CN1327761C - Method of breeding in vitro of small tuber for dioscorea nipponica Makino - Google Patents
Method of breeding in vitro of small tuber for dioscorea nipponica Makino Download PDFInfo
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- CN1327761C CN1327761C CNB200510016685XA CN200510016685A CN1327761C CN 1327761 C CN1327761 C CN 1327761C CN B200510016685X A CNB200510016685X A CN B200510016685XA CN 200510016685 A CN200510016685 A CN 200510016685A CN 1327761 C CN1327761 C CN 1327761C
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Abstract
The present invention belongs to a plant culture and propagation technology, particularly to an in-vitro tissue culture and micro-tuber propagation method for a medicinal plant(dioscorea nipponica makino). The present invention also discloses an in-vitro micro-tuber culture and outdoor culture method for a wild medicinal plant (dioscorea nipponica makino)by using technologies, such as in-vitro tissue culture, test-tube plantlet transplantation, etc. Under the condition of in-vitro culture, the overground spear segment of dioscorea nipponica makino, the sprout stem segment of a test-tube plantlet, a sprout tablet of a micro-tuber of test-tube plantlet, etc. are used as explants, test-tube plants are induced by concocting a culture medium. After special treatment, the test-tube plants generate micro-tubers. The present invention also discloses an outdoor culture method for micro-tubers. The present invention has the advantages of short propagation period, high transplantation survival rate, land saving, no seasonal limitation during production, etc. The present invention can be applied to the field of large-scale plant production.
Description
Technical field
The invention belongs to the plant cultivation propagation technique and be particularly related to the vitro tissue cultivation of medicinal plant dioscorea nipponica (Dioscorea nipponica Makino) and the propagation method of microtubers.
Background technology
Dioscorea nipponica is one of China's important Chinese medicine material kind commonly used, be used as medicine with root-like stock, its main active ingredient is the total saponin(e of Chinese yam, being the main medicine source of production for treating cardiovascular disease medicine, is again one of important medicine source that is used for the synthetic multiple steroid hormone class and the diosgenin (diosgenin) of contraception class medicine.Because the market demand increases year by year, wild resource reduces year by year, and dioscorea nipponica has become rare traditional Chinese medicine in imminent danger.
Seminal propagation is poor because of seed plumpness, the sterile grain rate height, and is difficult to solve the large-scale production needs; Conventional stem tuber breeding not only will consume a large amount of stem tubers, and reproduction coefficient is low, the difficulty of taking root, and growth rate and limited amount also are difficult to large-scale promotion.No matter be wild kind or cultivar, growth cycle is generally 3-5, and the stem tuber growth rate is slow, be vulnerable to influences such as environment, season, damage by disease and insect simultaneously, and most medicinal Chinese yam stem tuber is irregular, is unfavorable for results.
Utilizing the biotechnology means to cultivate dioscorea nipponica is the important channel that solves the shortage of Chinese herbal medicine germ plasm resource, is modern state-of-the-art Chinese herbal medicine breeding approach.It utilizes the totipotency of plant soma, and promptly each cell all contains a cover complete genome identical with its parent, and the potentiality that become a whole plant are all arranged, and can cultivate the intrinsic properties of the former kind of maintenance and the microtubers of characteristic.In order to realize the large-scale industrialized production of dioscorea nipponica, people are striving to find valid approach always.Method of breeding in vitro of small tuber for dioscorea nipponica Makino of the present invention has been created condition for realizing this goal.
Summary of the invention
The purpose of this invention is to provide a kind of method of cultivating the dioscorea nipponica microtubers by the biotechnology means.
Medium provided by the invention is the 6-benzyl purine (6-BA) that contains the 1.0-2.0 mg/litre, methyl (NAA) the MS medium (Murashige and SkoogShi medium) of 0.5-1.0 mg/litre.In inducing the root medium, the various compositions in the MS medium all reduce to half, and the active carbon of additional .1%-0.2%, are called for short the 1/2MS medium.
