material at 23-26 ° C for 2-4 days dark culture.

[0032] 步骤（2)的具体工艺步骤如下： [0032] Step (2) of the specific process steps are as follows:

(1)取出共培养后的材料，置于无菌的吸水纸上，吸去多余的菌液； (1) the material is removed after co-culture, put sterile absorbent paper, absorb excess bacteria;

(2)将材料接入筛选培养基C13KC进行培养，该筛选培养基C13KC是指MS培养基，含有1.0-3. Omg/L 6-苄基嘌呤、0.25-1. Omg/L 3-吲哚丁酸、l_50mg/L 卡那霉素、50-200 mg/ L 特美汀、20-30 g/L 蔗糖及7g/L 琼脂，pH 5. 8-6. O ； (2) access to the filter material C13KC cultured medium, the filter medium C13KC refers to MS medium containing 1.0-3. Omg / L 6- benzyladenine, 0.25-1. Omg / L 3- indole butyric acid, l_50mg / L kanamycin, 50-200 mg / L timentin, 20-30 g / L sucrose and 7g / L agar, pH 5. 8-6 O.;

(3)将接好的材料在23-26°C暗培养14-21天，获得抗性愈伤组织。 (3) will take good material at 23-26 ° C for 14-21 days dark culture, acquired resistance callus.

[0033] 步骤（3)的具体工艺步骤如下： [0033] Step (3) of the specific process steps are as follows:

(1)将获得的抗性愈伤组织，转入分化培养基SR13KC ；该分化培养基SR13KC是指MS培养基，含有0.25-1. 2 mg/L 6-苄基嘌呤、0. 1-1.0 mg/L 3-吲哚丁酸、0. 01-0. 2 mg/L 赤霉素、l-50mg/L 卡那霉素、50-200mg/L 特美汀、20-30 g/L 蔗糖以及7g/L 琼脂，pH 5. 8-6. O ； (1) resistant calluses obtained, transferred to differentiation medium SR13KC; SR13KC refers to the differentiation medium MS medium containing 0.25-1 2 mg / L 6- benzyladenine, 0 1-1.0. mg / L 3- indole butyric acid, 0. 01-0. 2 mg / L GA, l-50mg / L kanamycin, 50-200mg / L timentin, 20-30 g / L sucrose . and 7g / L agar, pH 5. 8-6 O;

(2)接好的材料在23-26 °C，光照培养30-60天即可得到不定芽。 (2) then good material at 23-26 ° C, light cultivation 30-60 days to get buds. 培养条件为，光照12-16小时/天，光照强度1800-2000 Ix0 Culture conditions, light 12-16 hours / day, light intensity 1800-2000 Ix0

[0034] 步骤（4)的具体工艺步骤如下： [0034] Step (4) of the specific process steps are as follows:

将分化出的不定芽在0. 1-1. Omg/L的3-吲哚丁酸溶液中浸泡2-10分钟后，再接种到生根培养基上30-60天即可生根。 After the differentiation of adventitious buds at 0. 1-1 indole-3-butyric acid solution Omg / L soak 2-10 minutes, then inoculated into the rooting medium 30-60 days to take root. 所述生根培养基是指1/2MS培养基，其含有0. 1-1.0 mg/L 3-吲哚丁酸、1-50 mg/L卡那霉素和50-200 mg/L特美汀。 The rooting medium refers to 1 / 2MS medium containing 0. 1-1.0 mg / L 3- indole butyric acid, 1-50 mg / L kanamycin and 50-200 mg / L timentin .

[0035] 生根培养条件为，23-¾ °C，光照12-16小时/天，光照强度1800-2000 Ix0 [0035] rooting conditions, 23-¾ ° C, light 12-16 hours / day, light intensity 1800-2000 Ix0

[0036] 通过上述步骤获得的转基因植株再进行移栽，通常是待再生苗根长到2〜3cm时进行炼苗移栽。 [0036] transgenic plants obtained through the above steps and then transplanting, usually grow to be regenerated rhizosphere 2~3cm be hardening transplanting.

