CN103535281B - Tissue culture medium of sealwort roots and stems and in-vitro regeneration method - Google Patents
Tissue culture medium of sealwort roots and stems and in-vitro regeneration method Download PDFInfo
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- CN103535281B CN103535281B CN201310532853.5A CN201310532853A CN103535281B CN 103535281 B CN103535281 B CN 103535281B CN 201310532853 A CN201310532853 A CN 201310532853A CN 103535281 B CN103535281 B CN 103535281B
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Abstract
The invention discloses a tissue culture medium of sealwort roots and stems and an in-vitro regeneration method of the tissue culture medium. The tissue culture medium comprises an inducing culture medium, a proliferation culture medium and a rooting culture medium. The method using the tissue culture medium of the sealwort roots and stems for carrying out in-vitro regeneration on the sealwort roots and stems comprises the three steps of induction of sprouts, proliferation culture of the sprouts and rooting culture. The tissue culture medium and the propagation method have the beneficial effects that a system of in-vitro regeneration of the sealwort roots and stems is established by utilizing a tissue culture technology, and the inducing culture medium, the proliferation culture medium and the rooting culture medium in the method are selected and optimized; and the tissue culture medium has the advantages of high sprout induction rate, high proliferation coefficient, high rooting rate, strong sprouts and seedlings, normal leaf shape and color and the like, and can meet the need of the market for the sealwort roots and stems.
Description
Technical field
The invention belongs to field of plant tissue culture technique, relate to a kind of tissue culture medium (TCM) and in-vitro regeneration method of sealwort rhizome.
Background technology
Sealwort (Polygonatum sibiricum Red.) is the general name of Liliaceae Polygonatum (Polygonatum Mill.) various plants rhizome, and be perennial root draft medicinal plant, its rhizome is traditional Chinese medicine.The Pharmacopoeia of the People's Republic of China version (one) regulation sealwort (Polygonatum sibiricum Delar.ex Redoute), David's-harp (P.cyrtonema Hua) and P. kingianum (P.kingianum Coll.et Hemsl.) 3 kind of plant in 2005 are its crude drug in whole source.Sealwort, containing the composition such as polysaccharide, amino acid, anthraquinone analog compound, has the effects such as antibacterial, step-down, anti-ageing and treatment rheumatalgia.
Along with the pharmaceutical use of sealwort and health value more and more be familiar with by people, the demand of sealwort raw material is also increasing, price constantly raises up, thus cause hill farmer to aggravate to gather a large amount of predatorinesses of wild sealwort, make sealwort natural resources exhausted rapidly, bring disadvantageous effect to the exploitation of sealwort plant and Sustainable development.Therefore, more and more higher to the cry ensureing sealwort raw material sources, implementation industrialization is planted.The wild change man of the people such as Lee's generation to sealwort plants cultivation and is studied, and some place also starts artificial growth sealwort, but above adopt root division owing to producing more, its breeding coefficient is low, plants rhizome consumption greatly, both uneconomical, again limit the yield potential of sealwort, inconvenience Planting management and popularization, and a long-term point root vegetative propagation is easy to cause sealwort deterioration of strains, therefore sealwort seedling problem has become the bottleneck of artificial establishing in large scale.And utilize plant tissue culture technique to set up effective way that sealwort vitro Regeneration System is production high-quality sealwort seedling, but realize sealwort Regeneration in Vitro by tissue culture have no relevant report.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of tissue culture medium (TCM) and in-vitro regeneration method of sealwort rhizome, the high-efficiency in-vitro regeneration of sealwort rhizome can be realized, production high-quality sealwort seedling.
