CN102228005A - Pinellia ternate tissue culture one-step speciation method - Google Patents

Abstract

The invention relates to a pinellia ternate tissue culture one-step speciation method. Pinellia ternate tissue culture seedlings, that are obtained from a one-step seedling and can be treated with hardening and transplanting, are injected with liquid medium without switch, and cultured sequentially at 25+- 2 DEG C, with a photoperiodicity of 12h/d and an illumination intensity of 1000-1500Lx. Pinellia ternates complete one growth period in a culture bottle, and are washed out from the medium to be transplanted. The invention is characterized in that the method simplifies hardening seedling steps, avoids problem of transplanting survival rate, and lowers the possibility of re-infection of virus in pinellia ternate first generation production. In addition, the method combines all merits of the one-step speciation method, so that pinellia ternate plants have good inductivity, differentiation rate, and number of tubers differentiated from each explant.

Description

Tuber of pinellia tissue culture one goes on foot kind of a method

Technical field

The present invention relates to tuber of pinellia group training one and go on foot kind of a method, this technology belongs to biotechnology engineering field.

Background technology

Tuber of pinellia Pinellia ternata (Thunb.) Breit. belongs to the Araeceae herbaceos perennial, be the non-irrigated tuber of pinellia again, mainly contain multiple compositions such as alkaloid, cupreol, polysaccharide, amino acid, volatile oil, pinellin and inorganic elements, clinical practice is very extensive, apart from modern existing more than 2000 year medicinal history, be to use conventional Chinese medicine very universal, that curative effect is more definite simply.

Tuber of pinellia wild resource is distributed more widely, but because excessively excavating of the change of cropping system and people causes the ecotope of suitable tuber of pinellia growth to dwindle year by year, along with the increase of market demand and people deepen continuously to the application study of the tuber of pinellia, its range of application enlarges day by day, and market supply is nervous always.In order to alleviate the imbalance between supply and demand of the tuber of pinellia, from the beginning of the eighties in last century, China just begins the tuber of pinellia is carried out the man experimental study of planting of wild change, but because tuber of pinellia natural propagation coefficient is low, artificial planting provenance wretched insufficiency, how to solve tuber of pinellia provenance becomes problem demanding prompt solution for this reason.

Plant Tissue Breeding regeneration approach is mainly the complete dedifferentiation of explant and forms callus, then callus is transferred to break up on the differential medium and sprouted, again it is transferred to root induction on the root media, but this approach is more loaded down with trivial details, germplasm easily produces variation, explant differentiation stem tuber number is not high yet, in recent years, has the researcher to create the forming seedling through one step culture method on the tissue culture of certain plants yet.And the forming seedling through one step culture method be exactly explant after dedifferentiation, do not need to transfer on the differential medium, but directly differentiation again on former medium, bud behind the normally first root of the order of differentiation is until forming healthy and strong full stand.Its feature that has is: the regeneration period is short, and explant differentiation stem tuber is counted height, and easy to operate, the cultivation program is simple, does not influence the rate of increase, can keep former specific character, is convenient to large-scale production.

Tuber of pinellia plant is the herbaceous plant of succulent stem, find in the research behind the tuber of pinellia forming seedling through one step culture at the tissue cultivating seedling bottle outlet, the wash-out medium, in the process of transplanting, because manual operation causes tuber of pinellia plant to be easy to be fractureed and be subjected to wound, cause after the transplanting of part tuber of pinellia tissue cultivating seedling directly dead, survival rate is only about 30%～50%, though also there is part to regrow at Miao Jishang, but the rising along with natural temperature in the tuber of pinellia growth has the normal phenomenon of falling the seedling, growth cycle is shorter, it is extremely low to cause the generation group of the tuber of pinellia to cultivate output, can not satisfy the actual needs of production far away.

