一种促进麻疯树外植体再生不定芽的方法 A Jatropha explant method of promoting regeneration of adventitious buds

技术领域 Technical Field

[0001] 本发明涉及植物生物技术领域，具体地，涉及一种促进麻疯树外植体再生不定芽的方法。 [0001] The present invention relates to the field of plant biotechnology, in particular, to a Jatropha explant method of promoting regeneration of adventitious buds.

背景技术 Background

[0002] 伴随着全球对化石燃料的需求日益增长，存储的化石燃料即将耗尽，人们越来越关注可再生的生物柴油。 [0002] With the growing global demand for fossil fuels, fossil fuel is about to run out of memory, there is a growing focus on renewable biodiesel. 在可产生生物柴油的候选植物中，属于大戟科的麻疯树CZairoAacurcas L.)具有明显的优势，它的种子含油量高，种仁含油量可达40%飞0%，同时，麻疯树油中含有活性成分多，如毒蛋白、麻疯酮等有着重要的农药和医药价值。 In the candidate can produce biodiesel plants belonging Euphorbiaceae Jatropha CZairoAacurcas L.) has obvious advantages, its high seed oil content, oil content of seeds fly up to 40% to 0%, at the same time, leprosy tree oil contains more active ingredients, such as toxic protein, ketone, leprosy has important agricultural and medical value.

[0003] 然而，大力推广麻疯树的种植却面临一系列问题，虽然麻疯树种子中含油量高，但种子产量不高；种油中活性成分多，但仍需要改变油的成分才能直接替代化石燃料；麻疯树对环境要求较高，耐寒能力弱，分布区域较窄。 [0003] However, to promote the cultivation of jatropha is facing a series of problems, although Jatropha seeds high oil content, but seed production is not high; seed oil, active ingredients and more, but still need to change the composition of oil to direct to replace fossil fuels; Jatropha for higher environmental requirements, weak cold, narrow distribution area. 麻疯树属包括175个种，可以通过种间杂交引入优良性状，但所需周期长，不能够获得特异的外源基因，故主要通过基因转化改变遗传背景。 Jatropha including 175 kinds, can be introduced through interspecific hybridization good traits, but required a long period, can not get specific exogenous gene, it is mainly by changing the genetic background genetic transformation. 高频率的植株再生体系是基因转化的基础。 Regeneration System is the basis for the high frequency of genetic transformation.

[0004] 麻疯树外植体根据来源不同可以分为不同的类型。 [0004] Jatropha explant according to different sources can be divided into different types. 从成年麻疯树植株上直接获得叶片、叶柄经处理后可以得到成年植株叶片外植体、成年植株叶柄外植体。 Obtained directly from the adult leaves jatropha plant, petioles can be treated adult explants of leaves, adult plant petiole explants. 将麻疯树种子进行无菌培养得到的无菌苗，从无菌苗上得到无菌下胚轴、无菌子叶叶片和无菌子叶叶柄，经处理后可以得到无菌下胚轴外植体、无菌子叶叶片外植体和无菌子叶叶柄外植体。 The Jatropha seeds sterile culture was no vaccine, never get the vaccine hypocotyl sterile, sterile and sterile cotyledon cotyledon leaves petioles, after treatment can be sterile hypocotyl explants sterile cotyledon leaves cotyledon explants and sterilized petiole explants. 将麻疯树种子经常规培育、萌发得到的萌发苗，从萌发苗上可以得到下胚轴和真叶叶柄，经处理后可以得到种子苗下胚轴外植体和真叶叶柄外植体。 The Jatropha seeds by conventional breeding, germination germination seedlings obtained from the germination of the seedlings can be hypocotyl and leaf petioles can be treated seed seedling hypocotyl explants, and leaf petiole explants.

[0005] 现有技术中诱导麻疯树外植体再生不定芽时，通常是在麻疯树外植体不定芽再生培养基中添加细胞分裂素，而最常用的细胞分裂素为6-苄氨基腺嘌呤(6-BA)，浓度为2 mg/1 ;6-糠氨基嘌呤(6-KT)，浓度为0.1-1.0 mg/1或苯基噻二唑基脲(TDZ)，浓度为 [0005] The prior art Jatropha explant inducing adventitious buds, usually adding cytokinins in Jatropha explant shoot regeneration medium, and the most commonly used cytokinin is 6-benzyl amino adenine (6-BA), at a concentration of 2 mg / 1; 6- bran aminopurine (6-KT), at a concentration of 0.1-1.0 mg / 1 or phenyl thiadiazolyl urea (TDZ), concentration

0.05、.5 mg/1。 0.05, .5 mg / 1. 在这种条件下叶柄外植体的不定芽再生效率很低，芽体的质量也较差。 Under such conditions buds petiole explants regeneration efficiency is very low, the quality is also poor buds. 同时，这些再生体系均存在周期长(80 d以上)、再生率不高的问题，严重制约了麻疯树遗传转化研究的开展。 At the same time, these regeneration system existed long period (80 d above), the regeneration rate is not high, which seriously hampered the conduct of Jatropha genetic transformation studies.

发明内容 DISCLOSURE

[0006] 本发明为了克服现有技术麻疯外植体再生不定芽时再生率低、再生芽体质量差、再生周期长的缺陷，提供一种促进麻疯树外植体再生不定芽的方法。 [0006] The present invention to overcome the prior art Jatropha explant regeneration of adventitious buds regeneration rate, poor regeneration bud quality, long regeneration cycle defects, provided a Jatropha explant method of promoting regeneration of adventitious buds . 所述方法简便，可以显著缩短整个培养周期，而且通过所述的方法可以得到数量更多、质量更好的不定芽。 The method is simple, can significantly shorten the culture period, but the number can be more, better quality buds by the methods described.

[0007] 本发明通过以下技术方案予以实现上述目的: [0007] The present invention is to be realized through the following technical solution above objects:

一种促进麻疯树外植体再生不定芽的方法，其特征在于，包括如下步骤: A Jatropha explant method of promoting regeneration of adventitious buds, characterized in that it comprises the following steps:

51.获得麻疯树外植体； 51. obtain Jatropha explant;

52.将步骤SI中的麻疯树外植体用10-60 mg/1苯基噻二唑基脲(TDZ)溶液浸泡处理5〜40 min ；S3.将步骤S2处理后的麻疯树外植体以横放方式接种至无激素的培养基上培养； 52. SI in step Jatropha explant with 10-60 mg / 1-phenyl thiadiazolyl urea (TDZ) Dipping Solution 5~40 min; S3 S2 after step process Jatropha outside. explants inoculated with horizontally to hormone-free culture medium;

所述横放方式为将外植体的横切面与培养基的水平表面相垂直； The way to the cross section horizontally explants and horizontal surface perpendicular to the medium;

所述麻疯树外植体为成年植株叶片外植体、无菌下胚轴外植体、无菌子叶叶片外植体、无菌子叶叶柄外植体、种子苗下胚轴外植体或真叶叶柄外植体。 The Jatropha explant for adult plant leaves explants, hypocotyl explants sterile, sterile blade cotyledon explants, cotyledon petiole explants sterile seed seedling hypocotyl explants or leaf petiole explants.

