麻疯树遗传转化苗的复壮及生根的培养方法 Cultivation of Jatropha genetic transformation method of rejuvenation and rooting seedlings

技术领域 Technical Field

[0001] 本发明涉及麻疯树植物遗传转化技术领域，尤其涉及一种麻疯树遗传转化苗的复壮及生根的培养方法。 [0001] The present invention relates to a Jatropha plant genetic transformation techniques, and more particularly relates to a method for cultivation of Jatropha genetic transformation of rejuvenation and rooting seedlings.

背景技术 Background

[0002] 麻疯树（Jatropha curcas),多年生木本油料植物，大戟科麻疯树属，原产美洲，广泛分布于热带亚热带地区。 [0002] Jatropha (Jatropha curcas), perennial woody oil plants, Euphorbiaceae Jatropha, native to the Americas, widely distributed in tropical and subtropical regions. 麻疯树耐干旱贫瘠，种子含油量高达60%，是目前重要的生物能源植物之一。 Jatropha resistant to arid, seed oil content of up to 60%, is one of the bio-energy plant currently important.

[0003] 然而，目前麻风树存在种植区域狭窄，产量低等问题，而传统育种周期长，要获得一个优良性状且稳定遗传的品种需要8-10年。 [0003] However, the presence of Jatropha planting area is narrow, and low yields, and long traditional breeding cycle, to get a good variety of genetic traits and stable needs 8--10 years. 近年来，植物基因工程技术的快速发展，在许多粮食和经济作物的种质改良方面发挥了重要的作用。 In recent years, the rapid development of plant genetic engineering techniques, germplasm improvement in many aspects of food and cash crops played an important role. 因此，成为改良麻风树抗逆性、提高产量、改善油质等性状的重要手段。 Therefore, it becomes modified jatropha resistance, increase yield and improve an important means of oil and other traits.

[0004] 根癌农杆菌介导法是最常用的植物基因转化方法之一。 [0004] 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。 Agrobacterium tumefaciens containing a Ti plasmid, on which there is a T-DNA region. 将目的基因插入到经过改造的T-DNA区，通过侵染植物伤口进入细胞后，可将目的基因插入到植物基因组中，通过减数分裂稳定地遗传给后代。 The target gene is inserted into the engineered T-DNA region, after the wound into the cells by infecting the plant, the target gene can be inserted into a plant genome to their offspring stably through meiosis.

[0005] 麻风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，且转化苗的生根率普遍较低。 [0005] Jatropha research started late, reports of jatropha tissue culture and genetic transformation technology is still limited, and the conversion of rooting is generally low. 李美茹等研究人员以麻风树子叶作为外植体，草胺膦作为筛选剂，建立了根癌农杆菌介导的遗传转化体系，转化苗生根率为78% (Li M., Li H., Jiang H.，et al. Transformation of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas. Plant Cell Tissue and Organ Culture, 2008, 92(2): 173-181)。 Limei Ru and other researchers jatropha cotyledon as explants, glufosinate as a screening agent, established Agrobacterium tumefaciens-mediated genetic transformation system conversion rooting was 78% (Li M., Li H., Jiang H., et al Transformation of an Agrobacterium-mediated cotyledon disc transformation method for Jatropha curcas Plant Cell Tissue and Organ Culture, 2008, 92 (2):.. 173-181). 而Kumar等研究人员以麻风树叶片作为外植体，潮霉素作为筛选剂，建立的根癌农杆菌介导麻风树的遗传转化中，转化苗的生根率只有40% (Kumar N., Anand KGV , Pamidimarri DVNS et al. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants. Industrial Crops and Products, 32: 41—47)。 And Kumar and other researchers to leprosy in leaves as explants, hygromycin as a screening agent, Agrobacterium-mediated genetic transformation of jatropha established tumefaciens, the conversion rooting rate of only 40% (Kumar N., Anand KGV, Pamidimarri DVNS et al Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants Industrial Crops and Products, 32:.. 41-47). Pan 等石开究人员以)¾风十片作为外植体，卡那霉素作为筛选剂，利用农杆菌介导法对麻风树进行转化中，获得的120 株再生植株只有1 株生根（Pan J. , Fu Q. , Xu F. Agrobacterium tumefaciens-mediated transformation of biofuel plant Jatropha curcas using kanamycin selection. African Journal of Biotechnology, 9 (39), 6477—6481)。 Pan and other stone open Researchers in) ¾ wind ten pieces as explants, kanamycin as a selection agent, by Agrobacterium-mediated transformation of Jatropha, a 120 regenerated plants obtained only one root (Pan J., Fu Q., Xu F. Agrobacterium tumefaciens-mediated transformation of biofuel plant Jatropha curcas using kanamycin selection. African Journal of Biotechnology, 9 (39), 6477-6481).

