CN104488710B - A kind of method of Calotropis gigantea tissue cultures - Google Patents

Abstract

The present invention relates to a kind of method of Calotropis gigantea tissue cultures, platymiscium field of tissue culture, comprise the steps: medium preparation, explant choose with sterilize, seed germination, adventitious bud inducing and propagation, root induction, plantlet in vitro tame and transplant.Does is minimal medium MS medium, sucrose 25 ~ 30g/L, agar powder 8 ~ 9g/L, PVP 0.1 ~ 0.5g/L, medium pH is 5.6 ~ 5.8; Does is seed germination medium MS or 1/2 MS; Does is adventitious bud induction culture base MS+6-BA 3.0 ~ 5.0mg/L+NAA 0.05 ~ 0.2mg/L+PVP 0.1 ~ 0.5g/L; Does is adventitious bud proliferation medium MS+6-BA 1.0 ~ 3.0mg/L+NAA 0.05 ~ 0.2mg/L+PVP 0.1 ~ 0.5g/L; Does is root media MS+IBA 0.01 ~ 0.5mg/L+PVP 0.1 ~ 0.5g/L or 1/2 MS+IBA 0.01 ~ 0.5mg/L+PVP 0.1 ~ 0.5g/L.Condition of culture is 25 ± 2 DEG C, 12 ~ 16h/d, 40 ~ 60 μm of olm ^-2s ^-1.Advantage of the present invention: the Calotropis gigantea group culturation rapid propagating technology system sapling multiplication speed of foundation is fast, evenly healthy and strong, and variation is few, and transplanting survival rate is high, is applicable to the large-scale production of excellent Calotropis gigantea kind seedling.

Description

A kind of method of Calotropis gigantea tissue cultures

Technical field

The present invention relates to plant tissue culture technique, be specifically related to a kind of method of Calotropis gigantea tissue cultures.

Background technology

Calotropis gigantea (Calotropisgigantea (Linn.) Dry.exAit.f), another name gelsemium elegan, sheep leaching tree, asthma tree, imperial crown flower etc., for Asclepiadaceae Calotropis herbaceos perennial, in Asia, all there is distribution in America, Africa, and China is mainly distributed in the provinces and regions such as Guangdong and Guangxi Provinces, Hainan, Fujian, Yunnan, Sichuan.Calotropis gigantea is drought-enduring, impoverishment tolerant ability is strong, often be born in arid or ground, semiarid wilderness, can be used as arid tract wasteland, poor and barren land greening pionner, and growth rapidly, grow weekly the most about 30 centimetres, plant up to 3m, can play windproof, soil conservation, water conservation effect, can be used as the pionner of barren land improvement.

Calotropis gigantea whole body is precious, has multiple value and wide market prospects.No matter always tender the whole plant of Calotropis gigantea is all can be used as fertilizer; Akund can be used for producing medium-to-high grade knit product, paper and synthetic cotton etc., and planting hair for making velvet raw material, can have broad application prospects at field of textiles; Containing a large amount of hydrocarbons and some hydrocarbons in the emulsion of Calotropis gigantea, close with natural crude oil composition, the Calotropis gigantea of 1 hectare can extract the crude oil of liter more than 10,000, also can be used for extracting biological pesticide preparation, can be made for weld, gummy raw material and tanning material etc. after emulsion drying; In addition Calotropis gigantea also has multiple medical value, the effects such as its cured leaf tool Eradicates phlegm, cough-relieving; The effects such as milk tool anti-inflammatory, anticoagulation, cardiac stimulant and expelling parasite, bark also can be used for treating favus.

At present, the conventional seed of Calotropis gigantea cultivation is bred, but seed amount is limited, and simultaneously due to growing environment restriction, under nature, seed germination rate is lower, cannot obtain a large amount of high quality seedling simultaneously.Plant Tissue Breeding is the method for the most effective plant seedling Fast-propagation at present, at present about the domestic rarely seen Li Kelie of Calotropis gigantea Study on tissue culture report etc. 1 section, it mainly utilizes axenic germination seedling leaf for starting material, obtains Calotropis gigantea regeneration plant by the first callus induction mode that callus induction breaks up again; External rarely seen AshisT.Roy etc. utilize cotyledon, young leaflet tablet and stem section to be starting material, utilizing liquid suspension culture technology to carry out expansion to callus after inducing callus numerous, then carrying out induction thus the research report obtaining Calotropis gigantea regeneration plant to expanding numerous callus.In plant tissue culture process, Progenies from Regenerated variation is obtained higher by callus approach, offspring's quality discrepancy is larger, with respect to the Regeneration Ways that callus induction obtains, variation that regeneration plant can cause in greatly reduction group training process occurs to utilize direct adventitious organogenesis to obtain, but there is not been reported to occur to obtain regeneration plant about Calotropis gigantea by direct organ.