[0008] 麻疯树外植体根据来源不同可以分为不同的类型。 [0008] Jatropha explant according to different sources can be divided into different types. 从成年麻疯树植株上直接获得叶片、叶柄经处理后可以得到成年植株叶片外植体、成年植株叶柄外植体。 Obtained directly from the adult leaves jatropha plant, petioles can be treated adult explants of leaves, adult plant petiole explants. 将麻疯树种子进行无菌培养得到的无菌苗，从无菌苗上得到无菌下胚轴、无菌子叶叶片和无菌子叶叶柄，经处理后可以得到无菌下胚轴外植体、无菌子叶叶片外植体和无菌子叶叶柄外植体。 The Jatropha seeds sterile culture was no vaccine, never get the vaccine hypocotyl sterile, sterile and sterile cotyledon cotyledon leaves petioles, after treatment can be sterile hypocotyl explants sterile cotyledon leaves cotyledon explants and sterilized petiole explants. 将麻疯树种子经常规培育、萌发得到的萌发苗，从萌发苗上可以得到下胚轴和真叶叶柄，经处理后可以得到种子苗下胚轴外植体和真叶叶柄外植体。 The Jatropha seeds by conventional breeding, germination germination seedlings obtained from the germination of the seedlings can be hypocotyl and leaf petioles can be treated seed seedling hypocotyl explants, and leaf petiole explants.

[0009] 优选地，所述无菌下胚轴外植体用20 mg/1苯基噻二唑基脲溶液浸泡处理20 min。 [0009] Preferably, the sterile hypocotyl explants soaking 20 min with 20 mg / 1-phenyl thiadiazolyl urea solution.

[0010] 优选地,所述成年植株叶片外植体或无菌子叶叶片外植体用20 mg/1苯基噻二唑基脲溶液浸泡处理40 min。 [0010] Preferably, the adult plant leaves cotyledon explants or sterile blade explants soaking 40 min with 20 mg / 1-phenyl thiadiazolyl urea solution.

[0011] 优选地，所述无菌子叶叶柄外植体或真叶叶柄外植体用20 mg/1苯基噻二唑基脲溶液浸泡处理20 min。 [0011] Preferably, the sterile cotyledon petiole or leaf petiole explants explants with 20 mg / 1-phenyl thiadiazolyl urea solution soaking 20 min.

[0012]同时，发明人在前期研究工作中发现:从成年麻疯树植株上获得的成年植株叶柄外植体最适合的处理方式为用3 mg/1苯基噻二唑基脲溶液浸泡处理5 min,不定芽的再生效果最好。 [0012] Meanwhile, the inventors previous study found that: adult Petiole explants obtained from jatropha plants grown the most suitable approach for the use of 3 mg / 1-phenyl thiadiazolyl urea solution soaking 5 min, regeneration buds best. 本发明同时也发现:而种子苗下胚轴外植体不适宜采用苯基噻二唑基脲溶液浸泡处理的方法来诱导不定芽再生。 The present invention also found that: The seed seedling hypocotyl explants suitable for the method of phenyl thiadiazolyl urea solution soaking treatment to induce adventitious buds regeneration. 由此可以看出:不同类型的麻疯树外植体的细胞的分裂能力各异，不同类型的麻疯树外植体对TDZ的敏感性也不同，决定了其再生不定芽的难易程度不同，所以，不是所有类型的麻疯树外植体都可以采用这种方法来诱导不定芽再生。 It can be seen: the ability of cells to divide different types of Jatropha explant different sensitivity of different types of Jatropha explants on TDZ is different, determines the degree of difficulty of regeneration of adventitious buds different, so, not all types of Jatropha explant can use this method to induce adventitious buds regeneration.

[0013] 优选地，所述成年植株叶片外植体或无菌子叶叶片外植体在以横放方式接种至无激素的培养基上培养时，采用叶片下表面朝上的接种放置方式，其不定芽的再生效果比较好。 [0013] Preferably, when the adult plant leaves cotyledon explants or sterile blade on explants inoculated with horizontally to hormone-free culture medium, using the lower blade surface up vaccination placement, its regeneration buds better. 所谓叶片下表面朝上的接种放置方式就是说让叶片的上表面紧贴培养基表面，叶片下表面朝上。 Under the so-called blade surface up vaccination placement that is close to the upper surface so that the blade surface of the medium and lower leaf surface facing up.

[0014] 步骤S2的TDZ溶液采用一般方法配制成所需即可，具体地，可以采用如下方法配制:称取一定量的TDZ粉末，用lmol/1 NaOH充分溶解，再用去离子水定容，采用I mol/1HCl溶液调整TDZ处理溶液的pH至5.8飞.0 ;使用前用0.22微米的水系滤膜对已配制好的TDZ处理溶液进行过滤灭菌。 TDZ solution [0014] Step S2, using the general method can be formulated as needed, in particular, can be formulated using the following method: Weigh a certain amount of TDZ powder fully dissolved lmol / 1 NaOH, deionized water volume using I mol / 1HCl TDZ treatment solution adjusting pH of the solution to 5.8 .0 fly; prior to use 0.22 micron filter to a previously prepared aqueous TDZ treatment solution sterilized by filtration.

[0015] 所述不同类型的麻疯树外植体的制备方法可以参考本技术领域常规方法得到即可。 Preparation [0015] The different types of Jatropha explant can refer to conventional methods present to give to.

[0016] 优选地，步骤S3中所述培养的条件为:光照强度为200-2500 lx，光照时间为12-16小时/天，培养温度为25±1°C。 [0016] Preferably, the conditions in the culturing step S3 is: light intensity 200-2500 lx, illumination time is 12-16 hours / day, the culture temperature was 25 ± 1 ° C.

[0017] 本发明的创造点在于先用高浓度的激素短暂处理，再用无激素的培养基培养再生不定芽，无激素培养基的种类对本发明来说不是着重点，只要是不含激素，而且能够为不定芽的再生提供充足营养成分的培养基都可以实现本发明。 [0017] The present invention is to create a point of first brief treatment with a high concentration of hormones, hormone-free culture medium and then regenerated adventitious buds, types of hormone-free medium is not the focus of the present invention, as long as it does not contain hormones, and can provide adequate nutrition for adventitious buds regeneration medium of the present invention can be achieved. 优选地，本发明步骤S3中所述无激素的培养基以无激素的MS培养基为主要成分。 Preferably, the culture medium of the present invention, the step S3 to hormone-free MS medium without hormones as a main component. 另外，所述无激素的培养基还含有25-35g/Ι蔗糖、8(Tl20 mg/1肌醇和6-8 g/Ι琼脂；培养基pH为5.8-6.0。所述培养基并不限定本发明的保护范围。[0018] 传统麻疯树外植体不定芽再生培养方法之所以存在不定芽再生效率很低，芽体的质量差、周期长的缺陷，首先是因为长时间培养外植体不能够用高浓度的细胞分裂素，因为长期高浓度激素培养外植体将导致外植体受伤甚至死亡，而低浓度的细胞分裂素的刺激不定芽再生的作用又不够强。其次是在不定芽形成以后残存在培养基和外植体组织中的细胞分裂素对不定芽的进一步生长有十分不利的影响。 In addition, the hormone-free medium also contains 25-35g / Ι sucrose, 8 (Tl20 mg / 1 inositol and 6-8 g / Ι agar; medium pH is 5.8-6.0 of the medium is not limited to this. the scope of the invention. [0018] Traditional Jatropha explant shoot regeneration culture method exists adventitious shoot regeneration efficiency is very low, poor quality of buds, long cycle defects, first, because prolonged incubation explants not enough high concentration of cytokinin, because long-term high concentrations of hormone explants cultured explants will lead to injury or even death, while low concentrations of cytokinins stimulate the regeneration of adventitious buds not strong enough. Second is uncertain After the buds are formed in the culture medium and the remaining tissue explants in cytokinins have a very negative impact on the further growth of adventitious buds.