发明内容 DISCLOSURE

[0006] 本发明实施例所要解决的技术问题在于，提供一种麻疯树遗传转化苗的复壮及生根的培养方法，以明显改善麻疯树遗传转化苗纤弱的生长状态，提高生根率。 Technical problems to be solved by the invention embodiment of [0006] The present invention is to provide a method for cultivation of Jatropha genetic transformation of rejuvenation and rooting seedlings to significantly improve Jatropha genetic transformation of state delicate seedlings and improve rooting rates.

[0007] 为解决上述技术问题，本发明提供如下技术方案：一种麻疯树遗传转化苗的复壮及生根的培养方法，包括如下步骤： [0007] In order to solve the above problems, the present invention provides the following technical solutions: culture of Jatropha genetic transformation method of rejuvenation and rooting seedlings, comprising the steps of:

准备步骤，选用合适的外植体由根癌农杆菌浸染处理后，再经抗性愈伤诱导及分化培养基培养后获得再生芽作为抗性芽备用； Preparation step, after the appropriate choice of explants from the Agrobacterium tumefaciens dip treatment, and then resistant callus induction and differentiation culture medium obtained after regeneration bud as resistant buds alternate;

壮苗培养步骤，将抗性芽从抗性愈伤组织上切取下来，切去褐化部位，转接到壮苗培养基P中进行壮苗培养； Seedlings incubation step, the resistance from the resistant callus buds cut down, cut browning site, transferred to seedlings seedling cultivation medium P is carried out;

生根培养步骤，挑取经过壮苗培养后高2-3厘米并有4-6片叶的抗性芽，切除基部愈伤组织，用10-20mg/L的生根粉溶液浸泡纩15分钟，转接至生根培养基R中进行生根培养，直至生根苗达到出罐炼苗的技术要求； Rooting step, picked after a 2-3 cm tall seedlings culture and have 4-6 leaves of resistant buds, callus removal of the base, with a rooting powder solution 10-20mg / L fine cotton soak for 15 minutes, turn R is connected to the rooting medium in rooting culture, until rooting out the technical requirements to achieve the hardening of the tank;

炼苗步骤，对达到出罐炼苗的技术要求的生根苗开盖炼苗，移栽至温室。 Hardening step, to meet the technical requirements of the tank lid rooted hardening of hardening, transplanted to the greenhouse.

[0008] 进一步地，准备步骤中选用的外植体为带有4-6片真叶的茎段，其长度为2-3cm。 [0008] Further, the preparation step is the selection of explants with stem segments 4-6 true leaves, the length of 2-3cm.

[0009] 进一步地，所述准备步骤中使用的根癌农杆菌是如下携带含有目的基因载体的根癌农杆菌菌株中的任意一种：EHA105、LBA4404、GV3101、MP90。 [0009] Further, the preparation step is the use of Agrobacterium tumefaciens strain carrying as containing the gene vector of any one of: EHA105, LBA4404, GV3101, MP90.

[0010] 进一步地，所述准备步骤中使用的抗性愈伤诱导及分化培养基含有1-3 mg/L草胺膦或20-50 mg/L卡那霉素或1-10 mg/L潮霉素。 [0010] Further, the preparation step using resistant callus and differentiation medium containing 1-3 mg / L glufosinate or 20-50 mg / L kanamycin or 1-10 mg / L hygromycin.