[0037] 并且，可对成活再生转基因植株进行⑶S染色检测，以确认基因转化效果，通常是取成活再生植株的叶片，置于⑶S染色液中，37°C过夜染色，蓝色即为转基因植株。 [0037] Also, it can survive regenerated transgenic plants were ⑶S staining to confirm the gene transfer effect, usually take leaves of regenerated plants survived, placed ⑶S staining solution, 37 ° C overnight staining, blue is the transgenic plants .

[0038] 通过以上的方法可以实现对麻疯树进行基因转化，从而使目的基因在植物细胞中得到表达。 [0038] The above method can be achieved on Jatropha genetic transformation, so that the target gene expression in plant cells. 携带各种不同目的基因的农杆菌，按上述方法进行介导，根据插入的目的基因不同，就可以实现对麻疯树进行定向改造，从而实现对麻疯树的遗传改良，以提高其各种特性，如提高油含量、产量，抗寒性等。 Carry a variety of different target genes of Agrobacterium mediated by the above method, depending on the target gene is inserted, you can achieve the transformation of Jatropha for orientation in order to achieve genetic improvement of Jatropha to improve its various characteristics, such as increased oil content, yield, cold and so on.

[0039] 下面结合实例详述描述本发明的实施方案。 [0039] The following detailed description is described with an example embodiment of the present invention. 需要说明的是，下述实例是说明性的， 不是限定性的，不能以下述实例来限定本发明的保护范围。 It should be noted that the following examples are illustrative and not limiting, the following examples should not limit the scope of the present invention.

[0040] 实例1 [0040] Example 1

本实例以农杆菌介导将GUS基因转入云南元谋野生麻疯树，具体阐述本发明创造的方法，样品的总数量以及经各处理步骤后的存活数量请参考表1。 This example Agrobacterium mediated GUS gene into wild jatropha Yunnan Province, the present invention is a method created specifically addressed, the total number of samples and the number of surviving after each processing step. See Table 1. [0041 ] 步骤（1 )，具体操作包括： [0041] Step (1), specific actions include:

1、采集云南元谋野生麻疯树种子。 1. Acquisition Yuanmou wild Jatropha seeds. 挑取饱满的种子，剥去外壳，75%乙醇中浸泡30秒， 无菌水冲洗2次，然后在0. 1%氯化汞中消毒3分钟，无菌水冲洗5次，然后用无菌水浸泡2 小时后剥去胚乳。 Picked full seed, stripped shell, 75% ethanol for 30 seconds, rinsed twice with sterile water, and then sterilized in 0.1% mercuric chloride for 3 minutes, rinsed 5 times in sterile water, and then with sterile After 2 hours water immersion stripped endosperm. 取完整的胚和子叶，接种于MS培养基中，每瓶接2颗，23°C暗培养2天后转入光照培养10天，得到实验用的无菌苗。 Take a complete embryo and cotyledon were inoculated in MS medium bottle then 2, 23 ° C for 2 days into light dark culture for 10 days to give the experiment with no vaccine.

[0042] 2、挑取农杆菌EHA105 (含有表达载体pBI121)的单克隆于含有抗生素利福平(20mg/L)，庆大霉素（20mg/L)和卡那霉素（50mg/L)的YEP培养基中，28°C，200rpm震荡培养20小时，然后按照1 :100体积比转接至300mL YEP培养基中，震荡培养对数生长期，农杆菌浓度为OD6tltl=O. 8-1. 0。 [0042] 2, picked Agrobacterium EHA105 (containing the expression vector pBI121) containing monoclonal antibiotic rifampicin (20mg / L), gentamicin (20mg / L) and kanamycin (50mg / L) The YEP medium, 28 ° C, 200rpm shaking culture for 20 hours and then a 1: 100 volume ratio of the culture transferred to 300mL YEP medium shock logarithmic growth phase, Agrobacterium concentration OD6tltl = O 8-1. 0. 将菌液转入50mL离心管，常温，4000rpm离心20分钟收集菌体，用C13A液体培养基(具体配方为：含有1.0mg/L 6-苄基嘌呤、0. 25mg/L 3-吲哚丁酸、50μΜ 乙酰丁香酮及20g/L蔗糖的MS培养基，pH5. 8)重新悬浮农杆菌，调至0D_=0. 3，培养1个小时，备用。 The broth into 50mL centrifuge tube, at room temperature, 4000rpm centrifuged for 20 minutes to collect the cells, with the C13A liquid medium (specific formula is: containing 1.0mg / L 6- benzyladenine, 0 25mg / L 3- indole Ding. acid, 50μΜ acetosyringone and 20g / L sucrose MS medium, pH5. 8) resuspended Agrobacterium, adjusted 0D_ = 0. 3, cultivate one hour and set aside.