For achieving the above object, the invention provides following technical scheme:
The invention discloses a kind of tissue culture medium (TCM) of sealwort rhizome, comprise inducing culture, proliferated culture medium and root media; Described inducing culture is with MS substratum for minimum medium, and is added with sucrose 30g/L, agar 4g/L, 6-benzyl aminopurine 4.0mg/L and naphthylacetic acid 0.2mg/L; Described proliferated culture medium is with MS substratum for minimum medium, and is added with 6-benzyl aminopurine 2.0mg/L, thidiazuron 1.0mg/L and indole-3-butyric acid 0.1mg/L; Described root media is with 1/2MS substratum for minimum medium, and is added with white sugar 20g/L, carrageenin 5g/L, naphthylacetic acid 0.5mg/L, indole-3-butyric acid 0.5mg/L and gac 50mg/L.
The invention also discloses a kind of method using the tissue culture medium (TCM) of above-mentioned sealwort rhizome to carry out sealwort rhizome Regeneration in Vitro, comprise the following steps:
1) induction of bud: after the leader cleaning and sterilizing sterilizing of sealwort rhizome, be inoculated in the induction carrying out bud in described inducing culture;
2) multiplication culture of bud: the bud derived is inoculated into the multiplication culture carrying out bud in described proliferated culture medium;
3) root culture: the root of Rhizoma Polygonati stem eye of multiplication culture is cut down, is inoculated in described root media and carries out root culture.
Further, in described step 1), when carrying out the induction of bud, first 7 days is light culture, then carries out illumination cultivation, and light application time is 12h/d, and intensity of illumination is 1500lx, control temperature 25 ± 2 DEG C.
MS substratum described in the present invention and 1/2MS substratum are minimum medium conventional in plant tissue culture, and it specifically fills a prescription as shown in the table:
Beneficial effect of the present invention is: the present invention utilizes plant tissue culture technique to establish sealwort vitro Regeneration System, and carried out selecting to optimize to the inducing culture related to, proliferated culture medium and root media, there is the advantages such as bud induction rate is high, growth coefficient is high, rooting rate is high, bud seedling healthy and strong, leaf leaf look normal, the needs of market to high-quality sealwort seedling can be met.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is the induction of root of Rhizoma Polygonati stem eye;
Fig. 2 is the multiplication culture of root of Rhizoma Polygonati stem eye;
Fig. 3 is root of Rhizoma Polygonati stem eye seedling rooting.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
The sealwort rhizome of test materials for being provided by forestry bureau of Peng River county of Chongqing City that the embodiment of the present invention is used.
1) induction of bud: select fine day to take sealwort rhizome, brush off sealwort rhizome earth, use running water 20-30min, the leader getting about 2.0cm is put in washing powder solution and soaked 3-5min, cleaner with running water; By the bud of wash clean with 0.1% mercury chloride process about 10min (the old tender degree depending on bud), by sterile water wash 4 times, by the rhizome tissue's excision be exposed in thimerosal, be inoculated on inducing culture, carry out the induction of bud: first 7 days is light culture, and then carry out illumination cultivation, light application time is 12h/d, intensity of illumination is 1500lx, control temperature 25 ± 2 DEG C.
Testing inducing culture used is with MS substratum for minimum medium, adds sucrose 30g/L, agar 4g/L, then adds mitogen and the growth hormone of dissimilar and different concns, and pH is adjusted to 5.8; In order to filter out the substratum of applicable root of Rhizoma Polygonati stem eye induction, be provided with 6 kinds of different combinations, each process inoculates 30 bottles, every 1, bottle graft kind rhizome, repeats 3 times; Observe after 30d and add up the bud ratio of its rhizome bud, the growing state of bud.
Test-results is in table 1, visible, the induction impact of substratum on root of Rhizoma Polygonati stem eye of the different mitogen type of same concentrations is very large, add the substratum inductivity of 6-BA higher than the substratum adding TDZ, when the concentration of 6-BA reaches 4mg/L, its inductivity reaches and is up to 82.22%, and the growth of bud is fast, as shown in Figure 1.The impact that the substratum of identical mitogen type different concns is induced root of Rhizoma Polygonati stem eye is very large, and along with the rising of mitogen concentration, its inductivity is in rising trend, and the speed of growth of bud is accelerated.Therefore, the substratum being applicable to the induction of root of Rhizoma Polygonati stem eye is MS+6-BA4.0+NAA0.2 combination, namely with MS substratum for minimum medium, and be added with sucrose 30g/L, agar 4g/L, 6-benzyl aminopurine (6-BA) 4.0mg/L and naphthylacetic acid (NAA) 0.2mg/L.