[0011] 进一步地，壮苗培养基P的配方为：MS基本培养基+30g/L蔗糖+0.2-1. 5 mg/L 6-苄氨基嘌呤+0. 005-0. 1 mg/L 吲哚丁酸+1-5 mg/L 硝酸银+100-300 mL/L 椰汁+5-10 g/L琼脂+1-3 mg/L草胺膦或20-50mg/L卡那霉素或l-10mg/L潮霉素+50-150 mg/L特美汀，pH=5. 6-6. 0。 [0011] Further, the seedling P medium recipe is:... MS basal medium + 30g / L sucrose + 0.2-1 5 mg / L 6- benzylaminopurine +0 005-0 1 mg / L indole IBA + 1-5 mg / L silver nitrate + 100-300 mL / L coconut + 5-10 g / L agar + 1-3 mg / L glufosinate or 20-50mg / L or kanamycin l-10mg / L hygromycin + 50-150 mg / L timentin, pH = 5. 6-6. 0.

[0012] 进一步地，壮苗培养的培养条件是：温度M_28°C，光照强度1000-2000 lx，光照时间12-18小时/天，培养时间为3-5周，每两周继代一次。 [0012] Further, seedling cultivation culture conditions are: temperature M_28 ° C, light intensity 1000-2000 lx, illumination time 12-18 hours / day, the incubation time is 3-5 weeks, once every two weeks subculture.

[0013] 进一步地，生根培养基R配方为：1/2MS基本培养基+ 0. 01-0. 1 mg/L吲哚_3_乙酸+ 0.01-0. 1 mg/L萘乙酸+ 0. 05-0. 25 mg/L吲哚丁酸+ 10-15 g/L蔗糖+ 4-8 g/L琼脂+ 1-3 mg/L草胺膦或20-50 mg/L卡那霉素或1-10 mg/L潮霉素+ 50-150 mg/L特美汀。 [0013] Further, the rooting medium R formula is:.. 1 / 2MS basic medium + 0. 01-0 1 mg / L indole acetic _3_ + 0.01-0 1 mg / L naphthalene acetic acid + 0. 05-0. 25 mg / L of IBA + 10-15 g / L sucrose + 4-8 g / L agar + 1-3 mg / L glufosinate or 20-50 mg / L kanamycin or 1-10 mg / L hygromycin + 50-150 mg / L timentin.

[0014] 进一步地，生根培养步骤中，培养条件是：温度M_28°C，光照强度1000-2000 Ix, 光照时间12-18小时/天，每两周继代一次。 [0014] Further, rooting step, the culture conditions are: temperature M_28 ° C, light intensity 1000-2000 Ix, illumination time 12-18 hours / day, once every two weeks subculture.

[0015] 进一步地，抗性芽在生根培养基R中培养7-10天开始生根，25-35天达到出罐炼苗的技术要求。 [0015] Further, resistant buds cultured for 7-10 days in the rooting medium R began to take root, 25-35 days to hardening of the technical requirements of the tank.

[0016] 进一步地，生根苗的根长为3-5 cm即可进行炼苗。 [0016] Further, rooted in the length of 3-5 cm root hardening can be carried out.

[0017] 通过采用上述技术方案，本发明至少具有如下有益效果： [0017] By using the techniques described above, the present invention has at least the following beneficial effects:

1.改善转化苗的生长状态。 1. To improve the growth state into seedlings. 经过根癌农杆菌浸染后的转化苗通常会更加纤细瘦弱，生根率会更低，尤其是经卡那霉素和潮霉素筛选所获得转化苗，叶片较小，茎细弱，植株发黄， 而本发明通过增加壮苗培养步骤，使转化苗叶片浓绿，茎段粗壮，生长旺盛，有利于后续的生根培养。 After a dip Agrobacterium transformation generally more slender thin seedlings, rooting rate will be lower, especially by kanamycin and hygromycin obtained transformed seedlings, small blade, thin stems, plant yellow, The present invention, by increasing the seedlings incubation step, so that the transgenic plant dark green leaves, stem stout, vigorous growth, in favor of subsequent rooting.