[0043] 3、取灭菌的盘，中间垫上两层滤纸。 [0043] 3, take the sterilization tray, two intermediate pad paper. 选取生长健壮，叶面平整均勻的无菌苗子叶， 加入少量水，切成0.5X0. 5cm (并不限于此尺寸）大小。 Select robust growth, leaf smooth uniform Cotyledons, adding a small amount of water, cut 0.5X0. 5cm (not limited to this size) size. 将切好的叶片放入50或IOOmL三角瓶中，加入准备好的菌液，置于摇床以100转/分的速度震荡10分钟。 The blade cut into 50 or IOOmL flask, add the prepared broth, placed in a shaker at 100 rev / min for 10 minutes concussion. 然后取出浸染过的材料，倒掉菌液，将叶片放在滤纸上，吸去多余的菌液，接入C13A固体培养基(具体配方为： 含1. Omg/L 6-苄基嘌呤、0. 25mg/L 3-吲哚丁酸、50 μ M乙酰丁香酮、20g/L蔗糖及7g/L琼脂的MS培养基，pH5. 8)，置于23°C暗培养2天。 Then remove the material disseminated over, drained broth, the leaves on filter paper to absorb excess bacteria, the access C13A solid medium (specific formula is: Includes 1. Omg / L 6- benzyladenine, 0 . 25mg / L 3- indole butyric acid, 50 μ M acetosyringone, 20g / L sucrose and 7g / L agar MS medium, pH5. 8), a dark place 23 ° C for 2 days.

[0044] 步骤（2)：取出共培养后的材料(外植体总数为1094块)，置于灭菌后的无菌滤纸上，吸去菌液，接入筛选培养基ClIC (具体配方为：含有1. Omg/L 6-苄基嘌呤、0.25mg/L 3-吲哚丁酸、lmg/L卡那霉素、50mg/L特美汀、20g/L蔗糖及7g/L琼脂的MS培养基，pH5. 8)， 230C暗培养14天，获得抗性愈伤组织936块，抗性愈伤组织率为85. 6%左右，见表1。 [0044] Step (2): Remove the material after co-culture (explants total body of 1094), placed on a sterile filter paper after sterilization, to absorb the broth, access screening medium ClIC (specific formula is : containing 1. Omg / L 6- benzyladenine, 0.25mg / L 3- IBA, lmg / L kanamycin, 50mg / L timentin, 20g / L sucrose and 7g / L agar MS medium, pH5. 8), 230C dark for 14 days to obtain resistant callus 936, resistant callus rate of about 85.6%, see Table 1.

[0045] 步骤（3)：将获得的抗性愈伤组织，转入分化培养基SR13KC (具体配方为：MS培养基含0. 25mg/L 6-苄基嘌呤、0. lmg/L 3-吲哚丁酸、0. Olmg/L赤霉素、lmg/L卡那霉素、50mg/L特美汀、20g/L蔗糖及7g/L琼脂，pH 5. 8)，23°C，光照培养30天即可得到抗性不定芽。 [0045] Step (3): resistant calluses obtained, transferred to differentiation medium SR13KC (specific formula is:. MS medium containing 0. 25mg / L 6- benzyladenine, 0 lmg / L 3- indole butyric acid, 0. Olmg / L GA, lmg / L kanamycin, 50mg / L timentin, 20g / L sucrose and 7g / L agar, pH 5. 8), 23 ° C, light cultured for 30 days to get resistant buds. 培养条件为，光照12小时/天，光照强度1800 Ix0分化出芽的抗性愈伤组织为422 块，抗性愈伤组织分化率为45. 1 %，见表1。 Culture conditions, light 12 hours / day, resistant callus differentiation light intensity 1800 Ix0 budding of 422, resistant callus rate of 45.1% (Table 1).