The selection of table 1 inducing culture
2) multiplication culture of bud: the budlet of the health derived is cut into simple bud, proceeds in proliferated culture medium, carries out the multiplication culture of bud.
Testing proliferated culture medium used is with MS substratum for minimum medium, adds plant-growth regulator 6-BA, TDZ, NAA and IBA of different concns combination; Each process inoculation 30 bottles, every 1, bottle graft kind rhizome, repeats 3 times; After inoculation, 40d switching once, adds up proliferation rate, the growing state of average seedling rate and bud seedling after contact subculture 3 times.
Test-results is in table 2, visible, root of Rhizoma Polygonati stem eye is that in the substratum of 6-BA2.0mg/L+TDZ1.0mg/L+IBA0.1mg/L, growing way is best in interpolation hormone combinations, its growth coefficient reaches and is up to 2.69, average plant height is up to 1.75cm, and the bud of propagation is healthy and strong, blade is normal, and the speed of growth is also fast.From table 2 add up result we can also find, in the rhizome Shoot propagation process of sealwort, 6-BA's and TDZ is of great impact, when 6-BA concentration increases gradually, when the concentration of TDZ reduces gradually, its proliferation rate is the downward trend again that first rises, when the concentration of 6-BA is 2.0mg/L, when the concentration of TDZ is 1.0mg/L, its proliferation rate is the highest, the average plant height of bud is the highest, and the upgrowth situation of bud is best, as shown in Figure 2.When increasing the concentration of 6-BA again, when reducing the concentration of TDZ, its proliferation rate declines gradually simultaneously, and the upgrowth situation of bud also dies down thereupon, the vitrifying of plant, and plant is downgraded.The growth conditions of comprehensive growth coefficient, average plant height and bud, the substratum being applicable to root of Rhizoma Polygonati shoot multiplication is combined as MS+6-BA2.0mg/L+TDZ1.0mg/L+IBA0.1mg/L, namely with MS substratum for minimum medium, and be added with 6-benzyl aminopurine (6-BA) 2.0mg/L, thidiazuron (TDZ) 1.0mg/L and indole-3-butyric acid (IBA) 0.1mg/L.
The selection of table 2 proliferated culture medium
3) root culture: a little old tissue of 3 ~ 5cm height indefinite bud band healthy and strong in multiplication culture is cut down, is inoculated in root media and carries out root culture.
Testing root media used is select MS and 1/2MS to be minimum medium respectively, adds NAA, IBA, 6-BA and AC of different concns, white sugar 20g/L, carrageenin 5g/L.Cultivating after 15d just has root to produce successively, continues on this substratum, cultivate 20d observation, and add up its rooting rate, mean elements, average root are long.Explant number × 100% of the explant number/inoculation of rooting rate=take root; Mean elements=sum of taking root/inoculation explant number; The sum of the total length/root of average root length=root.
Test-results is in table 3, visible, root of Rhizoma Polygonati stem eye seedling rooting can be made in the 8 kinds of substratum arranged, but with 1/2MS be situation of taking root in the substratum combination of minimum medium generally than MS for minimum medium is high, and the inorganic salt of high density affect the leaf look of root of Rhizoma Polygonati stem eye seedling.Its hormone is also very large on the impact of root of Rhizoma Polygonati stem eye seedling rooting, and only relatively high than the rooting rate only adding IBA during interpolation NNA, blade profile leaf look normal; Can show that root media, add a little mitogen is conducive to taking root of root of Rhizoma Polygonati stem eye seedling from process C7 and C8; By isocyatic for NAA and IBA mixing in process C3 and C6, and after adding a small amount of gac, rooting rate increases significantly; When process C3 take 1/2MS as basic cultivation, when interpolation NAA0.5mg/L, IBA0.5mg/L and AC50mg/L, its rooting rate reaches and is up to 88.89%, and mean elements is 4.17, average root is long is 1.13cm, and its bud is healthy and strong, blade profile leaf look normal, as shown in Figure 3.Therefore, the substratum being applicable to root of Rhizoma Polygonati stem eye seedling rooting is combined as 1/2MS+NAA0.5mg/L+IBA0.5mg/L+AC50mg/L, namely with 1/2MS substratum for minimum medium, and be added with white sugar 20g/L, carrageenin 5g/L, naphthylacetic acid (NAA) 0.5mg/L, indole-3-butyric acid (IBA) 0.5mg/L and gac (AC) 50mg/L.