[0018] 2.生根率高。 [0018] 2. rooting rate. 麻风树组织培养中一直存在着生根困难的问题，生根率普遍较低。 Jatropha tissue culture there has been a difficult problem rooting, rooting rate is generally low. 经过根癌农杆菌浸染后的转化苗状态较差，生根率更低，而本发明的方法中，不论经过草胺膦，卡那霉素还是潮霉素筛选得到的转化苗，经壮苗培养后，生根率均可达80-90 %，为麻风树的转基因育种奠定了良好的基础。 After Agrobacterium tumefaciens dip after poor seedling status, lower rooting rate, and the method of the present invention, whether through glufosinate, kanamycin or hygromycin was transformed seedlings, the seedling cultivation After rooting rate up to 80-90%, and laid a good foundation for transgenic breeding jatropha.

[0019] 3.有效筛选出转基因苗。 [0019] 3. Effective screening transgenic seedlings. 现有技术都是在抗性愈伤组织阶段和分化阶段加入筛选剂，将得到的抗性芽转入不含筛选剂的生根培养基；本发明不仅在抗性愈伤组织阶段和分化阶段加入筛选剂，生根阶段也选择了合适的筛选剂浓度，这样有助于避免非转化苗的逃逸，有效筛选出转化苗，减少后期的鉴定工作。 Art screening agents are added in resistant callus stage and differentiation stage, the resulting shoots transferred to rooting medium without resistance screening agent; the present invention is not added to the resistant callus stage and differentiation stage screening agents, rooting stage also chose suitable screening agent concentration, which helps to prevent the escape of non-transgenic plants, effectively screen out transgenic plants, reducing appraisal work late. 具体实施方式 DETAILED DESCRIPTION

[0020] 下面结合实例详述描述本发明的实施方案。 [0020] 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.

[0021] 本发明提供一种麻疯树遗传转化苗的复壮及生根的培养方法，步骤如下： [0021] The present invention provides a genetic transformation of Jatropha seedling cultivation method rejuvenation and rooting, as follows:

准备步骤，选用合适的外植体由根癌农杆菌浸染处理后，再经抗性愈伤诱导及分化培养基培养后获得再生芽作为抗性芽备用； Preparation step, after the appropriate choice of explants from the Agrobacterium tumefaciens dip treatment, and then resistant callus induction and differentiation culture medium obtained after regeneration bud as resistant buds alternate;

壮苗培养步骤，将抗性芽从抗性愈伤组织上切取下来，切去褐化部位，转接到壮苗培养基P中进行壮苗培养； Seedlings incubation step, the resistance from the resistant callus buds cut down, cut browning site, transferred to seedlings seedling cultivation medium P is carried out;

生根培养步骤，挑取经过壮苗培养后高2-3厘米并有4-6片叶的抗性芽，切除基部愈伤组织，用10-20mg/L的生根粉溶液浸泡纩15分钟，转接至生根培养基R中进行生根培养，直至生根苗达到出罐炼苗的技术要求； Rooting step, picked after a 2-3 cm tall seedlings culture and have 4-6 leaves of resistant buds, callus removal of the base, with a rooting powder solution 10-20mg / L fine cotton soak for 15 minutes, turn R is connected to the rooting medium in rooting culture, until rooting out the technical requirements to achieve the hardening of the tank;

炼苗步骤，对达到出罐炼苗的技术要求的生根苗开盖炼苗，移栽至温室。 Hardening step, to meet the technical requirements of the tank lid rooted hardening of hardening, transplanted to the greenhouse.

[0022] 其中，所述准备步骤中，使用的外植体优选为带有4-6片真叶的小桐子茎段，其长度为2-3cm;而所用的根癌农杆菌是如下携带含有目的基因载体的根癌农杆菌菌株中的任意一种疋拟105、1^六4404、6¥3101、]\^90，而目的基因载体可以是？ [0022] wherein the preparation step, explant preferably Jatropha stem sections with 4-6 true leaves, the length of 2-3cm; and Agrobacterium tumefaciens is used as portable contain Agrobacterium tumefaciens strain in a gene vector for any purpose intended 105,1 Cloth ^ six 4404,6 ¥ 3101,] \ ^ 90, and the target gene carrier can be? 81121呼11或？ 81121 or call 11? 0六1^认1301 等。 0 1 ^ recognize six 1301 and so on. 使用的抗性愈伤诱导及分化培养基中含有1-3 mg/L草胺膦或20-50 mg/L卡那霉素或1-10 mg/L潮霉素。 Resistant callus induction and differentiation medium used contained 1-3 mg / L glufosinate or 20-50 mg / L kanamycin or 1-10 mg / L hygromycin.