[0046] 步骤（4)：将分化出的抗性不定芽(共81株）在0. 5mg/L的3_吲哚丁酸溶液中浸泡5分钟后，接种到生根培养基1/2MS培养基上光照培养30天即可生根。 [0046] Step (4): The differentiation of resistant buds (of 81) soaked in a solution of IBA 3_ 0. 5mg / L for 5 minutes after inoculation to rooting medium 1 / 2MS culture light culture to take root on the base 30 days.

[0047] 待再生苗根长到2〜3 cm时进行炼苗移栽至温室。 [0047] rhizosphere grow to be regenerated 2~3 cm were transplanted to the greenhouse hardening.

[0048] 取成活再生植株的叶片，置于⑶S染色液中，37°C过夜染色，蓝色即为转基因植株。 [0048] to take leaves of regenerated plants survived, placed ⑶S staining solution, 37 ° C overnight staining, blue is the transgenic plants.

[0049] 表1本发明实例中的抗性愈伤组织得出率及分化率 [0049] Table Example 1 of the present invention results in resistant callus rate and differentiation rate

实例2 Example 2

本实例以农杆菌介导将GUS基因转入云南元谋野生麻疯树，具体阐述本发明创造的方法，样品的总数量以及经各处理步骤后的存活数量请参考表2。 This example Agrobacterium mediated GUS gene into wild jatropha Yunnan Province, the present invention is a method created specifically addressed, the total number of samples and the number of surviving after each processing step. See Table 2.

[0050] 步骤（1 )，包括如下具体操作： [0050] Step (1), comprising the following specific actions:

1、采集云南元谋野生麻疯树种子，挑取饱满的种子，剥去外壳，75%乙醇中浸泡30秒， 无菌水冲洗3次，然后在0. 1%氯化汞中消毒5分钟，无菌水冲洗7次，然后用无菌水浸泡4 小时后剥去胚乳。 1. Acquisition Yuanmou wild Jatropha seeds, picked full seed, stripped shell, 75% ethanol for 30 seconds, washed three times with sterile water and then sterilized in 0.1% mercuric chloride in 5 minutes, sterile water 7 times with sterile water and then soaked for 4 hours stripped endosperm. 取完整的胚和子叶，接种于MS培养基中，每瓶接3颗，暗培养4天后转入光照培养12天，得到实验用的无菌苗。 Take a complete embryo and cotyledon were inoculated in MS medium bottle then three, four days later transferred to light dark culture for 12 days to give the experiment with no vaccine.

[0051] 2、挑取农杆菌GV3101 (含有表达载体pBI121)的单克隆于含有抗生素利福平(20mg/L)，庆大霉素（20mg/L)和卡那霉素（50mg/L)的YEP培养基中，，200rpm震荡培养对小时，然后按照1 :100体积比转接至300mL YEP培养基中，震荡培养对数生长期，农杆菌浓度为0D_=0. 8-1. 0。 [0051] 2, picked Agrobacterium GV3101 (containing the expression vector pBI121) containing monoclonal antibiotic rifampicin (20mg / L), gentamicin (20mg / L) and kanamycin (50mg / L) The YEP medium ,, 200rpm culture shock for hours, then a 1: 100 volume ratio of calls to 300mL YEP medium, shake culture in logarithmic growth phase, Agrobacterium concentration 0D_ = 0 8-1 0... 将菌液转入50mL离心管，常温，4000rpm离心20分钟收集菌体， 用C13A液体培养基(具体配方为MS培养基含3.0mg/L 6-苄基嘌呤、1. Omg/L 3-吲哚丁酸、 200 μ M乙酰丁香酮及30g/L蔗糖，pH6. O )重新悬浮农杆菌，调至OD6tltl=O. 6，培养2个小时，备用。 The broth into 50mL centrifuge tube, at room temperature, 4000rpm centrifuged for 20 minutes to collect the cells, with the C13A liquid medium (MS medium containing specific formulations of 3.0mg / L 6- benzyladenine, 1. Omg / L 3- indole IBA, 200 μ M acetosyringone and 30g / L sucrose, pH6. O) resuspended Agrobacterium, transferred OD6tltl = O. 6, cultured 2 hours, spare.

