细胞-组织培养大规模生产膏桐植株的方法 Cell - Tissue culture for mass production methods curcas plant

技术领域 FIELD

[0001] 本发明属于植物种苗的培养技术，具体涉及通过细胞培养和组织培养大规模生产膏桐植株的方法。 [0001] The present invention pertains to a nursery plant culture technology, particularly to large-scale production of Jatropha curcas plant cell culture method and tissue culture.

背景技术 BACKGROUND

[0002] 膏桐（Jatropha curcas L.)又名麻疯树、小桐子，分布于热带、亚热带地区，我国有栽培或半野生。 [0002] Jatropha (Jatropha curcas L.), also known as Jatropha, Jatropha, distributed in tropical and subtropical regions, China has cultivated or semi-wild. 膏桐为灌木或小乔木，高2-5m，多分枝，速生，喜沃土壤，酸性土或钙质土均宜生长，耐0°C左右的低温及轻霜，抗风力强。 Curcas shrubs or small trees, high 2-5m, multi-branched, fast-growing, hi fertile soil, acidic soil or calcareous soils are advised to grow, resistant to low temperatures around 0 ° C and a light frost, wind resistance. 以前膏桐多栽培作围篱及供观赏，种仁含油脂约50%，可作润滑油、制皂等用，并可催吐下泻，但有毒，忌食。 Before curcas cultivation for more fences and ornamental, seeds contain about 50% oil, can be used as lubricants, soap, etc. to use, and can induce vomiting tumble, but toxic, not eat. 近年来，工业上用膏桐籽油或经改性的膏桐籽油作为生物柴油用于各种柴油发动机，因此大规模生产膏桐植株对生物能源的发展具有重要作用。 In recent years, the industry curcas seed oil or a modified curcas seed oil as biodiesel used in a variety of diesel engines, so the large-scale production of Jatropha plants for bioenergy development plays an important role.

[0003] 现在栽培或半野生的膏桐繁殖方法，一般是通过种子繁殖或扦插繁殖，繁殖周期长，成本高。 [0003] Now cultivation of Jatropha curcas or semi-wild breeding methods, generally propagated by seed or cuttings propagation through a long reproductive cycle, high cost. 植物组织培养，就是分离植物体的一部分组织，如根、茎段、叶、花、幼胚等，在无菌试管中，配合一定的营养、激素、温度、光照等条件，使其产生完整植株。 Plant tissue culture is part of the organizational separation plants, such as roots, stems, leaves, flowers, and other immature embryos, in a sterile test tube, with certain nutrients, hormones, temperature, light and other conditions, to produce a complete plant . 由于其条件可以严格控制，生长迅速，一个月左右即为一个周期，因此在植物的生产上有重要应用价值。 Conditions can be strictly controlled because of its rapid growth, that is, a period of about a month, so there are important applications in plant production. 专利号为200510048718. 9的中国发明专利提供了一种通过组织培养大规模生产膏桐种苗的方法，可缩短周期降低成本，但其繁殖速度还有进一步提高的必要和可能。 Patent No. 200,510,048,718.9 of Chinese patent provides a culture by organizing large-scale production of Jatropha curcas seed method can shorten the cycle to reduce costs, but also to further improve the speed of propagation of the necessary and possible. 植物细胞大量培养技术是在植物组织培养快速繁殖的基础上发展起来的。 A large number of plant cell culture technology is developed on the basis of plant tissue culture and rapid propagation on up. 具体做法就是把植物细胞从试管通过三角瓶转移到微生物发酵的大型发酵罐里，给予适当的条件进行培养，使植物细胞像微生物一样在发酵罐里大量繁殖，但细胞培养方法成本高。 Specific approach is to plant cells are transferred from the test tube by microbial fermentation flask to a large fermentation tank, given the right conditions were cultured plant cells as microorganisms like blooms in the fermentation tank, but the high cost of cell culture methods. 虽然申请号为03135614. 1的中国专利文件公布了一种“细胞-组织的培养方法”，以求提高增殖率，降低培养成本，但其技术方案只能用于铁皮石斛。 Although the Chinese Patent Application No. 03135614.1 published file a "cell - tissue culture methods", in order to increase the growth rate, lower training costs, but it can only be used Dendrobium technical solutions.