[0023] 在壮苗培养步骤中，壮苗培养基P的配方为：MS基本培养基+10_30g/L蔗糖+0.2-1.5 mg/L 6-苄氨基嘌呤+0. 005-0. 1 mg/L 吲哚丁酸+1-5 mg/L硝酸银+100-300 mL/ L椰汁+5-10 g/L琼脂+1-3 mg/L草胺膦或20-50mg/L卡那霉素或Ι-lOmg/L潮霉素+50-150 mg/L特美汀，pH=5. 6-6.0。 [0023] In step culture seedlings, seedlings P in the formula of the medium: MS basal medium + 10_30g / L sucrose + 0.2-1.5 mg / L 6- benzylaminopurine +0 005-0 1 mg /.. L indole butyric acid + 1-5 mg / L silver nitrate + 100-300 mL / L coconut + 5-10 g / L agar + 1-3 mg / L glufosinate or 20-50mg / L Kanamycin plain or Ι-lOmg / L hygromycin + 50-150 mg / L timentin, pH = 5. 6-6.0. 壮苗培养条件是：培养温度M_28°C，光照强度1000-2000 Ix, 光照时间12-18小时/天，培养时间为3-5周，每两周继代一次。 Seedling cultivation conditions are: culture temperature M_28 ° C, light intensity 1000-2000 Ix, illumination time 12-18 hours / day, the incubation time is 3-5 weeks, once every two weeks subculture.

[0024] 而生根培养步骤中，生根培养基R配方为：1/2MS基本培养基+ 0. 01-0. 1 mg/L吲哚-3-乙酸+ 0.01-0.1 mg/L 萘乙酸+ 0. 05-0. 25 mg/L 吲哚丁酸+ 10-15 g/L 蔗糖+ 4-8 g/L琼脂+ 1-3 mg/L草胺膦或20-50 mg/L卡那霉素或1-10 mg/L潮霉素+ 50-150 mg/L 特美汀。 [0024] and rooting step rooting medium R formula is:. 1 / 2MS basic medium + 0. 01-0 1 mg / L indole-3-acetic acid + 0.01-0.1 mg / L naphthalene acetic acid + 0 . 05-0. 25 mg / L IBA + 10-15 g / L sucrose + 4-8 g / L agar + 1-3 mg / L glufosinate or 20-50 mg / L kanamycin or 1-10 mg / L hygromycin + 50-150 mg / L timentin. 生根培养条件是：培养温度M_28°C，光照强度1000-2000 lx，光照时间12-18小时/天，每两周继代一次。 Rooting conditions are: culture temperature M_28 ° C, light intensity 1000-2000 lx, illumination time 12-18 hours / day, once every two weeks subculture. 一般地，抗性芽在生根培养基R中培养7-10天开始生根，25-35 天达到出罐炼苗的技术要求。 In general, resistant buds cultured in rooting medium R 7-10 days begin to take root, 25-35 days to hardening of the technical requirements of the tank.

[0025] 根据目前的技术水平状况，生根苗的根长为3-5 cm即可进行炼苗。 [0025] According to the current level of technology status, rooted in the length of 3-5 cm root hardening can be carried out.

[0026] 下面结合实例详述描述本发明的实施方案。 [0026] 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.