[0019] 本发明对通常的麻疯树外植体不定芽再生培养方法进行了创新，即在诱导不定芽再生培养基中都不添加TDZ，取而代之的是利用高浓度的TDZ溶液对麻疯树外植体进行短期的浸泡处理，给予外植体具有分化能力的细胞相对于普通添加TDZ的培养基更强的刺激，诱导形成更多的不定芽。 [0019] The present invention is generally Jatropha explant shoot regeneration culture methods were innovative, ie the induction of adventitious shoot regeneration medium not add TDZ, replaced by the use of high concentrations of TDZ solution of Jatropha explants short soaking, giving explant cells have differentiation with respect to general media add TDZ stronger stimulation to induce the formation of more buds. 同时，由于仅是局部短时的浸泡处理，TDZ在细胞中的浓度随后很快降低，减少了在培养后期对所形成的不定芽的发育的负面影响，可以达到一次培养操作就可以得到完整植株的高效目的。 Also, because only a short time of soaking locally, TDZ concentration in the cells and then quickly reduced, reducing the negative impact on the buds are formed in the culture and development of late stage, can reach a train you can get a complete plant operation Efficient purposes.

[0020] 本发明的发明人在前期工作中只研究了高浓度苯基噻二唑基脲短期处理方法促进成年植株叶柄外植体不定芽再生的效果。 [0020] The present inventors in previous work studied only high concentrations of phenyl thiadiazolyl urea short-term treatment of adult plant petiole explants shoot regeneration effect. 但是，后来经过大量的创造性试验发现:不同类型的麻疯树外植体的细胞的分裂能力各异，不同类型的麻疯树外植体对TDZ的敏感性也不同，决定了其再生不定芽的难易程度不同，所以，不是所有类型的麻疯树外植体都可以采用这种方法来诱导不定芽再生。 Later, however, after a lot of creative study found: cell division capacity of different types of Jatropha explant different sensitivity of different types of Jatropha explant of TDZ different, determines the regeneration of adventitious buds different degree of difficulty, so not all types of Jatropha explant can use this method to induce adventitious buds regeneration.

[0021] 本发明的有益效果: [0021] The present invention beneficial effects:

本发明所述麻疯树外植体直接再生不定芽的方法简便，可以显著缩短整个培养周期，传统方法的再生周期为80天以上，而本方法只需70天；通过本发明所述的麻疯树外植体直接再生不定芽的方法可以得到数量更多、质量更好的不定芽，现有方法的不定芽再生率一般为16.08 9-55.11 %，而采用本发明的方法的不定芽再生率为75.67 9-92.98 %。 The present invention Jatropha explant direct adventitious buds regeneration method is simple, can significantly shorten the culture period, the traditional method of regeneration cycle is 80 days, but this method only 70 days; through the Commission of the present invention crazy tree explants direct adventitious buds regeneration method can be more numerous, better quality of adventitious buds, adventitious buds regeneration rate of existing methods generally 9-55.11 16.08%, while the method of the present invention adventitious buds regeneration 9-92.98 rate of 75.67%.

[0022] 说明书附图 [0022] The accompanying drawings

图1.不同浓度的TDZ溶液处理无菌下胚轴外植体20分钟后不定芽再生培养35天的效果；A: 10 mg/1 ；B:20 mg/1 ；C:30 mg/1 ；D:60 mg/1 ；bar =1 cm。 Figure 1. Different concentrations of TDZ was treated under sterile buds after 20 minutes hypocotyl explants cultured regenerative effect 35 days; A: 10 mg / 1; B: 20 ​​mg / 1; C: 30 mg / 1; D: 60 mg / 1; bar = 1 cm.

[0023] 图2.无菌下胚轴外植体再生不定芽在伸长培养基上培养15天后的效果；A:再生不定芽在伸长培养基中的生长效果图；B:将再生不定芽从伸长培养基中取出来的效果图；bar =1 cm。 [0023] FIG. 2. Sterile hypocotyl explants cultured adventitious buds regeneration effect after 15 days on elongation medium; A: regeneration buds elongation growth medium renderings; B: the adventitious shoot elongation medium taken out from the renderings; bar = 1 cm.

[0024] 图3.采用20 mg/1 TDZ溶液浸泡处理2种类型下胚轴外植体20 min后以两种方式接种至不定芽再生培养基上培养35天后的效果；A:无菌下胚轴外植体横放方式；B:无菌下胚轴外植体竖插方式；C:种子苗下胚轴外植体横放方式；D:种子苗下胚轴外植体竖插方式；bar =1 cm。 [0024] Figure 3. After 20 mg / 1 TDZ solution soak for 2 types of hypocotyl explants were inoculated 20 min in two ways to the culture medium of adventitious buds regeneration effect 35 days; A: Under sterile hypocotyl explants way horizontally; B: Sterile hypocotyl explants vertical interpolation mode; C: seed seedling hypocotyl explants horizontally way; D: seed seedling hypocotyl explants vertical interpolation method ; bar = 1 cm.

[0025] 图4.采用20 mg/1的TDZ溶液浸泡处理2种类型叶柄外植体20 min后以两种方式接种至不定芽再生培养基上培养35天后的效果；A:真叶叶柄外植体横放方式；B:真叶叶柄外植体竖插方式；C:无菌子叶叶柄外植体横放方式；D:无菌子叶叶柄外植体竖插方式；bar =1 cm。 [0025] Figure 4. TDZ solution 20 mg / 1 of 2 types after soaking petiole explants 20 min in two ways inoculated culture medium adventitious buds regeneration effect 35 days; A: leaf petiole explants way horizontally; B: leaf petiole explants vertical interpolation mode; C: Sterile cotyledon petiole explants horizontally way; D: Sterile cotyledon petiole explants vertical interpolation mode; bar = 1 cm.

[0026] 图5.2种类型叶柄外植体再生不定芽在伸长培养基上培养15天后的效果；A、B:真叶叶柄外植体再生不定芽伸长效果；C、D:无菌子叶叶柄外植体再生不定芽伸长效果；bar =1 cm。 [0026] Figure 5.2 types petiole explants cultured adventitious buds regeneration effect after 15 days on elongation medium; A, B: leaf petiole explant regeneration buds elongation effect; C, D: sterile cotyledon petiole explant regeneration buds elongation effect; bar = 1 cm.