[0052] 取灭菌的盘，中间垫上两层滤纸。 [0052] to take sterile tray, two intermediate pad paper. 选取生长健壮，叶面平整均勻的无菌苗子叶，加入少量水，切成0. 5X0. 5cm大小。 Select robust growth, leaf smooth uniform Cotyledons, adding a small amount of water, cut into 0. 5X0. 5cm size. 将切好的叶片放入50或IOOmL三角瓶中，加入准备好的菌液，置于摇床以90转/分的速度震荡10分钟。 The blade cut into 50 or IOOmL flask, add the prepared broth, placed in a shaker at 90 rev / min for 10 minutes concussion. 然后取出浸染过的材料，倒掉菌液，将叶片放在滤纸上，吸去多余的菌液，接入C13A固体培养基(具体配方为：含有3. Omg/L 6-苄基嘌呤、1. Omg/L 3-吲哚丁酸、200 μ M乙酰丁香酮、30g/L蔗糖及7g/L琼脂的MS培养基， PH6. 0)，置于暗培养4天。 Then remove the material disseminated over, drained broth, the leaves on filter paper to absorb excess bacteria, the access C13A solid medium (specific formula is: containing 3. Omg / L 6- benzyladenine, 1 . Omg / L 3- indole butyric acid, 200 μ M acetosyringone, 30g / L sucrose and 7g / L agar MS medium, PH6. 0), dark place for 4 days.

[0053] 步骤（2):取出共培养后的材料(外植体总数为958块)，置于灭菌后的无菌滤纸上， 吸去菌液，接入筛选培养基C13KC (具体配方为：MS培养基含3. Omg/L 6-苄基嘌呤、1. Omg/ L 3-吲哚丁酸、50mg/L卡那霉素、200mg/L特美汀、30g/L蔗糖及7g/L琼脂，pH 6.0),26°C 暗培养21天，获得抗性愈伤组织762块，抗性愈伤组织率为79. 5 %左右，见表2。 [0053] Step (2): Remove the material after co-culture (explants total body of 958), placed on a sterile filter paper after sterilization, to absorb the broth, access screening medium C13KC (specific formula is : MS medium containing 3. Omg / L 6- benzyladenine, 1 Omg / L 3- indole butyric acid, 50mg / L kanamycin, 200mg / L timentin, 30g / L sucrose and 7g /. L agar, pH 6.0), 26 ° C the dark for 21 days to obtain resistant callus 762, resistant callus rate of about 79.5% (Table 2).

[0054] 步骤（3)：将获得的抗性愈伤组织，转入分化培养基SR13KC (具体配方为：MS培 [0054] Step (3): The resistant callus obtained, transferred to differentiation medium SR13KC (specific formula is: MS training

养基含l.ang/L 6-苄基嘌呤、1. Omg/L 3-吲哚丁酸、0. ang/L赤霉素、10mg/L卡那霉素、 Support groups contain l.ang / L 6- benzyladenine, 1. Omg / L 3- indole butyric acid, 0. Ang / L GA, 10mg / L kanamycin,

200mg/L特美汀、30g/L蔗糖及7g/L琼脂，pH6. 0)J6°C，光照培养30天即可得到抗性不定 200mg / L timentin, 30g / L sucrose and 7g / L agar, pH6. 0) J6 ° C, for 30 days to give light resistance uncertain

芽。 Bud. 培养条件为，光照16小时/天，光照强度2000 Ix0分化出芽的抗性愈伤组织数为432块，抗性愈伤组织分化率为56.7 %，见表2。 Culture conditions, light 16 hours / day, the number of resistant callus differentiation light intensity 2000 Ix0 budding of 432, resistant callus rate of 56.7% (Table 2).

[0055] 步骤（4)：将分化出的抗性不定芽(共76株）在0. 5mg/L的3_吲哚丁酸溶液中浸泡5分钟后，接种到生根培养基1/2MS培养基上光照培养30天即可生根。 [0055] Step (4): The differentiation of resistant buds (of 76) soaked in a solution of IBA 3_ 0. 5mg / L for 5 minutes after inoculation to rooting medium 1 / 2MS culture light culture to take root on the base 30 days.

[0056] 待再生苗根长到2〜3 cm时进行炼苗移栽至温室。 [0056] rhizosphere grow to be regenerated 2~3 cm were transplanted to the greenhouse hardening.