Summary of the invention

The object of the invention is to the deficiency overcoming prior art existence, and a kind of method of Calotropis gigantea tissue cultures is provided.The sterilizable material that quasi-solution Calotropis gigantea of the present invention utilizes seed asepsis sprouting to obtain, sets up good Calotropis gigantea tissue culture regeneration system by direct evoking adventive bud approach, expands numerous, rearing new variety and genetic improvement etc. carry out technical support for the excellent plantation of Calotropis gigantea.

The object of the invention is to have come by following technical solution.The method of this Calotropis gigantea tissue cultures, the method comprises the steps:

1), the choosing and processing of explant: choose Calotropis gigantea seed as starting material, from Calotropis gigantea fruit, seed is taken out, after removing the surface of the seed fiber after seed soaking 2h, select the seed sinking to container bottom as group training explant;

2), seed germination: on superclean bench, Calotropis gigantea seed through surface sterilization is transferred in aseptic inoculation dish, explant surface moisture is sucked with aseptic filter paper, be inoculated in seed germination medium MS or 1/2MS medium and carry out seed germination, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

3) adventitious bud inducing: on aseptic working platform, the aseptic seedling obtained by seed germination every two buds below axillalry bud are a unit segment, stem with bud is inoculated in the induction that adventitious bud induction culture base carries out indefinite bud, adventitious bud induction culture base is in MS+6-BA3.0 ~ 5.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L, carry out the induction of indefinite bud, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

4) adventitious bud proliferation: on aseptic working platform, indefinite bud of growing thickly high for 1 ~ 2cm of induction acquisition is one from base portion with 2 ~ 3 indefinite buds connected together and inoculates unit separation, parting material is inoculated in adventitious bud proliferation medium and breeds, adventitious bud proliferation medium is MS+6-BA1.0 ~ 3.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

5), adventitious bud rooting induction: by Seedling height to the indefinite bud of growing thickly of 3 ~ 4cm from the single separation of base portion, the induction of adventive root is carried out in access root media, root media is MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L or 1/2MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

6), plantlet in vitro domestication and transplanting: after indefinite bud base portion of growing thickly grows the adventive root of 4 ~ 62 ~ 3cm, seedling of taking root moves to greenhouse, scattered light is opened bottle cap again and is cultivated 1 ~ 2d after cultivating 3 ~ 5d, Calotropis gigantea plantlet in vitro is carefully connected from blake bottle root to take out, clean the medium on plantlet in vitro surface with running water, then after plantlet in vitro surface moisture is slightly dried in ventilating and cooling place by Calotropis gigantea plantlet in vitro, plant in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse;

Seed germination, adventitious bud inducing and propagation, adventitious bud rooting induction minimal medium are MS medium, dosage of sucrose is 25 ~ 40g/L, and coagulating agent is agar powder, and consumption is 8 ~ 9g/L, PVP consumption is 0.1 ~ 0.5g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing; Seed germination medium is MS or 1/2MS medium; Adventitious bud induction culture base is MS+6-BA3.0 ~ 5.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L; Adventitious bud proliferation medium is MS+6-BA1.0 ~ 3.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L; Root media is MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L or 1/2MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L; Seed germination, adventitious bud inducing and propagation, adventitious bud rooting condition of culture are that cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

Before the selection of initial explant and process, seed germination, adventitious bud inducing, adventitious bud proliferation, root induction, transplanting domestication process and after transplanting moisturizing more than 90% cultivate 15d.

Beneficial effect of the present invention is:

1, Calotropis gigantea quick reproduction technique is carried out by tissue culture technique, produce by the restriction of area, season, weather and maternal plant growth year, be convenient to factorial seedling growth, can produce according to order, production time and scale controlled, for applying of Calotropis gigantea provides sufficient high quality seedling guarantee.

2, reproduction coefficient reaches 3.0 ~ 3.5, rooting rate 100%, transplanting survival rate 100%, reaches the requirement that seedling factorial seedling growth is produced.

3, the present invention adopts the Reproduction methods of the direct evoking adventive bud of Calotropis gigantea axillalry bud, and without more organizing wound induction and callus induction link, greatly can reduce the generation of progeny variation in plant tissue culture process, seedling can keep maternal merit to greatest extent.

4, be conducive to Calotropis gigantea fine individual plant, new varieties tissue-culturing rapid propagation Establishing is used for reference, promote.

5, set up good tissue-culturing rapid propagation system, for utilizing Plant Biotechnology from now on, the research work such as rearing new variety, genetic improvement is carried out to Calotropis gigantea and lay the foundation.

Embodiment

Below by embodiment, the present invention is further elaborated, and help is understood the present invention by embodiment better, but the present invention is not limited only to following embodiment.