[0027] 图6.采用20 mg/1 TDZ溶液浸泡处理2种类型叶片外植体40 min后以两种方式接种至不定芽再生培养基上培养35天后的效果；A:成年植株叶片外植体上表面朝上，B:成年植株叶片外植体下表面朝上；C:无菌子叶叶片外植体上表面朝上；D:无菌子叶叶片外植体下表面朝上；bar =1 cm ο [0027] Figure 6. The use of 20 mg / 1 TDZ solution after soaking two types of explants were inoculated 40 min in two ways to the culture medium of adventitious buds regeneration effect 35 days; A: adult plant leaves explants On the surface upward, B: under adult explants of leaves surface up; C: Sterile cotyledon leaves explants surface up; D: sterile cotyledon leaves explants surface up; bar = 1 cm ο

[0028]图7.无菌子叶叶片外植体再生不定芽在伸长培养基上培养15天后的效果图；bar=1 cm。 [0028] Figure 7. sterile cotyledon leaves explants cultured adventitious bud regeneration after 15 days of renderings on elongation medium; bar = 1 cm.

具体实施方式 DETAILED DESCRIPTION

[0029] 下面结合附图和具体实施例进一步详细说明本发明。 [0029] The accompanying drawings and the following specific examples further illustrate the present invention. 除非特别说明，实施例中采用的试剂和方法为本领域常规使用的试剂和方法。 Unless otherwise specified, reagents and methods reagents and methods used in the examples are known to those conventionally used.

[0030] 实施例1: [0030] Example 1:

S1.对外植体进行短期细胞分裂素溶液处理的容器的准备 S1. Explants were treated container short-term solution to prepare cytokinin

选取直径为9 Cm的有盖培养皿，置于高压蒸汽灭菌锅中，在121°C，0.1 MPa的条件下灭菌20 min。 Select the diameter of 9 Cm petri dish, place the pot autoclaving at 121 ° C, 0.1 MPa conditions sterilized 20 min.

[0031] S2.苯基噻二唑基脲(TDZ)处理溶液的配制。 [0031] S2. Phenyl thiadiazolyl urea (TDZ) processing solution preparation.

[0032] 准确称取一定量的TDZ粉末，用I mol/1 NaOH溶液充分溶解，再用去离子水定容，配制成0、10、20、30、40、50、60 mg/1的TDZ处理溶液(对照组O mg/1 TDZ溶液为无菌去离子水)。 [0032] Accurately weigh a certain amount of TDZ powder, with I mol / 1 NaOH solution is fully dissolved, with deionized water volume, the preparation of TDZ 0,10,20,30,40,50,60 mg / 1 of treating solution (control group O mg / 1 TDZ solution is sterile deionized water). 采用I mol/1 HCl溶液调整TDZ处理溶液的pH至5.8飞.0 ;使用前用0.22微米的水系滤膜对已配制好的TDZ处理溶液进行过滤灭菌。 Using I mol / 1 HCl solution to adjust the pH of the solution to deal with TDZ 5.8 .0 fly; prior to use 0.22 micron filter to a previously prepared aqueous TDZ treatment solution sterilized by filtration.

[0033] S3.麻疯树无菌下胚轴外植体的获取将浸泡了2天的麻疯树种子去壳，经过0.1%升汞溶液灭菌后，在超净工作台中用无菌手术刀剥离胚胎，将剥离的胚胎接种至经过高温蒸汽灭菌的MS培养基(MS配方成分+30 g/I蔗糖+100 mg/1肌醇+6 g/Ι琼脂；pH5.8-6.0)上，培养12天后，获取种子苗上的下胚轴，用无菌手术刀将下胚轴切成长度约为0.5 cm的下胚轴切段，即可作为无菌下胚轴外植体。 [0033] S3. Jatropha obtained under sterile soaking up two days of Jatropha seed hypocotyl explants shelled, after 0.1% mercuric chloride solution was sterilized in a laminar flow hood using sterile surgery peeling knife embryo, the embryo peeled seeded to high temperature steam sterilization of MS medium (MS recipe ingredients +30 g / I sucrose +100 mg / 1 inositol +6 g / Ι agar; pH5.8-6.0) on cultured for 12 days, get on the hypocotyl seedling seed with a sterile scalpel to cut hypocotyl length of about 0.5 cm hypocotyl segments, it can be used as a sterile hypocotyl explants.

[0034] S4.细胞分裂素(TDZ)溶液处理麻疯树种子苗下胚轴外植体 [0034] S4. Cytokinin (TDZ) was treated jatropha seed seedling hypocotyl explants

在超净工作台中，把切好的无菌下胚轴外植体分别置于上述步骤Si准备好的灭过菌的培养皿中，向各个培养皿中分别倒入上述步骤S2配置的各个浓度的TDZ处理溶液至浸没叶柄外植体为止，盖上培养皿盖，静置20 min后，倒掉TDZ溶液，保留无菌下胚轴，用灭过菌的镊子从培养皿中取出所有的无菌下胚轴外植体，放置在无菌吸水纸上，吸去无菌下胚轴外植体表面多余的水分，每个浓度的TDZ溶液分别有3个平行处理。 In a laminar flow hood, the cut sterile hypocotyl explants were placed above steps Si prepared sterilized Petri dish, respectively, into each of the concentration of the step S2 configuration to each dish TDZ treatment solution to post-immersion petiole explant date, cover the dish cover and let stand 20 min, drained TDZ solution, sterile reserved hypocotyl with sterilized forceps removed from the dish all the non- Bacteria hypocotyl explants, placed in a sterile absorbent paper to absorb the sterile hypocotyl explants excess surface water, each concentration of TDZ has three solutions were processed in parallel.

[0035] S5.将切好的无菌下胚轴外植体置于细胞分裂素(TDZ)溶液处理的容器中，向该容器的广口玻璃瓶中倒入不同浓度的TDZ溶液(O、10、20、30、60 mg/1)至浸没所有的无菌下胚轴外植体为止(对照组O mg/1 TDZ溶液为无菌去离子水),盖上瓶盖,静置20 min后，倒掉TDZ溶液，保留下胚轴，用灭过菌的镊子从广口玻璃瓶中取出所有的无菌下胚轴外植体，放置在无菌吸水纸上，吸去下胚轴外植体表面多余的液体，最后把处理后的无菌下胚轴外植体以横放方式(将下胚轴外植体放置在培养基上，使下胚轴外植体与培养基表面轻轻接触，并且使下胚轴外植体的横切面与培养基的水平表面相垂直)接种在无激素MS培养基(MS配方成分+30 g/Ι蔗糖+100 mg/1肌醇+6 g/Ι琼脂；ρΗ5.8〜6.0)上培养35天，所获得实验结果如表I和图1所不。 [0035] S5. The cut sterile hypocotyl explants placed in a container cytokinin (TDZ) solution treatment, to the container glass jar in different concentrations of TDZ into the solution (O, 10,20,30,60 mg / 1) to all sterile immersed hypocotyl explants up (control group O mg / 1 TDZ solution is sterile deionized water), cover cap and let stand 20 min After drained TDZ solution, reservations hypocotyl, remove all sterile hypocotyl explants from a wide-mouth glass jar with sterilized forceps, placed in a sterile absorbent paper to absorb the hypocotyl explant surface excess liquid, and finally sterile hypocotyl explants treated with horizontally way (the hypocotyl explants placed on media, make hypocotyl explants and culture medium surface light Lite-Touch, and the horizontal surface of the medium hypocotyl explants cross section perpendicular) were seeded in hormone-free MS medium (MS recipe ingredients +30 g / Ι sucrose +100 mg / 1 inositol +6 g Training 35 days ρΗ5.8~6.0) on the experimental results shown in Table I and Figure 1 is not approved; / Ι agar.