发明内容 SUMMARY

[0004] 本发明的目的在于提供一种细胞-组织培养大规模生产膏桐植株的方法，既具有细胞培养和组织培养的优点，又能克服细胞培养和组织培养缺点，为膏桐的人工繁殖、脱毒苗生产、培育优良品种开辟高效的途径。 [0004] The present invention is to provide a cell - a large-scale production of tissue culture methods curcas plant, both in cell culture and tissue culture of merit, but also overcome the shortcomings of cell culture and tissue culture, artificial propagation of Jatropha curcas , virus-free seedling production, cultivation of improved varieties open and efficient way.

[0005] 本发明方法包括以下步骤： [0005] The present invention comprises the steps of:

[0006] 1、膏桐细胞悬浮培养：将膏桐组织研磨后接种，加入1〜3倍重量的细胞悬浮液体培养基，容器放入旋转式摇床，于温度25 士2°C，黑暗条件下进行膏桐细胞悬浮培养2〜4 天，摇床转速为80〜130r/min，细胞悬浮液体培养基是：MS+6_BA 0. 2〜0. 5mg/L+NAA 1〜 6mg/L+LH 0. 5〜1. 5mg/L+蔗糖19〜22g/L，PH值5. 8〜6。 [0006] 1 curcas cell suspension cultures: After inoculation curcas tissue grinder, add 1 ~ 3 times the weight of the cell suspension liquid medium, the container into a rotary shaker at a temperature of 25 ± 2 ° C, the dark carried out under curcas cell suspension culture ~ 4 days, shaking speed 80~130r / min, the cell suspension liquid medium is:. MS + 6_BA 0. 2~0 5mg / L + NAA 1~ 6mg / L + LH 0. 5~1. 5mg / L + sucrose 19~22g / L, PH value 5. 8~6. 膏桐组织的研磨最好使用砚体。 Abrasive paste Tong Yan body tissue is best to use.

[0007] 2、对培养的膏桐细胞进行细胞生长测定。 [0007] 2, the cultured cells curcas cell growth assay. 可将增殖的细胞悬浮液倒入有滤纸的布式漏斗，抽去培液，再用蒸馏水洗涤，然后进行细胞生长测定。 Can be proliferated cells suspension was poured into a Buchner funnel filter paper, removing culture fluid, and then washed with distilled water and then subjected to cell growth assay.

[0008] 3、丛芽增殖培养：选取生长进入静止期的膏桐细胞，接入固体的丛芽增殖培养基，光照强度1000〜2000LX，丛芽增殖培养基是：MS+6-BA 0. 1〜lmg/L+NAA 0. 1〜1. 5mg/L+ 蔗糖20〜30g/L，用质量比为0. 5%〜0. 8%琼脂固化，PH值5. 8〜6，培养时间为15〜20 天。 [0008] 3, buds proliferation culture: Select curcas grown into the quiescent cells, access to solid buds proliferation medium, light intensity 1000~2000LX, buds proliferation medium is: MS + 6-BA 0. 1~lmg / L + NAA 0. 1~1. 5mg / L + sucrose 20~30g / L, with a mass ratio of 0.5% ~ 0. 8% agar solidified, PH value 5. 8~6, incubation time 15 ~ 20 days. 最佳的丛芽增殖培养基是：MS+6-BA 0. 9〜lmg/L+NAA 0. 4〜0. 6mg/L+蔗糖20〜30g/ L0 Best buds proliferation medium is: MS + 6-BA 0. 9~lmg / L + NAA 0. 4~0 6mg / L + sucrose 20~30g / L0.

[0009] 4、生根培养：选择生长良好的丛芽接入生根培养基进行生根培养，最后生产出膏桐苗，生根培养基是：MS+NAA 0. 1〜lmg/L。 [0009] 4, rooting: selective growth good access buds were rooting rooting medium, the final production of Jatropha curcas seedlings, rooting medium is: MS + NAA 0. 1~lmg / L. 最佳的生根培养基是：MS+NAA 0. 4〜0. 6mg/L。 The best rooting medium is:. MS + NAA 0. 4~0 6mg / L.

[0010] 5、出瓶栽培：出瓶栽培的苗先保湿15〜20天，保持温度不低于17°C。 [0010] 5, a bottle cultivation: a bottle of moisturizer before planting seedlings 15 ~ 20 days, keeping the temperature of not less than 17 ° C.