Existing patent documentation: the quick quick-breeding method of non-irrigated tuber of pinellia tissue culture (application number: 200910218386), may further comprise the steps: 1) preparation medium, each component and every liter of institute's content of minimal medium and other group training stage medium are: minimal medium: use the MS medium, additional saccharose 15-45g/L, agar 10g/L regulates pH5.8; Callus inducing medium: add KT1.0-3.0mg/L and 2,4-D1.0-3.0mg/L among the MS; Differential medium: add KT1.0-3.0mg/L and 2,4-D1.0-3.0mg/L among the MS; Proliferated culture medium: add KT0.2-2.0mg/L and 2,4-D0.2-2.0mg/L among the MS; Root media: add KT1.0-2.0mg/L and NAA0-1.0mg/L among the MS; 2) cultivate aseptic seedling: after non-irrigated tuber of pinellia stem tuber is carried out sterilization treatment, be cut into the fritter about 5mm, it is moved to MS+KT2.0mg/L+2,4-D2.0mg/L medium on produce callus, change MS+KT3.0mg/L+2 thereafter, 4-D1.0mg/L medium on make its differentiation, with the clump sprout tuber that produces, be cut into the simple bud piece again and move to that growth obtains aseptic seedling on the MS minimal medium; 3) evoked callus: under aseptic condition, with step 2) blade, petiole and the callus lines of the aseptic seedling of turning out are cut into the tissue of 5mm size, are seeded on the callus inducing medium, induce the formation callus; 4) differentiation culture: induce the callus of formation to move to step 3) its differentiation is sprouted; 5) enrichment culture: the clump bud that step 4) is generated is cut into simple bud (callus lines that comprises base portion), is seeded to and carries out enrichment culture on the proliferated culture medium, it is broken up again sprout; 6) culture of rootage: the clump sprout tuber that step 5) forms is cut into simple bud (callus lines that comprises base portion), is seeded in and carries out culture of rootage in the root media; 7) transplant: when the tissue cultivating seedling of taking root of step 6) grows to 3-5cm, radical is transplanted in the matrix when above at 5, is cultured to be a seedling in the greenhouse.

A kind of hybridizing pinellia tuber and F ↓ [1] expanding propagation method (application number: 201019026001), it may further comprise the steps: step 1, tuber of pinellia variety collection and germ plasm resource garden are set up: at first collect dissimilar tuber of pinellia kind stems, select healthy, of the same size kind of stem, the branch sub-district is colonizated in the tuber of pinellia germ plasm resource garden, inserts card mark and carries out water and fertilizer management; Step 2, the hybrid strain screening: from the dissimilar tuber of pinellia population in tuber of pinellia germ plasm resource garden, it is good to select proterties, and representative population is as hybrid strain; Step 3, hybridization technique: the organ bract of tuber of pinellia flower is parted a little the column cap tool mucus of turning white, stamen smooth surface, double summer flower artificial emasculation pollination during loose powder not, bagging was isolated after the artificial emasculation pollination finished, and the mark of listing is treated to receive hybridization F ↓ [1] for mature seed behind the seed maturity; Step 4, multiplication technique: one, seedling obtains: the hybridization F that will collect ↓ [1] is inoculated in hybridization F ↓ [1] after the sterilization in the 1/2MS medium for mature seed cultivation tuber of pinellia F1 seedling under certain condition of culture for the mature seed sterilization that carries out disinfection; Two, seedling and propagating grows directly from seeds: the tuber of pinellia F1 seedling of choosing the health that obtains through seed culture, cut petiole base, petiole top, blade, the stem tuber of tuber of pinellia F1 seedling respectively, petiole segment after cutting, blade fritter and stem tuber fritter are inoculated into MS+2, evoked callus in the 4-D0.5mg/L+6-BA1.0mg/L solid culture medium; Three, orbicule propagation: choose quality, the callus that differentiates orbicule of color and luster unanimity is inoculated into that increment produces tuber of pinellia orbicule in the MS liquid nutrient medium of improvement; Four, F ↓ [1] is for plant regeneration: will rise in value produces tuber of pinellia orbicule and is inoculated in the MS+NAA0.1mg/L+6-BA1.0mg/L differential medium, induces it to take root and is differentiated to form complete F ↓ [1] for plant, can carry out acclimatization and transplants; Step 5, tuber of pinellia F ↓ [1] is for variety comparative test: respectively the different parents of the tuber of pinellia and F ↓ [1] the cauline leaf form for plant is compared, adopt spectrophotometer method to measure each F1 for the pigment content in the blade, and its photoresponse curve, CO ↓ [2] response curve, chlorophyll fluorescence parameters measured comparison, comprehensive relatively each F ↓ [1] is for photosynthesis characteristics, detect the content of F ↓ [1], determine all excellent hybridization F of photosynthesis characteristics and active component ↓ [1] generation for guanosine in the tuber of pinellia and total free organic acids.