表I不同浓度的TDZ溶液浸泡处理麻疯树无菌下胚轴外植体诱导不定芽直接再生的效果 Table I soaked in different concentrations of TDZ treatment Jatropha sterile hypocotyl explants to induce adventitious buds regeneration direct effect

注:数据采用SPSS Statistics 17.0统计分析软件进行方差分析和邓肯多重比较(P含0.05)，数据后字母不同表示处理间差异显著。 Note: The data using SPSS Statistics 17.0 software for statistical analysis ANOVA and Duncan multiple comparison (P containing 0.05), after the data is different letters represent significant differences among treatments. 再生率(％)=(再生出不定芽的外植体数/总外植体数)X 100% ;平均每个外植体的芽数(颗)=再生不定芽总数/再生出不定芽的外植体数。 Regeneration rate (%) = (regenerated adventitious buds explants / total number of explants) X 100%; the average number of buds (pieces) of each explant = total number of adventitious buds regeneration / regeneration of adventitious buds the number of explants.

[0036] 从表I中可知，未用TDZ溶液处理的无菌下胚轴外植体没有不定芽产生，而用不同浓度TDZ溶液处理的无菌下胚轴外植体均能再生出不定芽，但不同浓度TDZ溶液处理诱导无菌下胚轴外植体再生不定芽的频率和每个外植体的平均再生芽数多表现显著差异。 [0036] From Table I, sterile hypocotyl explants treated with TDZ not a solution not produce buds, but with sterile hypocotyl explants treated with different concentrations of TDZ solution can regenerate adventitious buds , but was treated with different concentrations of TDZ-induced sterility hypocotyl explants average adventitious buds regeneration frequency and the number of each explant regeneration showed more significant differences. 此外，随着TDZ浓度的增加，不定芽再生率和平均每个无菌下胚轴外植体的再生芽数表现出先增加后降低的趋势；其中TDZ溶液浓度为20 mg/1时，不定芽的再生率和平均每个外植体的芽数均为最高，分别为81.91%和10.16颗。 In addition, with the increase of TDZ concentration, adventitious bud regeneration rate and the average number of buds each sterile hypocotyl explants exhibited decreased after the first increase; wherein when TDZ concentration in the solution 20 mg / 1, adventitious buds buds regeneration rates and average per explant are highest, respectively, 81.91 percent and 10.16.

[0037] 同时，采用传统方法诱导麻疯树无菌下胚轴外植体不定芽再生:把切好的下胚轴外植体直接以横放方式(将下胚轴外植体放置在培养基上，使下胚轴外植体与培养基表面轻轻接触，并且使下胚轴外植体的横切面与培养基的水平表面相垂直)接种于添加了不同浓度TDZ (0,0.1,0.3,0.6,1.2 mg/1)的MS培养基上培养35天(对照组O mg/1 TDZ溶液为无菌去离子水)，所获得实验结果如表2所示。 [0037] At the same time, using the traditional method of inducing sterility Jatropha hypocotyl explants shoot regeneration: to cut hypocotyl explants manner directly horizontally (the hypocotyl explants placed in culture on the base, so that the hypocotyl explants light contact with the surface of the medium, and the horizontal surface of the medium hypocotyl explants cross section perpendicular) inoculated in different concentrations of TDZ (0,0.1, Training 35 days on 0.3,0.6,1.2 mg / 1) MS medium (control group O mg / 1 TDZ solution is sterile deionized water), the obtained results are shown in Table 2.

[0038] 表2采用传统方法诱导麻疯树无菌下胚轴外植体不定芽再生 [0038] Table 2 using the traditional method of inducing sterility Jatropha hypocotyl explants shoot regeneration

注:数据采用SPSS Statistics 17.0统计分析软件进行方差分析和邓肯多重比较(P含0.05)，数据后字母不同表示处理间差异显著。 Note: The data using SPSS Statistics 17.0 software for statistical analysis ANOVA and Duncan multiple comparison (P containing 0.05), after the data is different letters represent significant differences among treatments. 再生率(％)=(再生出不定芽的外植体数/总外植体数)X 100% ;平均每个外植体的芽数(颗)=再生不定芽总数/再生出不定芽的外植体数。 Regeneration rate (%) = (regenerated adventitious buds explants / total number of explants) X 100%; the average number of buds (pieces) of each explant = total number of adventitious buds regeneration / regeneration of adventitious buds the number of explants.

[0039] 从表2可知，当添加的TDZ浓度为1.2 mg/1时，不定芽再生率和平均每个外植体的芽数均为最高，分别为30.97%和3.03颗。 [0039] From Table 2, when TDZ was added at a concentration of 1.2 mg / 1 when the number of buds bud regeneration rates and average per explant are highest, respectively, 30.97 percent and 3.03.

[0040] 综上所述，由两个表中的数据可知，采用短期高浓度TDZ浸泡处理麻疯树无菌下胚轴外植体的不定芽再生效果显著优于采用传统方法的效果。 [0040] In summary, the data in the two tables shows that short-term high concentrations of TDZ soaking Jatropha under aseptic adventitious buds regeneration explants significantly better than traditional methods results.

[0041] 按照实施例所述高浓度TDZ处理得到的无菌下胚轴外植体再生不定芽在不定芽伸长培养基(MS配方成分+30 g/Ι蔗糖+100 mg/1肌醇+0.5 mg/1 BA(苄氨基腺嘌呤)+0.2mg/1 KT (激动素)+0.4 mg/1 GA3 (赤霉素)+0.2 mg/1 IAA (口引哚乙酸)+10 mg/1 精氨酸+5 g/Ι琼脂；pH5.8飞.0)上培养15天后，可获得较多生长状态良好的再生不定芽芽条，结果见图2。 [0041] The Sterile hypocotyl explants example of the high concentration of TDZ treatment obtained in regeneration of adventitious buds in shoot elongation medium (MS recipe ingredients +30 g / Ι sucrose +100 mg / 1 Inositol + 0.5 mg / 1 BA (benzyl-amino-adenine) + 0.2mg / 1 KT (KT) +0.4 mg / 1 GA3 (GA) +0.2 mg / 1 IAA (indole acetic mouth) +10 mg / 1 fine acid +5 g / Ι agar; pH5.8 fly .0) for 15 days, the availability of more growth in good condition adventitious buds bud sticks, results shown in Figure 2.

[0042] 实施例2 TDZ溶液浸泡处理麻疯树无菌下胚轴外植体的时间对直接再生不定芽效果的影响 [0042] Example 2 TDZ Jatropha sterile solution soaking time under the influence of hypocotyl explants direct effect of adventitious buds

S1.对外植体进行短期细胞分裂素溶液处理的容器的准备:同实施例1。 . S1 vessel explants were short-term treatment of cytokinin solution preparation: same as in Example 1.