[0011] 本发明方法生成的膏桐组培苗可以直接移栽至苗圃地，不需要在苗床上进行练 [0011] The present invention can be produced curcas plantlets transplanted directly to the nursery, do not need to practice in the seedbed

田ο Tian ο

[0012] 本发明的有益效果：本发明将膏桐细胞培养和组织培养有机的结合起来，充分发挥细胞培养和组织培养的优点，周期短、成本低，完全能满足膏桐大面积栽培需要，为膏桐的人工繁殖、脱毒苗生产、培育优良品种提供了一种高效途径，对膏桐的开发利用具有良好前景。 [0012] the beneficial effects of the invention: The present invention will curcas cell culture and tissue culture organic combination, give full play to the advantages of cell culture and tissue culture, short cycle, low cost, fully able to meet the large scale cultivation of Jatropha curcas needs, artificial propagation of Jatropha curcas, virus-free seedling production, cultivation of improved varieties provides an efficient way for the development and utilization of Jatropha curcas has good prospects.

具体实施方式 DETAILED DESCRIPTION

[0013] 见如下实施例： [0013] See the following examples:

[0014] 实施例1 ： [0014] Example 1:

[0015] 1、膏桐外植体的选取及培养：选取膏桐茎段，用洗洁精洗干净后，在流水下冲洗IOmin,日后转到洁净工作台上，吸干水分，先用70% (体积比）酒精灭菌30秒，再转入0.1% (体积比）的HgCl2溶液中灭菌5分钟，无菌蒸馏水冲洗5次，将茎段剪成Icm长的小段，接种于愈伤组织诱导培养基MS+6-BAang/L+NAAlmg/L+蔗糖30g/L，用0. 8% (质量比） 琼脂固化，高温灭菌前PH调为5. 8，培养温度25°C。 [0015] 1, the selection and cultivation of Jatropha curcas explants: Select curcas stems, wash with detergent after washing under running water IOmin, later to clean the table, sucking up water, first with 70 subparagraph% (by volume) ethanol sterilized 30 seconds, and then transferred to 0.1% (by volume) HgCl2 solution was sterilized for 5 minutes, rinsed five times in sterile distilled water, cut into Icm length of the stem section of inoculated callus organizations induction medium MS + 6-BAang / L + NAAlmg / L + sucrose 30g / L, with a 0.8% (by mass) agar solidified, autoclaved before PH adjusted to 5.8, temperature 25 ° C. 外植体接种20天后可见明显芽。 20 days after inoculation of explants showed obvious buds.

[0016] 2、膏桐细胞悬浮培养：取上述培养的芽，先在研体中加少许细胞悬液体培养基研磨接种。 [0016] 2 curcas cell suspension cultures: Take these cultured buds, before adding a few cells suspended in a liquid medium in a mortar grind vaccination. 用三角瓶内盛研磨后的膏桐组织和2倍重量的细胞悬浮液体培养基，放入旋转式摇床，于温度25V，黑暗条件下进行膏桐细胞悬浮培养2天，摇床转速为125r/min，细胞悬浮液体培养基是：MS+6-BA 0. 2mg/L+NAA2mg/L+LH 0. 5mg/L+ 蔗糖20g/L，PH 值6。 Within a flask filled with milled curcas tissues and cells suspended two times the weight of the liquid medium, into a rotary shaker, make curcas cell suspension cultures 2 days at a temperature of 25V, dark conditions, the rotation speed of 125r / min, the cell suspension liquid medium is: MS + 6-BA 0. 2mg / L + NAA2mg / L + LH 0. 5mg / L + sucrose 20g / L, PH value of 6.

[0017] 3、将培养后的细胞悬浮液倒入有滤纸的布式漏斗，抽去培养液，再用蒸馏水洗涤若干次，再抽干，进行细胞生长测定，备用。 [0017] 3, the cultured cell suspension was poured into a Buchner funnel filter paper, removing the culture medium, washed several times with distilled water, and then drained, the cell growth assay is performed, the standby.

[0018] 4、丛芽增殖培养：选取生长进入静止期的膏桐细胞，接入固体的丛芽增殖培养基， 光照强度1000LX，丛芽增殖培养基是：MS+6-BA lmg/L+NAA 0. 5mg/L+蔗糖25g/L，用0. 8% 琼脂固化，PH值5. 8。 [0018] 4, buds proliferation culture: Select curcas grown into the quiescent cells, access to solid buds proliferation medium, light intensity 1000LX, buds proliferation medium is: MS + 6-BA lmg / L + NAA 0. 5mg / L + sucrose 25g / L, with a 0.8% agar solidified, PH value of 5.8. 当植株生长到2cm左右将植株移至生根培养基中，生根培养基是： MS+NAA 0. 5mg/L。 When the plants grow to about 2cm to move the plants rooting medium, rooting medium is: MS + NAA 0. 5mg / L.