The dioscorea nipponica explant that the present invention uses is dioscorea nipponica tender stem segment on the ground, the stem with bud of dioscorea nipponica test-tube plantlet and the band bud fritter of the little microtubers of test-tube plantlet.
Cultivate the method for dioscorea nipponica microtubers, comprise following step;
(1) gets the plant of eugonic dioscorea nipponica, deduct the blade of the tender stem of gained, aseptic routinely method is inoculated on superclean bench in methyl (NAA) the MS inducing culture of the 6-benzyl purine (6-BA) that contains the 1.0-2.0 mg/litre, 0.5-1.0 mg/litre.Rein in light intensity with 1000-10000, illumination every day 14-20 hour is cultivated under temperature 18-24 ℃ the condition, induces the no cingula root test-tube plantlet that has stem and leaf.
(2) the no cingula root test-tube plantlet that quilt is induced in previous step is rapid is implanted in the above-mentioned MS medium that the composition that contains active carbon reduces by half, rein in light intensity with 1000-6000, illumination every day 8-16 hour is cultivated under temperature 18-24 ℃ the condition, expands the formation microtubers until main root; Perhaps: with its stem with bud as the explant cultivation of regenerating, to continue to induce into aseptic seedling.
(3) aseptic seedling that will have than big microtubers moves to after hardening in the soil, and first indoor cultivation goes to outdoor then.
(4) the gained microtubers also can be used as explant and is used to continue to induce aseptic seedling, its method is, on aseptic superclean bench, take out microtubers, clean solid culture medium with sterile distilled water, be cut into small pieces, every contains a bud point at least as an explant, promptly be inoculated in the MS medium, rein in light intensity with 1000-10000, illumination every day 14-20 hour is cultivated under temperature 18-24 ℃ the condition.
Facts have proved that the medium that the present invention adopts comparatively is fit to cultivate the dioscorea nipponica explant and forms microtubers, make and induce, differentiation, Cheng Miao, take root and once to finish; Various compositions in the MS medium are all reduced to half, and the active carbon of additional .1%-0.2%, promptly can be used as the 1/2MS medium that impels the microtubers growth.Method is easy, is easy to grasp, and also is convenient to batch production production and large tracts of land and promotes.
The present invention will be further described below in conjunction with embodiment, but scope of the present invention is not limited to following embodiment.
Embodiment
1. the preparation of material and inoculation
Cut the tender stem section of newly sending from eugonic dioscorea nipponica plant, with running water flushing 2 hours, again with after the distillation washing 2 times; On superclean bench about 40 seconds earlier then, use 0.1%HgCl again with 70% alcohol immersion
2Sterilized 4 minutes, aseptic washing 6 times, blot water with aseptic filter paper after, be cut into the stem section (all being with an axillalry bud for every section) about 0.5 centimetre, stand on the MS medium.
2. induce and breed
Be seeded in the explant on the MS medium, after 2～3 weeks, begin to grow 1～2 budlet from original axillalry bud, after, the bud that newly grows grows to the seedling of 2～3 axillalry buds.
3. take root
Treat that seedling on the MS medium grows tall on 3～4 centimetres (about 3～4 leaves) and change on the 1/2MS medium when a small amount of fibrous root is arranged, grow several roots after 5～15 days.
4. generation microtubers
On the MS medium, grow seedling, minority has microtubers, and is less; After moving to the 1/2MS medium, form bigger, more microtubers gradually.
5. differentiation again
Seedling with a plurality of axillalry buds can be cut into individual plant, be seeded on the MS medium and cultivate; Also can cut the terminal bud of seedling, be seeded on the MS medium, allow its induced bud again; To cut off fibrous root for band root seedling, and be cut into stem with bud, and be seeded on the MS medium, recycle just can obtain a large amount of aseptic seedling, carries out microtubers again and induces.
6. outdoor transplanting
The microtubers of band bud point is cultivated in medium, behind the plant that regenerates, opened lid,, carefully take out seedling, plant in the sterilized soil with the clean medium of sterile water indoor hardening 2～3 days.After strong sprout, move on to field production.