[0043] S2.配置20 mg/1的苯基噻二唑基脲(TDZ)溶液:同实施例1。 . [0043] S2 configuration 20 mg / 1 phenyl thiadiazolyl urea (TDZ) solution: same as in Example 1.

[0044] S3.麻疯树无菌下胚轴外植体的获取:同实施例1 . [0044] S3 Jatropha sterile hypocotyl explants of acquisition: Example 1

S4.细胞分裂素(TDZ)溶液处理麻疯树无菌下胚轴外植体:将切好的无菌下胚轴外植体置于细胞分裂素(TDZ)溶液处理的容器中，向该容器的广口玻璃瓶中倒入浓度为20 mg/1的TDZ溶液至浸没所有的下胚轴外植体为止(对照组O mg/1 TDZ溶液为无菌去离子水)，盖上瓶盖，对下胚轴外植体进行不同时间(0、5、20、40 min)的TDZ溶液浸泡处理后，倒掉TDZ溶液，保留下胚轴外植体，用灭过菌的镊子从广口玻璃瓶中取出所有的下胚轴外植体，放置在无菌吸水纸上，吸去下胚轴外植体表面多余的液体，最后把处理后的下胚轴外植体以横放方式(将下胚轴外植体放置在培养基上，使下胚轴外植体与培养基表面轻轻接触，并且使下胚轴外植体的横切面与培养基的水平表面相垂直)接种在无激素MS培养基(MS配方成分+30 g/Ι蔗糖+100 mg/1肌醇+6 g/Ι琼脂；pH5.8〜6.0)上培养35天，所获得实验结果如表3所示。 . S4 cytokinin (TDZ) was treated jatropha sterile hypocotyl explants: chopped Sterile hypocotyl explants placed in a container cytokinin (TDZ) solution treated, to the wide-mouth glass containers into the concentration of 20 mg / 1 of TDZ immersion solution to all of hypocotyl explants up (control group O mg / 1 TDZ solution is sterile deionized water), cover caps After hypocotyl explants for different time (0,5,20,40 min) of TDZ soaking solution, drained TDZ solution retained hypocotyl explants, using sterilized forceps from the wide-mouth Remove any glass hypocotyls explants, placed in a sterile absorbent paper to absorb hypocotyl explants excess surface liquid, and finally the hypocotyl explants treated with horizontally way ( The hypocotyl explants placed on media, make hypocotyl explants light contact with the surface of the medium, and the medium level surface and cross section of the hypocotyl explants perpendicular) Seeding hormone-free MS medium (MS recipe ingredients +30 g / Ι sucrose +100 mg / 1 inositol +6 g / Ι agar; pH5.8~6.0) on day 35 of culture, the obtained results are shown in Table 3.

[0045] 表3 TDZ溶液浸泡处理时间对麻疯树无菌下胚轴外植体直接再生不定芽的影响 [0045] Table 3 TDZ solution soaking time of Jatropha sterile hypocotyl explants directly affect the regeneration of adventitious buds

注:数据采用SPSS Statistics 17.0统计分析软件进行方差分析和邓肯多重比较(P含0.05)，数据后字母不同表示处理间差异显著。 Note: The data using SPSS Statistics 17.0 software for statistical analysis ANOVA and Duncan multiple comparison (P containing 0.05), after the data is different letters represent significant differences among treatments. 再生率(％)=(再生出不定芽的外植体数/总外植体数)X 100% ;平均每个外植体的芽数(颗)=再生不定芽总数/再生出不定芽的外植体数。 Regeneration rate (%) = (regenerated adventitious buds explants / total number of explants) X 100%; the average number of buds (pieces) of each explant = total number of adventitious buds regeneration / regeneration of adventitious buds the number of explants.

[0046] 从表3中可知，随着浸泡处理无菌下胚轴外植体的时间增加，不定芽再生率和平均每个外植体的再生芽数表现出先增加后降低的趋势，以浸泡时间为20 min时的不定芽再生效果最佳。 [0046] From Table 3, with the soaking time sterile hypocotyl explants increased regeneration rate of adventitious buds and the average number of buds per explant showed decreased after the first increase, to soak adventitious buds regeneration time is 20 min at best.

[0047] 实施例3 [0047] Example 3

S1.对外植体进行短期细胞分裂素溶液处理的容器的准备:同实施例1。 . S1 vessel explants were short-term treatment of cytokinin solution preparation: same as in Example 1.

[0048] S2.配置20 mg/1的苯基噻二唑基脲(TDZ)溶液，具体配置方法同实施例1。 [0048] S2. Configure phenyl thiadiazolyl urea 20 mg / 1 of (TDZ) solution, the specific configuration described in Example 1.

[0049] S3.麻疯树无菌下胚轴外植体和种子苗下胚轴外植体的获取:具体方法同实施例1o . [0049] S3 Jatropha sterile hypocotyl explants and seed seedling hypocotyl explants get: the specific method described in Example 1o

[0050] S4.细胞分裂素(TDZ)溶液分别处理麻疯树无菌下胚轴外植体和种子苗下胚轴外植体:将切好的2种类型的下胚轴外植体置于细胞分裂素(TDZ)溶液处理的容器中，向该容器的广口玻璃瓶中倒入浓度为20 mg/1的TDZ溶液至浸没所有的下胚轴外植体为止，盖上瓶盖，对下胚轴外植体进行20 min的浸泡处理后，倒掉TDZ溶液，保留下胚轴外植体，用灭过菌的镊子从广口玻璃瓶中取出所有的下胚轴外植体，放置在无菌吸水纸上，吸去下胚轴外植体表面多余的液体，最后把处理后的下胚轴外植体以两种放置方式(横放方式:将下胚轴外植体放置在培养基上，使下胚轴外植体与培养基表面轻轻接触，并且使下胚轴外植体的横切面与培养基的水平表面相垂直。竖插方式:将下胚轴外植体放置在培养基上，使得下胚轴外植体的中轴与培养基表面相垂直，下胚轴外植体插入培养基中的深度为0.1-0.2cm。)接种在无激素MS培养基(MS配方成分+30 g/Ι鹿糖+100 mg/1肌醇+6 g/Ι琼脂；pH5.8飞.0)上培养35天，所得实验结果如表4所示。 [0050] S4 cytokinin (TDZ) solutions were sterile processing Jatropha hypocotyl explants and seed seedling hypocotyl explants: chopped 2 types of hypocotyl explants set in containers cytokinin (TDZ) solution treatment, to the container glass jar in a concentration of TDZ solution was poured into 20 mg / 1 to the immersion of all hypocotyl explants far, close the bottle on hypocotyl explants treated within 20 min and after immersion, drained TDZ solution retained hypocotyl explants, using sterilized forceps remove all the hypocotyl explants from a wide-mouth glass jar, placed in a sterile absorbent paper to absorb hypocotyl explants excess surface liquid, and finally the hypocotyl explants treated in two placement (horizontally way: the hypocotyl explants placed On the medium, so that the hypocotyl explants light contact with the surface of the medium, and the cross-section of the hypocotyl explants and culture medium horizontal surface perpendicular vertical interpolation method: the hypocotyl explants It was placed on the media, so that the central axis of the surface of the medium hypocotyl explants perpendicular hypocotyl explants insert medium depth of 0.1-0.2cm.) were seeded in MS medium without hormones (MS recipe ingredients +30 g / Ι deer sugar +100 mg / 1 inositol +6 g / Ι agar; pH5.8 fly 0.01) on day 35 of culture, the experimental results obtained are shown in Table 4.