Summary of the invention

The objective of the invention is: at the tuber of pinellia lower problem of transplanting survival rate in the forming seedling through one step culture process, we have carried out further improvement to the method for tuber of pinellia group training, propose tuber of pinellia group training one and go on foot kind of a method.

Tuber of pinellia group training one goes on foot the technical scheme of kind of method to be: earlier with tuber of pinellia group training forming seedling through one step culture, medium is MS+NAA 0.1mg/L+6-BA 0.5mg/L+ sucrose 10～50g/L+ agar 5～10g/L, at pH value 5～6, temperature is 25 ± 2 ℃, periodicity of illumination is 12h/d, intensity of illumination is to cultivate under the condition of 1500～2500Lx, obtain can acclimatization and transplants tissue cultivating seedling.

Do not carry out acclimatization and transplants in this link, continue to allow its growth, MS+NAA 0.1mg/L+6-BA 0.5mg/L+ sucrose 10～50g/L liquid nutrient medium if oligotrophy can be annotated on medium, make the tuber of pinellia in blake bottle, finish a growth cycle, that treats is grown to serve as tuber of pinellia tubercle, and last bottle outlet, wash-out medium are transplanted.

Effect of the present invention: this method has reduced the step of hardening and the problem of having avoided transplanting survival rate, has reduced the tuber of pinellia simultaneously and suffer the possibility of virus infection once more in the production process of generation kind.

This method has been taken into account all advantages of forming seedling through one step culture method simultaneously, and the stem tuber number that breaks up on tuber of pinellia plant induction rate, differentiation rate, each explant all shows better.

Description of drawings

Fig. 1 is a process chart of the present invention.

Embodiment

The present invention is described further below in conjunction with embodiment,

Tuber of pinellia group training one goes on foot the technological process of kind of method:

In conjunction with Fig. 1, in this technology, improved the approach that the increment of tuber of pinellia group training subculture is cultivated, cultivate again from the blade petiole and the stem tuber that become seedling, reason is in traditional tissue culture shoot proliferation, and repeatedly switching causes the variation of material, makes it normally to form complete plant.By this improvement, the stem tuber number that breaks up on plant differentiation rate, each explant still becomes all be better than the former evening morning of seedling time, and variation does not produce simultaneously, can fully keep all hereditary capacities of the tuber of pinellia.

Claims (1)

1. a tuber of pinellia tissue culture one goes on foot kind of a method, it is characterized in that, earlier with tuber of pinellia group training forming seedling through one step culture, medium is MS+NAA 0.1mg/L+6-BA 0.5mg/L+ sucrose 10～50g/L+ agar 5～10g/L, and at pH value 5～6, temperature is 25 ± 2 ℃, periodicity of illumination is 12h/d, intensity of illumination is to cultivate under the condition of 1500～2500Lx, obtain can acclimatization and transplants tissue cultivating seedling

Do not carry out acclimatization and transplants in this link, continue to allow its growth, MS+NAA 0.1mg/L+6-BA 0.5mg/L+ sucrose 10～50g/L liquid nutrient medium if oligotrophy can be annotated on medium, make the tuber of pinellia in blake bottle, finish a growth cycle, that treats is grown to serve as tuber of pinellia tubercle, and last bottle outlet, wash-out medium are transplanted.

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