[0027]实例 1 [0027] Example 1

本实例是以根癌农杆菌GV3101介导将GUS基因转入云南元谋野生麻风树进行培养，各工艺步骤具体如下： This example is Agrobacterium tumefaciens GV3101 mediated GUS gene into wild jatropha Yuanmou culturing process steps as follows:

准备步骤，将外植体经携带含有目的基因载体PCAMBIA1301的根癌农杆菌GV3101浸染后，再在含有2 mg/L草胺膦的抗性愈伤诱导及分化培养基上培养获得再生芽作为抗性芽备用； Preparatory steps, the explants by Agrobacterium tumefaciens GV3101 carrying containing the gene vector PCAMBIA1301 after dip, then regenerated shoots containing cultured on callus induction and differentiation medium Resistance 2 mg / L glufosinate as an anti- of buds alternate;

壮苗培养步骤，将抗性芽从抗性愈伤组织上小心切取下来，切去褐化部位，转接到壮苗培养基P中进行壮苗培养，所述壮苗培养基P配方为：MS基本培养基+30g/L蔗糖+ 0.2 mg/ L 6-苄氨基嘌呤+ 0. 005 mg/L吲哚丁酸+1 mg/L硝酸银+200 mL/L椰汁+ 7 g/L琼脂+ 2 mg/L草胺膦+ 50 mg/L特美汀，pH=5. 6-6. 0,培养条件是：温度，光照强度2000 Ix, 光照时间16小时/天。 Seedlings incubation step, the resistance from the resistant callus buds carefully cut down, cut browning site, transferred to seedlings seedlings were cultured in medium P, the seedling medium P formula is: MS medium + 30g / L sucrose + 0.2 mg / L 6- benzylaminopurine + 0. 005 mg / L indole butyric acid +1 mg / L silver nitrate +200 mL / L coconut + 7 g / L agar + 2 mg / L glufosinate + 50 mg / L timentin, pH = 5 6-6 0, culture conditions: temperature, light intensity 2000 Ix, light 16 hours / day. 培养时间为四周，每两周继代一次； Training time is four weeks, once every two weeks subculture;

生根培养步骤，挑取经过壮苗培养后高2-3厘米并有4-6片叶的抗性芽，切除基部愈伤组织，用由北京艾比蒂研究开发中心研制及销售的ABT-I号生根粉配制得到的10 mg/L的生根粉溶液浸泡10分钟，转接至生根培养基R中，生根培养基R配方为：1/2MS基本培养基+ 0.01 mg/L 吲哚-3-乙酸+ 0.01 mg/L 萘乙酸+ 0. 05 mg/L 吲哚丁酸+ 10 g/L 蔗糖+ 7 g/L琼脂+ 2 mg/L草胺膦+ 50 mg/L特美汀，培养条件是：温度，光照强度2000 Ix, 光照时间16小时/天，每两周继代一次，一般地，7-10天开始生根，25-35天可以达到出罐炼苗要求； Rooting step, picked after a 2-3 cm tall seedlings culture and have 4-6 leaves of resistant buds, callus removal of the base, with the Beijing Research and Development Center Yi Bidi development and sales of ABT-I 10 mg / ABT ABT solution to prepare a number of L of water for 10 minutes and transferred to rooting medium R, the rooting medium R formula is: 1 / 2MS basic medium + 0.01 mg / L indole-3 acetic acid + 0.01 mg / L naphthalene acetic acid + 0. 05 mg / L indole butyric acid + 10 g / L sucrose + 7 g / L agar + 2 mg / L glufosinate + 50 mg / L timentin, culture conditions They are: temperature, light intensity 2000 Ix, light 16 hours / day, once every two weeks subculture, in general, began to take 7-10 days, 25-35 days to reach the tank hardening requirements;

炼苗步骤，将生长25-35天，根长3-5 cm的生根苗开盖炼苗，移栽至温室。 Hardening step, it will grow 25-35 days, root length of 3-5 cm rooted openings hardening, transplanted to the greenhouse.