[0051] 表4下胚轴外植体接种放置方式对不定芽再生效果的影响 [0051] Table 4 affect hypocotyl explants inoculation placement of adventitious buds regeneration effect

注:数据采用SPSS Statistics 17.0统计分析软件进行方差分析和邓肯多重比较(P≤0.05)，数据后字母不同表示处理间差异显著。 Note: The data using SPSS Statistics 17.0 software for statistical analysis ANOVA and Duncan multiple comparisons (P≤0.05), treatment difference between different letters represent significant after data. 再生率(％)=(再生出不定芽的外植体数/总外植体数)X 100% ;平均每个外植体的芽数(颗)=再生不定芽总数/再生出不定芽的外植体数。 Regeneration rate (%) = (regenerated adventitious buds explants / total number of explants) X 100%; the average number of buds (pieces) of each explant = total number of adventitious buds regeneration / regeneration of adventitious buds the number of explants. 外植体来源I为来源于麻疯树去壳种子的胚胎在无菌的MS培养基上培养12天后获取的无菌下胚轴外植体；外植体来源2为来源于疯树种子在培养基质(土:沙砾=1:1)上以自然条件培养12天后获取的种子苗下胚轴外植体。 Explants I was shelled from Jatropha seeds sterile after 12 days of embryo culture hypocotyl explants obtained in sterile MS medium; explant source 2 is derived from the crazy tree seeds culture medium (soil: sand = 1: 1) at a natural condition for 12 days seed seedling hypocotyl explants obtained.

[0052] 从表4中的数据可知，无论是哪种来源的下胚轴外植体，采用横放方式接种的外植体不定芽再生效果均显著优于竖插方式的再生效果。 [0052] Data from Table 4 shows that both hypocotyl explants which sources are used horizontally inoculated explants shoot regeneration effects were significantly better than the vertical interpolation method of regeneration. 而种子苗下胚轴外植体不定芽再生率仅为25.70%,因此不适宜采用苯基噻二唑基脲溶液浸泡处理的方法来诱导种子苗下胚轴外植不定芽再生。 The seed seedling hypocotyl explants shoot regeneration rate was 25.70%, and therefore not suitable for the method of phenyl thiadiazolyl urea solution soaking treatment to induce lower seed seedling hypocotyl explants of adventitious buds regeneration.

[0053] 实施例4 [0053] Example 4

分别参考实施例1、2和3的步骤，研究不同浓度TDZ溶液处理成年植株叶片外植体、无菌子叶叶片外植体、种子苗下胚轴外植体、无菌子叶叶柄外植体和真叶叶柄外植体的不定芽再生的情况。 Reference Example were steps 2 and 3, the study was treated with different concentrations of TDZ adult explants of leaves, sterile explants cotyledon leaves, seeds, seedlings hypocotyl explants, sterile and cotyledon petiole explants Indefinite leaf petiole explants shoot regeneration conditions.

[0054] 由结果得知:成年植株叶片外植体、无菌子叶叶片外植体最适合的TDZ溶液的浓度为20 mg/1，处理时间为40 min ;无菌子叶叶柄外植体和真叶叶柄外植体最适合的TDZ溶液的浓度为20 mg/1，处理时间为20 min。 [0054] from the results that: the concentration of adult explants of leaves cotyledon leaves of sterile explants TDZ most suitable solution is 20 mg / 1, the processing time was 40 min; sterile cotyledon petiole explants and true the concentration of leaf petiole explants TDZ solution best suited to 20 mg / 1, the processing time was 20 min. 而种子苗下胚轴外植体不适宜采用高浓度TDZ溶液短期浸泡处理的方法来诱导不定芽再生。 The seed seedling hypocotyl explants suitable for the method of short-term high concentrations of TDZ soaking solution to induce adventitious buds regeneration.

[0055] 实施例5:不同接种方式对麻疯树两种类型叶柄外植体再生不定芽效率的影响本实施例所述的两种类型叶柄外植体分别为无菌子叶叶柄外植体和真叶叶柄外植体。 [0055] Example 5: Two different inoculation leprosy type of tree petiole plant regeneration adventitious buds efficiency of the present embodiment are two types of cases the petiole explants were sterile and cotyledon petiole explants leaf petiole explants.

无菌子叶叶柄外植体来源于自然萌发种子苗真叶叶柄；真叶叶柄外植体来源于无菌萌发种子苗子叶叶柄。 Sterile cotyledon petiole explants germinating seedlings from natural leaf petiole; leaf petiole explants from sterile seeds germinated seedling leaf petioles.

[0056] 将切好的2种类型的叶柄外植体置于细胞分裂素(TDZ)溶液处理的容器中，向该容器的广口玻璃瓶中倒入浓度为20 mg/1的TDZ溶液至浸没所有的叶柄外植体为止，盖上瓶盖,对叶柄外植体进行20分钟的浸泡处理后，倒掉TDZ溶液，保留叶柄外植体，用灭过菌的镊子从广口玻璃瓶中取出所有的叶柄外植体，放置在无菌吸水纸上，吸去叶柄外植体表面多余的液体，最后把处理后的叶柄外植体以两种放置方式(横放方式:将叶柄外植体放置在培养基上，使叶柄外植体与培养基表面轻轻接触，并且使叶柄外植体的横切面与培养基的水平表面相垂直。竖插方式:将叶柄外植体放置在培养基上，使得叶柄外植体的中轴与培养基表面相垂直，叶柄外植体的形态学下端插入培养基中的深度为0.1-0.2 cm。)接种在无激素MS培养基(MS配方成分+30 g/Ι蔗糖+100 mg/1肌醇+6 g/Ι琼脂；ρΗ5.8-6.0)上培养35天，所得实验结果如表5和图4所示。 [0056] The chopped 2 types of petiole explants placed cytokinin (TDZ) was treated container, to the wide-mouth glass containers into the concentration of TDZ solution 20 mg / 1 to After the immersion of the petiole explant all up, covered with caps on petiole explants soaking for 20 minutes, drained TDZ solution, retention petiole explants with sterilized forceps from a wide-mouth glass jar Remove all of the petiole explants, placed in a sterile absorbent paper to absorb excess petiole explant surface of the liquid, and finally the petiole explants treated in two placement (horizontally way: the petiole explants was placed on the media, so petiole explants light contact with the surface of the medium, and the horizontal surface of the medium petiole explants cross section perpendicular to the vertical interpolation method: the petiole explants were placed in culture On the base, so that the central axis of the petiole explant culture medium surface perpendicular, morphology of the lower end of the petiole explant medium insertion depth of 0.1-0.2 cm.) were seeded in hormone-free MS medium (MS formulation ingredients +30 g / Ι sucrose +100 mg / 1 myo-inositol +6 g / Ι Agar; culture 35 days ρΗ5.8-6.0), the obtained results are shown in Table 5 and Figure 4.