[0057] 取成活再生植株的叶片，置于⑶S染色液中，37°C过夜染色，蓝色即为转基因植株。 [0057] to take leaves of regenerated plants survived, placed ⑶S staining solution, 37 ° C overnight staining, blue is the transgenic plants.

[0058] 表2本发明实例中的抗性愈伤组织得出率及分化率 [0058] Table Example 2 of the present invention results in resistant callus rate and differentiation rate

本实例以农杆菌介导将GUS基因转入云南元谋野生麻疯树，具体阐述本发明创造的方法，样品的总数量以及经各处理步骤后的存活数量请参考表3。 This example Agrobacterium mediated GUS gene into wild jatropha Yunnan Province, the present invention is a method created specifically addressed, the total number of samples and the number of surviving after each processing step, refer to Table 3.

[0059] 步骤（1 )，包括如下具体操作： [0059] Step (1), comprising the following specific actions:

1、采集云南元谋野生麻疯树种子。 1. Acquisition Yuanmou wild Jatropha seeds. 挑取饱满的种子，剥去外壳，75%乙醇中浸泡30秒， 无菌水冲洗3次，然后在0. 1%氯化汞中消毒4分钟，无菌水冲洗6次，然后用无菌水浸泡3 小时后剥去胚乳。 Picked full seed, stripped shell, 75% ethanol for 30 seconds, rinsed 3 times with sterile water, and then sterilized in 0.1% mercuric chloride in 4 minutes, rinsed six times with sterile water, and then with sterile After three hours of water immersion stripped endosperm. 取完整的胚和子叶，接种于MS培养基中，每瓶接3颗，暗培养3天后转入光照培养11天，得到实验用的无菌苗。 Take a complete embryo and cotyledon were inoculated in MS medium, then 3 bottle dark into light culture cultured for 3 days for 11 days to get the experimental vaccine free.

[0060] 2、挑取农杆菌LBA4404 (含有表达载体pBI121)的单克隆于含有抗生素利福平(20mg/L)，庆大霉素（20mg/L)和卡那霉素（50mg/L)的YEP培养基中，28 °C，200 rpm震荡培养22小时，然后按照1 :100体积比转接至300mL YEP培养基中，震荡培养对数生长期，农杆菌浓度为OD6tltl=O. 8-1. 0。 [0060] 2, picked Agrobacterium LBA4404 (containing the expression vector pBI121) containing monoclonal antibiotic rifampicin (20mg / L), gentamicin (20mg / L) and kanamycin (50mg / L) The YEP medium, 28 ° C, 200 rpm shaking culture for 22 hours and then a 1: 100 volume ratio of calls to 300mL YEP medium, shake culture in logarithmic growth phase, Agrobacterium concentration OD6tltl = O 8-. 1.0. 将菌液转入50mL离心管，常温，4000rpm离心20分钟收集菌体，用C13A液体培养基(具体配方为：含有2.0mg/L 6-苄基嘌呤、0. 5mg/L 3-叼丨哚丁酸、100 μ M 乙酰丁香酮及25g/L蔗糖的MS培养基，pH5. 9)重新悬浮农杆菌，调至0D_=0. 5，培养1个小时，备用。 The broth into 50mL centrifuge tube, at room temperature, 4000rpm centrifuged for 20 minutes to collect the cells, with the C13A liquid medium (specific formula is: containing 2.0mg / L 6- benzyladenine, 0 5mg / L 3- Diao 丨 indole acid, 100 μ M acetosyringone and 25g / L sucrose MS medium, pH5. 9) resuspended Agrobacterium, transferred 0D_ = 0. 5, cultured one hour, standby.