[0028]实例 2 [0028] Example 2

本实例是以根癌农杆菌EHA105介导将GUS基因转入云南元谋野生麻风树进行培养，各工艺步骤具体如下： This example is Agrobacterium EHA105 mediated GUS gene into wild jatropha Yuanmou culturing process steps as follows:

准备步骤，选取合适的外植体经携带含有目的基因载体pBI121-Hyg的根癌农杆菌EHA105浸染后，在含有5 mg/L潮霉素的抗性愈伤诱导及分化培养基上培养获得再生芽作为抗性芽备用； Preparation steps, select the appropriate explants by Agrobacterium tumefaciens EHA105 carrying containing the gene vector pBI121-Hyg after dip in the resistant callus induction and differentiation medium containing 5 mg / L hygromycin cultured regeneration bud as resistant shoots back;

壮苗培养步骤，将抗性芽从抗性愈伤组织上小心切取下来，切去褐化部位，转接到壮苗培养基P中进行壮苗培养，壮苗培养基P配方为：MS基本培养基+ 30g/L蔗糖+ 1.5 mg/ L 6-苄氨基嘌呤+ 0. 1 mg/L吲哚丁酸+ 5 mg/L硝酸银+200 mL/L椰汁+ 7 g/L琼脂+ 5 mg/L潮霉素+ 150 mg/L特美汀，pH=5. 6-6. 0,培养条件是：温度，光照强度2000 Ix, 光照时间16小时/天，培养时间为四周，每两周继代一次； Seedling cultivation steps to carefully cut resistant buds removed from the resistant callus, browning cut parts, transferred to seedling culture medium P is carried out seedlings, seedlings medium P formula is: MS basic medium + 30g / L sucrose + 1.5 mg / L 6- benzylaminopurine + 0. 1 mg / L IBA + 5 mg / L silver nitrate +200 mL / L Coconut + 7 g / L agar + 5 .. mg / L hygromycin + 150 mg / L timentin, pH = 5 6-6 0, culture conditions: temperature, light intensity 2000 Ix, light 16 hours / day incubation period was four weeks every two ZHOU Ji-generation time;

生根培养步骤，挑取经过壮苗培养后高2-3厘米并有4-6片叶的抗性芽，切除基部愈伤组织，用由北京艾比蒂研究开发中心研制及销售的ABT-I号生根粉配制得到的20 mg/L的生根粉溶液浸泡10分钟，转接至生根培养基R中，生根培养基R配方为：1/2MS基本培养基+ 0. 1 mg/L 吲哚-3-乙酸+ 0. 1 mg/L 萘乙酸+ 0. 25 mg/L 吲哚丁酸+ 15 g/L 蔗糖+ 7 g/ L琼脂+ 5 mg/L潮霉素+ 150 mg/L特美汀，培养条件是：温度，光照强度1800 Ix,光照时间16小时/天，每两周继代一次，一般7-10天开始生根，25-35天可以达到出罐炼苗要求； Rooting step, picked after a 2-3 cm tall seedlings culture and have 4-6 leaves of resistant buds, callus removal of the base, with the Beijing Research and Development Center Yi Bidi development and sales of ABT-I 20 mg / ABT ABT solution to prepare a number of L of water for 10 minutes and transferred to rooting medium R, the rooting medium R formula is: 1 / 2MS basic medium + 0. 1 mg / L indole - acetoxy + 0. 1 mg / L naphthalene acetic acid + 0. 25 mg / L indole butyric acid + 15 g / L sucrose + 7 g / L agar + 5 mg / L hygromycin + 150 mg / L US special Ting, culture conditions: temperature, light intensity 1800 Ix, light 16 hours / day, once every two weeks subculture, generally began to take 7-10 days, 25-35 days to reach the tank hardening requirements;

炼苗步骤，将生长25-35天，根长3-5 cm的生根苗开盖炼苗，移栽至温室。 Hardening step, it will grow 25-35 days, root length of 3-5 cm rooted openings hardening, transplanted to the greenhouse.

[0029]实例 3 [0029] Example 3

本实例是以根癌农杆菌MP90介导将FT基因转入云南元谋野生麻风树进行培养，各工艺步骤具体如下： This example is Agrobacterium mediated MP90 Yuanmou FT gene into cultured wild jatropha, various process steps as follows:

准备步骤，选取合适的外植体经携带含有目的基因载体PBI121-FT的根癌农杆菌MP90 浸染后，在含有20 mg/L卡那霉素的抗性愈伤诱导及分化培养基上获得再生芽作为抗性芽备用； Preparation steps, select the appropriate explants by Agrobacterium tumefaciens carrying MP90 containing the gene vector PBI121-FT after dip, reborn in the resistant callus induction and differentiation medium containing 20 mg / L kanamycin bud as resistant shoots back;