[0057] 表5叶柄外植体接种放置方式对不定芽再生效果的影响 [0057] Table 5 Effect of inoculation petiole explants placement of adventitious buds regeneration effect

注:数据采用SPSS Statistics 17.0统计分析软件进行方差分析和邓肯多重比较(P含0.05)，数据后字母不同表示处理间差异显著。 Note: The data using SPSS Statistics 17.0 software for statistical analysis ANOVA and Duncan multiple comparison (P containing 0.05), after the data is different letters represent significant differences among treatments. 再生率(％)=(再生出不定芽的外植体数/总外植体数)X 100% ;平均每个外植体的芽数(颗)=再生不定芽总数/再生出不定芽的外植体数。 Regeneration rate (%) = (regenerated adventitious buds explants / total number of explants) X 100%; the average number of buds (pieces) of each explant = total number of adventitious buds regeneration / regeneration of adventitious buds the number of explants.

[0058] 从表5中的数据可知，无论是哪种类型的叶柄外植体，采用横放接种方式的不定芽再生效果均显著优于竖插方式的效果。 [0058] The data in Table 5 shows that, no matter what type of petiole explants inoculation using horizontally adventitious buds regeneration were significantly better than the vertical interpolation method results.

[0059] 2种类型的叶柄外植体再生不定芽在不定芽伸长培养基(MS配方成分+30 g/Ι蔗糖+100 mg/1 肌醇+0.5 mg/1 BA (苄氨基腺嘌呤)+0.2 mg/1 KT (激动素)+0.4 mg/1 GA3(赤霉素)+0.2 mg/1 IAA (吲哚乙酸)+10 mg/1精氨酸+5 g/Ι琼脂；ρΗ5.8〜6.0)上培养15天后，可获得较多生长状态良好的再生不定芽芽条，结果见图5。 [0059] 2 types of petiole explant regeneration adventitious buds buds elongation medium (MS recipe ingredients +30 g / Ι sucrose +100 mg / 1 inositol +0.5 mg / 1 BA (benzylamino adenine) +0.2 mg / 1 KT (KT) +0.4 mg / 1 GA3 (GA) +0.2 mg / 1 IAA (indole acetic acid) +10 mg / 1 arginine +5 g / Ι agar; ρΗ5.8 ~6.0 culture) 15 days, get more growth in good condition adventitious buds bud sticks, results shown in Figure 5.

[0060] 实施例6:不同接种方式对2种类型麻疯树叶片外植体再生不定芽效率的影响本次实验，选用如说明书c中所述经表面灭菌后成年树龄的麻疯树茎顶端叶片和无菌 [0060] Example 6: Different inoculation methods on two types of leaf explants leprosy adventitious buds regeneration efficiency of this experiment, after the selection as described in the specification c surface-sterilized Jatropha age of adult stem tips of the blades and sterile

条件下培养12天的种子苗上的子叶叶片为实验材料，在超净工作台中用无菌手术刀将叶片切成大小约为0.5X0.5 cm的叶片小块作为外植体，将切好的叶片外植体置于细胞分裂素溶液处理的容器中，向该容器的广口玻璃瓶中倒入浓度为20 mg/1的TDZ溶液至浸没所有的叶片外植体为止，盖上瓶盖，对叶片外植体进行40 min的TDZ溶液浸泡处理后，倒掉TDZ溶液，保留叶片，用灭过菌的镊子从广口玻璃瓶中取出所有的叶片外植体，放置在无菌吸水纸上，吸去叶片外植体表面多余的液体，最后把处理后的叶片外植体以两种放置方式接种(使得叶片外植体的下表面朝上，其上表面与培养基表面接触；使得叶片外植体的上表面朝上，其下表面与培养基表面接触)在无激素MS培养基上培养35天，所获得实验结果如表6和图6所不。 Culture conditions for 12 days on the cotyledon leaves seedling seed as experimental material, in a laminar flow hood with a sterile scalpel blade cut into a size of about 0.5X0.5 cm blade pieces as explants, the chopped leaf explants were placed cytokinin was treated container, to the wide-mouth glass containers into the concentration of TDZ solution 20 mg / 1 to the submerged leaves all explants far, covered with caps on leaf explants were TDZ solution after soaking 40 min, drained TDZ solution, retention leaves with sterilized forceps remove all leaves explants from a wide-mouth glass jar, placed in a sterile absorbent paper on the surface to absorb excess liquid Leaf explants, and finally the leaf explants treated in two placement inoculation (so that the lower surface of the blade explants upward, its upper surface in contact with the surface of the medium; make on the surface of the blade explants upward, the lower surface in contact with the surface of the medium) were cultured in 35 days on MS medium without hormones, the results are shown in Table 6 and Figure 6 is not approved.

[0061] 表6叶片外植体的接种方式对不定芽再生效果的影 [0061] Table 6 inoculation of explants of adventitious buds regeneration effect of shadow

注:数据采用SPSS Statistics 17.0统计分析软件进行方差分析和邓肯多重比较(P含0.05)，数据后字母不同表示差异显著。 Note: The data using SPSS Statistics 17.0 software for statistical analysis ANOVA and Duncan multiple comparison (P containing 0.05), after the data is different letters represent significant differences. [0062] 再生率(％)=(再生出不定芽的外植体数/总外植体数)X 100% ;平均芽数(颗)=再生不定芽总数/出芽的总外植体数。 [0062] regeneration rate (%) = (regenerated adventitious buds explants / total number of explants) X 100%; the average number of buds (pieces) = total number of adventitious buds regeneration / sprouting total number of explants.

[0063] 从表6的数据可知，对麻疯树叶片外植体进行再生不定芽培养时，采用叶片下表面朝上的接种放置方式的再生不定芽的效果明显优于采用上表面朝上的接种放置方式的效果。 [0063] Table 6 shows the data from, for leprosy of leaf explants cultured adventitious buds regeneration, using the blade lower surface up vaccination placement of adventitious buds on the surface better than using upward placement inoculation effect.

[0064] 叶片外植体再生不定芽在不定芽伸长培养基(MS配方成分+30 g/Ι鹿糖+100 mg/I肌醇+0.5 mg/1 BA (苄氨基腺嘌呤)+0.2 mg/1 KT (激动素)+0.4 mg/1 GA3 (赤霉素)+0.2 mg/1 IAA (吲哚乙酸)+10 mg/Ι精氨酸+5 g/Ι琼脂；pH5.8〜6.0)上培养15天后，可获得较多生长状态良好的再生不定芽芽条。 [0064] The explants regenerated adventitious buds buds elongation medium (MS recipe ingredients +30 g / Ι deer sugar +100 mg / I inositol +0.5 mg / 1 BA (benzyl-amino-adenine) +0.2 mg / 1 KT (KT) +0.4 mg / 1 GA3 (GA) +0.2 mg / 1 IAA (indole acetic acid) +10 mg / Ι arginine +5 g / Ι agar; pH5.8~6.0) After 15 days on the train, get more growth in good condition adventitious buds bud sticks. 无菌苗子叶叶片在不定芽伸长培养基培养15天后的结果见图7。 Cotyledons blades buds elongation medium and incubated for 15 days results shown in Figure 7.