The selection of table 3 root media
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (3)
1. the tissue culture medium (TCM) of sealwort rhizome, is characterized in that: comprise inducing culture, proliferated culture medium and root media; Described inducing culture is with MS substratum for minimum medium, and is added with sucrose 30g/L, agar 4g/L, 6-benzyl aminopurine 4.0mg/L and naphthylacetic acid 0.2mg/L; Described proliferated culture medium is with MS substratum for minimum medium, and is added with 6-benzyl aminopurine 2.0mg/L, thidiazuron 1.0mg/L and indole-3-butyric acid 0.1mg/L; Described root media is with 1/2MS substratum for minimum medium, and is added with white sugar 20g/L, carrageenin 5g/L, naphthylacetic acid 0.5mg/L, indole-3-butyric acid 0.5mg/L and gac 50mg/L.
2. use the tissue culture medium (TCM) of the sealwort rhizome described in claim 1 to carry out the method for sealwort rhizome Regeneration in Vitro, it is characterized in that: comprise the following steps:
1) induction of bud: after the leader cleaning and sterilizing sterilizing of sealwort rhizome, be inoculated in the induction carrying out bud in described inducing culture;
2) multiplication culture of bud: the bud derived is inoculated into the multiplication culture carrying out bud in described proliferated culture medium;
3) root culture: the root of Rhizoma Polygonati stem eye of multiplication culture is cut down, is inoculated in described root media and carries out root culture.
3. the method for sealwort rhizome Regeneration in Vitro according to claim 2, is characterized in that: in described step 1), when carrying out the induction of bud, first 7 days is light culture, then carries out illumination cultivation, and light application time is 12h/d, intensity of illumination is 1500lx, control temperature 25 ± 2 DEG C.
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| CN104472353B (en) * | 2014-11-21 | 2016-08-17 | 广西中医药大学 | A kind of set up the method that Rhizoma Polygonati breeds system soon |
| CN105010135B (en) * | 2015-06-29 | 2019-01-18 | 贵州信邦中药材发展有限公司 | A kind of tissue culture and rapid propagation method of rhizoma polygonati |
| CN105580734B (en) * | 2016-01-14 | 2017-09-15 | 遵义市龙驰生物科技有限公司 | A kind of highly effective revulsion induction method of the in vitro rhizome of sealwort |
| CN107409736A (en) * | 2017-09-12 | 2017-12-01 | 衡阳市九龙生态农业有限公司 | The implantation methods of sealwort |
| CN108541594A (en) * | 2018-06-28 | 2018-09-18 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | A kind of tissue culture and rapid propagation method of polygonatum cirrhifolium Royle |
| CN112042533A (en) * | 2020-08-10 | 2020-12-08 | 阳山县三连阳生态农林开发有限公司 | Universal culture medium for cultivating and seedling growing of rhizome tissue and preparation method thereof |
| CN113100057B (en) * | 2021-04-02 | 2022-09-13 | 安徽省林业高科技开发中心 | Seedling strengthening and rapid propagation method for polygonatum cyrtonema |
| CN115581202B (en) * | 2022-09-29 | 2023-11-14 | 中国科学院合肥物质科学研究院 | Method for regenerating new variety of Polygonatum cyrtonema Fabricius and in vitro seedling |
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