壮苗培养步骤，将抗性芽从抗性愈伤组织上小心切取下来，切去褐化部位，转接到壮苗培养基P中进行壮苗培养，壮苗培养基P配方为：MS基本培养基+ 30g/L蔗糖+ 1.0 mg/L6-苄氨基嘌呤+ 0. 05 mg/L吲哚丁酸+ 3 mg/L硝酸银+200 mL/L椰汁+ 7 g/L琼脂+ 20 mg/L卡那霉素+ 100 mg/L特美汀，pH=5. 6-6.0，培养条件是：温度25°C，光照强度1500 Ix, 光照时间16小时/天，培养时间为四周，每两周继代一次； Seedling cultivation steps to carefully cut resistant buds removed from the resistant callus, browning cut parts, transferred to seedling culture medium P is carried out seedlings, seedlings medium P formula is: MS basic medium + 30g / L sucrose + 1.0 mg / L6- benzylaminopurine + 0. 05 mg / L of IBA + 3 mg / L silver nitrate +200 mL / L coconut + 7 g / L agar + 20 mg / L kanamycin + 100 mg / L timentin, pH = 5 6-6.0, culture conditions: temperature 25 ° C, light intensity 1500 Ix, light 16 hours / day, incubation time around, every two weeks subculture once;

生根培养步骤，挑取经过壮苗培养后高2-3厘米并有4-6片叶的抗性芽，切除基部愈伤组织，用由北京艾比蒂研究开发中心研制及销售的ABT-I号生根粉配制得到的15 mg/L的生根粉溶液浸泡10分钟，转接至生根培养基R中，生根培养基R配方为：1/2MS基本培养基+ 0. 05 mg/L 吲哚-3-乙酸+ 0. 05 mg/L 萘乙酸+ 0.1 mg/L 吲哚丁酸+ 12 g/L 蔗糖+ 7 g/L琼脂+ 20 mg/L卡那霉素+ 100 mg/L特美汀，培养条件是：温度，光照强度2000 Ix,光照时间16小时/天，每两周继代一次，一般地，7-10天开始生根，25-35天可以达到出罐炼苗程度； Rooting step, picked after a 2-3 cm tall seedlings culture and have 4-6 leaves of resistant buds, callus removal of the base, with the Beijing Research and Development Center Yi Bidi development and sales of ABT-I 15 mg / ABT ABT solution to prepare a number of L of water for 10 minutes and transferred to rooting medium R, the rooting medium R formula is: 1 / 2MS basic medium + 0. 05 mg / L indole - acetoxy + 0. 05 mg / L naphthalene acetic acid + 0.1 mg / L indole butyric acid + 12 g / L sucrose + 7 g / L agar + 20 mg / L kanamycin + 100 mg / L timentin culture conditions: temperature, light intensity 2000 Ix, light 16 hours / day, once every two weeks subculture, in general, began to take 7-10 days, 25-35 days to reach the degree of hardening of the tank;

炼苗步骤，将生长25-35天，根长3-5cm的生根苗开盖炼苗，移栽至温室。 Hardening step, will grow 25-35 days, the roots of rooted 3-5cm long openings hardening, transplanting to the greenhouse. [0030] 以上三个实例所得的抗性芽的生根率统计如下表：_ [0030] The above three examples of the resulting resistant buds rooting rate statistics in the following table: _

实例序号I存活抗性芽数(棵） I生根苗(棵）I生根率~ Example No. I survived resistant buds (trees) I rooted (trees) I rooting rate ~

实例1 11_10_90. 90_ Example 1 11_10_90. 90_

实例2 12 10 83. 33 “ Examples 21210 83.33 "

实例3 1126 IlOl |80. 16 Examples 3 1126 IlOl |. 80 16

由上表可看出，采用本发明的培养方法，抗性芽的生根率能达到80、0%，远高于现有技术的生根率，实用性强。 From the above table it can be seen, the use of culture method of the present invention, the resistance shoots rooting rate can reach 80,0%, well above the rooting rate of the prior art, and practical.