[0019] 5、出瓶移栽：当根长0.5cm左右，叶片长到3〜5片时，将生根苗直接移至苗圃地， 先保湿保温15天，保持温度不低于17°C，随后苗木生长良好。 [0019] 5, a bottle transplant: When the root length of about 0.5cm, leaf length to 3 to 5 films will move directly rooted nursery, the first 15 days moisture insulation, keeping the temperature of not less than 17 ° C, Then the seedlings grow well.

[0020] 实施例2 ： [0020] Example 2:

[0021] 1、膏桐外植体的选取及培养同实施例1。 [0021] 1 curcas explant selection and cultivation same as in Example 1.

[0022] 2、膏桐细胞悬浮培养：取上述培养的芽，先在研体中加少许细胞悬液体培养基研磨接种。 [0022] 2 curcas cell suspension cultures: Take these cultured buds, before adding a few cells suspended in a liquid medium in a mortar grind vaccination. 用三角瓶内盛研磨后的膏桐组织和2倍重量的细胞悬浮液体培养基，放入旋转式摇床，于温度25°C，黑暗条件下进行膏桐细胞悬浮培养2天，摇床转速为lOOr/min，细胞悬 Tong Sheng abrasive paste for organizations within the flask and the cell suspension after 2 times the weight of the liquid medium, into a rotary shaker, make curcas cell suspension cultures 2 days at a temperature of 25 ° C, dark conditions, rotation speed To lOOr / min, the cell suspension

4浮液体培养基是：MS+6-BA 0. 5mg/L+NAA 6mg/L+LH 1. 5mg/L+ 蔗糖2 2g/L, PH 值5. 8。 4 floating liquid medium is: MS + 6-BA 0. 5mg / L + NAA 6mg / L + LH 1. 5mg / L + sucrose 2 2g / L, PH value of 5.8.

[0023] 3、将培养后的细胞悬浮液倒入有滤纸的布式漏斗，抽去培养液，再用蒸馏水洗涤若干次，再抽干，进行细胞生长测定，备用。 [0023] 3, the cultured cell suspension was poured into a Buchner funnel filter paper, removing the culture medium, washed several times with distilled water, and then drained, the cell growth assay is performed, the standby.

[0024] 4、丛芽增殖培养：选取生长进入静止期的膏桐细胞，接入固体的丛芽增殖培养基， 光照强度1800LX，丛芽增殖培养基是：MS+6-BA 0. 2mg/L+NAAl. 5mg/L+蔗糖30g/L，用0. 6% 琼脂固化，PH值5. 8。 [0024] 4, buds proliferation culture: Select curcas grown into the quiescent cells, buds proliferation medium access solid, light intensity 1800LX, buds proliferation medium is: MS + 6-BA 0. 2mg / L + NAAl. 5mg / L + sucrose 30g / L, with 0.6% agar-solidified, PH value of 5.8. 当植株生长到2cm左右将植株移至生根培养基中，生根培养基是： MS+NAA 0. 2mg/L。 When the plants grow to about 2cm to move the plants rooting medium, rooting medium is: MS + NAA 0. 2mg / L.

[0025] 5、出瓶移栽同实施例1。 [0025] 5, the same as in Example 1 bottle transplanting. 苗木生长良好。 Seedlings grow well.

[0026] 实施例3 ： [0026] Example 3:

[0027] 1、膏桐外植体的选取及培养同实施例1。 [0027] 1 curcas explant selection and cultivation same as in Example 1.

[0028] 2、膏桐细胞悬浮培养：取上述培养的芽，先在研体中加少许细胞悬液体培养基研磨接种。 [0028] 2 curcas cell suspension cultures: Take these cultured buds, before adding a few cells suspended in a liquid medium in a mortar grind vaccination. 用三角瓶内盛研磨后的膏桐组织和倍重量的细胞悬浮液体培养基，放入旋转式摇床，于温度27 V，黑暗条件下进行膏桐细胞悬浮培养3天，摇床转速为130r/min，细胞悬浮液体培养基是：MS+6-BA 0. 2mg/L+NAA lmg/L+LH 0. 5mg/L+ 蔗糖19g/L。 Tong Sheng abrasive paste for organizations within the flask and the cell suspension after times the weight of the liquid medium, into a rotary shaker curcas cell suspension were cultured for 3 days at a temperature of 27 V, the dark, shaking speed 130r / min, the cell suspension liquid medium is: MS + 6-BA 0. 2mg / L + NAA lmg / L + LH 0. 5mg / L + sucrose 19g / L.