[0061] 3、取灭菌的盘，中间垫上两层滤纸。 [0061] 3, take the sterilization tray, two intermediate pad paper. 选取生长健壮，叶面平整均勻的无菌苗子叶， 加入少量水，切成0. 5X0. 5cm大小。 Select robust growth, leaf smooth uniform Cotyledons, adding a small amount of water, cut into 0. 5X0. 5cm size. 将切好的叶片放入50或IOOmL三角瓶中，加入准备好的菌液，置于摇床以95转/分的速度震荡10分钟。 The blade cut into 50 or IOOmL flask, add the prepared broth, placed in a shaker at 95 rev / min for 10 minutes concussion. 然后取出浸染过的材料，倒掉菌液，将叶片放在滤纸上，吸去多余的菌液，接入C13A固体培养基(具体配方为：含有2. 0mg/L6-苄基嘌呤、0. 5mg/L 3-吲哚丁酸、100 μ M乙酰丁香酮、25g/L蔗糖及7g/L琼脂的MS培养基， PH5.9)，置于25°C暗培养3天。 Then remove the material disseminated over, drained broth, the leaves on filter paper to absorb excess bacteria, the access C13A solid medium (specific formula is: containing 2. 0mg / L6- benzyladenine, 0. 5mg / L 3- indole butyric acid, 100 μ M acetosyringone, 25g / L sucrose and 7g / L agar MS medium, PH5.9), dark place 25 ° C for 3 days.

[0062] 步骤（2)：取出共培养后的材料(外植体总数为22M块)，置于灭菌后的无菌滤纸上，吸去菌液，接入筛选培养基ClIC (具体配方为：MS培养基含2.0mg/L 6-苄基嘌呤、 0. 5mg/L 3-吲哚丁酸、5mg/L卡那霉素、150mg/L特美汀、25g/L蔗糖及7g/L琼脂，ρΗ5· 9)， 25°C暗培养18天，获得抗性愈伤组织1767块，抗性愈伤组织率为78. 4 %左右，见表3。 [0062] Step (2): Remove the material after co-culture (explants total body of 22M blocks), placed on a sterile filter paper after sterilization, to absorb the broth, access screening medium ClIC (specific formula is : MS medium containing 2.0mg / L 6- benzyladenine, 0. 5mg / L 3- indole butyric acid, 5mg / L kanamycin, 150mg / L timentin, 25g / L sucrose and 7g / L Agar, ρΗ5 · 9), 25 ° C the dark for 18 days to obtain resistant callus 1767, resistant callus rate of about 78.4%, see Table 3.

[0063] 步骤（3)：将获得的抗性愈伤组织，转入分化培养基SR13KC (具体配方为：MS培养基含0. 5mg/L6-苄基嘌呤、0. 5mg/L 3-吲哚丁酸、0. 5mg/L赤霉素、5mg/L卡那霉素、150mg/ L特美汀、25g/L蔗糖及7g/L琼脂，pH5.9)，25°C，光照培养30天即可得到抗性不定芽。 [0063] Step (3): resistant calluses obtained, transferred to differentiation medium SR13KC (specific formula is:. MS medium with 0. 5mg / L6- benzyladenine, 0 5mg / L 3- indole IBA, 0. 5mg / L GA, 5mg / L kanamycin, 150mg / L timentin, 25g / L sucrose and 7g / L agar, pH5.9), 25 ° C, for 30 light days to get resistant buds. 培养条件为，光照14小时/天，光照强度1900 Ix0分化出芽的抗性愈伤数为1169块，抗性愈伤组织分化率为66. 2 %，见表1。 Culture conditions, light 14 hours / day, the number of resistant callus differentiation light intensity 1900 Ix0 budding of 1169, resistant callus was 66.2% (Table 1).

[0064] 步骤（4):将分化出的抗性不定芽(共192株）在0. 5mg/L的3_吲哚丁酸溶液中浸泡5分钟后，接种到生根培养基1/2MS培养基上光照培养30天即可生根。 [0064] Step (4): The differentiation of resistant buds (of 192) was immersed in a solution of IBA 3_ 0. 5mg / L for 5 minutes after inoculation to rooting medium 1 / 2MS culture light culture to take root on the base 30 days.

[0065] 待再生苗根长到2〜3cm时进行炼苗移栽至温室。 [0065] to be regenerated rhizosphere were transplanted to the greenhouse hardening grow to 2~3cm.

[0066] 取成活再生植株的叶片，置于⑶S染色液中，37°C过夜染色，蓝色即为转基因植株。 [0066] to take leaves of regenerated plants survived, placed ⑶S staining solution, 37 ° C overnight staining, blue is the transgenic plants.

[0067] 表3本发明实例中的抗性愈伤组织得出率及分化率 [0067] Table Example 3 of the present invention results in resistant callus rate and differentiation rate