Although so far, described content of the present invention and embodiment in detail, those skilled in the art might propose to improve or change on the basis of above-mentioned disclosure to the present invention, yet, should understand these improvement or the change do not depart from the scope of the present invention yet and design.
Claims (2)
1, a kind of method of breeding in vitro of small tuber for dioscorea nipponica Makino, it is characterized in that by the various compositions in the conditioned media, explant is cultivated, induce the generation of plant regeneration and microtubers, detailed process is: the plant of getting eugonic dioscorea nipponica, deduct the blade of the tender stem of gained, aseptic routinely method is inoculated in the 6-benzyl purine that contains the 1.0-2.0 mg/litre on superclean bench, 0.5-1.0 in the MS inducing culture of the methyl of mg/litre, rein in light intensity with 1000-10000, illumination every day 14-20 hour, cultivate under temperature 18-24 ℃ the condition, induce the no cingula root test-tube plantlet that has stem and leaf, the various compositions that the no cingula root test-tube plantlet that is induced in previous step is rapid is implanted in the MS medium all reduce in the medium of half, and the additional activity charcoal, rein in light intensity with 1000-6000, illumination every day 8-16 hour, cultivate under temperature 18-24 ℃ the condition, expand the formation microtubers until main root.
2,, it is characterized in that explant is dioscorea nipponica tender stem segment on the ground, the stem with bud of dioscorea nipponica test-tube plantlet and the band bud fritter of the little microtubers of test-tube plantlet according to the described method of breeding in vitro of small tuber for dioscorea nipponica Makino of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510016685XA CN1327761C (en) | 2005-03-31 | 2005-03-31 | Method of breeding in vitro of small tuber for dioscorea nipponica Makino |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB200510016685XA CN1327761C (en) | 2005-03-31 | 2005-03-31 | Method of breeding in vitro of small tuber for dioscorea nipponica Makino |
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| CN1692713A CN1692713A (en) | 2005-11-09 |
| CN1327761C true CN1327761C (en) | 2007-07-25 |
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| CNB200510016685XA Expired - Fee Related CN1327761C (en) | 2005-03-31 | 2005-03-31 | Method of breeding in vitro of small tuber for dioscorea nipponica Makino |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101248760B (en) * | 2008-04-03 | 2010-06-09 | 浙江大学 | Cultivation method of rhizoma corydalis stem tuber |
| CN103891619A (en) * | 2014-04-20 | 2014-07-02 | 上饶师范学院 | Organic induction method of miniature dioscorea bulbifera tuber |
| KR101883284B1 (en) * | 2016-09-30 | 2018-07-31 | (주)이그린글로벌 | Pretreatment method for direct seeding of in vitro microtuber |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335053A (en) * | 2001-08-29 | 2002-02-13 | 中国科学院昆明植物研究所 | Method of bacteria-free germination and fast propagation of peltate yam |
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2005
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335053A (en) * | 2001-08-29 | 2002-02-13 | 中国科学院昆明植物研究所 | Method of bacteria-free germination and fast propagation of peltate yam |
Non-Patent Citations (4)
| Title |
|---|
| 盾叶薯蓣组织培养及微块茎的离体诱导 徐向丽，刘选明，周朴华，易克，朱至清，湖南农业大学学报，第26卷第4期 2000 * |
| 穿龙薯蓣的组织培养与快速繁殖 罗凤霞，祝朋芳，周广柱，等，植物生理学通讯，第40卷第3期 2004 * |
| 穿龙薯蓣的资源开发及栽培技术 张继福，庞忠义，黄朝晖，杨育文，人参研究，第3卷 2003 * |
| 穿龙薯蓣的资源开发及栽培技术 张继福，庞忠义，黄朝晖，杨育文，人参研究，第3卷 2003;穿龙薯蓣的组织培养与快速繁殖 罗凤霞，祝朋芳，周广柱，等，植物生理学通讯，第40卷第3期 2004;盾叶薯蓣组织培养及微块茎的离体诱导 徐向丽，刘选明，周朴华，易克，朱至清，湖南农业大学学报，第26卷第4期 2000 * |
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