[0029] 3、将培养后的细胞悬浮液倒入有滤纸的布式漏斗，抽去培养液，再用蒸馏水洗涤若干次，再抽干，进行细胞生长测定，备用。 [0029] 3, the cultured cell suspension was poured into a Buchner funnel filter paper, removing the culture medium, washed several times with distilled water, and then drained, the cell growth assay is performed, the standby.

[0030] 4、丛芽增殖培养：选取生长进入静止期的膏桐细胞，接入固体的丛芽增殖培养基， 光照强度1 200LX，丛芽增殖培养基是：MS+6-BA lmg/L+NAAO. 2mg/L+蔗糖20g/L，用0. 5% 琼脂固化，PH值6。 [0030] 4, buds proliferation culture: Select curcas grown into the quiescent cells, access to solid buds proliferation medium, light intensity 1 200LX, buds proliferation medium is: MS + 6-BA lmg / L + NAAO. 2mg / L + sucrose 20g / L, with 0.5% agar-solidified, PH value 6. 当植株生长到2cm左右将植株移至生根培养基中，生根培养基是：MS+NAA 0. 8mg/L。 When the plants grow to about 2cm to move the plants rooting medium, rooting medium is: MS + NAA 0. 8mg / L.

[0031] 5、出瓶移栽同实施例1。 [0031] 5, the same as in Example 1 bottle transplanting. 苗木生长良好。 Seedlings grow well.

[0032] 实施例4 ： [0032] Example 4:

[0033] 1、膏桐外植体的选取及培养同实施例1。 [0033] 1 curcas explant selection and cultivation same as in Example 1.

[0034] 2、膏桐细胞悬浮培养：取上述培养的芽，先在研体中加少许细胞悬液体培养基研磨接种。 [0034] 2 curcas cell suspension cultures: Take these cultured buds, before adding a few cells suspended in a liquid medium in a mortar grind vaccination. 用三角瓶内盛研磨后的膏桐组织和1倍重量的细胞悬浮液体培养基，放入旋转式摇床，于温度23°C，黑暗条件下进行膏桐细胞悬浮培养3天，摇床转速为80r/min，细胞悬浮液体培养基是：MS+6-BA 0. 5mg/L+NAA6mg/L+LH 1. 5mg/L+ 蔗糖2 2g/L。 Tong Sheng abrasive paste for organizations within the flask and the cell suspension after a times the weight of the liquid medium, into a rotary shaker curcas cell suspension were cultured for 3 days at a temperature of 23 ° C, dark conditions, rotation speed To 80r / min, the cell suspension liquid medium is: MS + 6-BA 0. 5mg / L + NAA6mg / L + LH 1. 5mg / L + sucrose 2 2g / L.

[0035] 3、将培养后的细胞悬浮液倒入有滤纸的布式漏斗，抽去培养液，再用蒸馏水洗涤若干次，再抽干，进行细胞生长测定，备用。 [0035] 3, the cultured cell suspension was poured into a Buchner funnel filter paper, removing the culture medium, washed several times with distilled water, and then drained, the cell growth assay is performed, the standby.

[0036] 4、丛芽增殖培养：选取生长进入静止期的膏桐细胞，接入固体的丛芽增殖培养基，光照强度1 900LX，丛芽增殖培养基是：MS+6-BA 0. 2mg/L+NAAl. 5mg/L+蔗糖30g/L，用0. 8%琼脂固化，PH值6。 [0036] 4, buds proliferation culture: Select curcas grown into the quiescent cells, access to solid buds proliferation medium, light intensity 1 900LX, buds proliferation medium is: MS + 6-BA 0. 2mg / L + NAAl. 5mg / L + sucrose 30g / L, with 0.8% agar-solidified, PH value 6. 当植株生长到2cm左右将植株移至生根培养基中，生根培养基是： MS+NAA 0. 5mg/L。 When the plants grow to about 2cm to move the plants rooting medium, rooting medium is: MS + NAA 0. 5mg / L.

[0037] 5、出瓶移栽：同实施例1。 [0037] 5, a bottle Transplanting: same as in Example 1. 苗木生长良好。 Seedlings grow well.

[0038] 以上实施例仅对发明做进一步的说明，而本发明的范围不受所举实施例的局限。 Example only invention [0038] The above embodiment further explanation, and the scope of the present invention is not confined to the illustrated embodiment.