The method of this Calotropis gigantea tissue cultures, the method comprises the steps:

1, medium and condition of culture: seed germination, adventitious bud inducing, adventitious bud proliferation propagation, adventitious bud rooting induction minimal medium are MS medium, dosage of sucrose is 25 ~ 30g/L, coagulating agent is agar powder, consumption is 8 ~ 9g/L, PVP consumption is 0.1 ~ 0.5g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing; Seed germination medium is MS or 1/2MS medium; Adventitious bud induction culture base is MS+6-BA3.0 ~ 5.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L; Adventitious bud proliferation medium is MS+6-BA1.0 ~ 3.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L; Root media is MS+IBA0 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L or 1/2MS+IBA0 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L; Seed germination, adventitious bud inducing and propagation, adventitious bud rooting condition of culture are that cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

2, the process of explant and sterilization: choose Calotropis gigantea seed as starting material, by the Calotropis gigantea seed removing kind of hair collected after water seed soaking 2h, select the seed being deposited on container bottom as explant, the explant liquid detergent solution cleaning 20min of select, period constantly stirs gently, then running water is through the residual liquid detergent cleaning solution of clean the surface of the seed, on the superclean bench of sterilization, the Calotropis gigantea seed cleaned up is being transferred in aseptic conical flask, with 70 ~ 75% alcohol-pickled 60s, period constantly rocks conical flask gently, removal is attached to Calotropis gigantea the surface of the seed bubble, pour out 75% alcohol, use 0.1%HgCl again ₂solution soaks seed 6 ~ 10min, or is that 1%NaClO solution soaks seed 20 ~ 30min with effective chlorine density, constantly rocks conical flask gently, remove and be attached to Calotropis gigantea the surface of the seed bubble, pour out 0.1%HgCl between soak period ₂solution or 1%NaClO solution, with the seed 4 ~ 5 time of sterile water wash through surface sterilization process, a small amount of sterile water of the seed after process soaks, for subsequent use.

3, seed germination: on superclean bench, Calotropis gigantea seed through surface sterilization is transferred in aseptic inoculation dish, the surface of the seed moisture is blotted with aseptic filter paper, be inoculated in seed germination medium MS or 1/2MS medium and carry out seed germination, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

4, adventitious bud inducing: seed being sprouted the high aseptic seedling of 2 ~ 3cm of acquisition on aseptic working platform is 1 unit with each armpit (top) bud, be cut into the segment of about 1cm, be inoculated in adventitious bud induction culture base MS+6-BA3.0 ~ 5.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L, carry out the induction of indefinite bud, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

5, adventitious bud proliferation: on aseptic working platform, induction is obtained cluster, indefinite bud that 1 ~ 2cm is high from base portion with 2 ~ 3 disjunctor indefinite buds for inoculation unit is separated, be inoculated in adventitious bud proliferation medium and breed, adventitious bud proliferation medium is MS+6-BA1.0 ~ 3.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

6, adventitious bud rooting induction: on aseptic working platform, Seedling height is cut into single to the indefinite bud of growing thickly of 3 ~ 4cm from base portion, the induction of adventive root is carried out in access root media, root media is MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L or 1/2MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

7, plantlet in vitro domestication and transplanting: after indefinite bud base portion grows the adventive root of 4 ~ 62 ~ 3cm, seedling of taking root moves to greenhouse, scattered light opens bottle cap after cultivating 5d, cultivate 1 ~ 2d again, the plant that taken root by Calotropis gigantea is carefully taken out from blake bottle, cleans the medium on plantlet in vitro surface with running water, by Calotropis gigantea plantlet in vitro, plant in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse.

Claims (1)

1. a method for Calotropis gigantea tissue cultures, is characterized in that: the method comprises the steps:

1), the choosing and processing of explant: choose Calotropis gigantea seed as starting material, from Calotropis gigantea fruit, seed is taken out, after removing the surface of the seed fiber after seed soaking 2h, select the seed sinking to container bottom as group training explant;

2), seed germination: on superclean bench, Calotropis gigantea seed through surface sterilization is transferred in aseptic inoculation dish, explant surface moisture is sucked with aseptic filter paper, be inoculated in seed germination medium MS or 1/2MS medium and carry out seed germination, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

3), adventitious bud inducing: on aseptic working platform, the aseptic seedling obtained by seed germination every two buds below axillalry bud are a unit segment, stem with bud is inoculated in the induction that adventitious bud induction culture base carries out indefinite bud, adventitious bud induction culture base is in MS+6-BA3.0 ~ 5.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L, carry out the induction of indefinite bud, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

4), adventitious bud proliferation: on aseptic working platform, indefinite bud of growing thickly high for 1 ~ 2cm of induction acquisition is one from base portion with 2 ~ 3 indefinite buds connected together and inoculates unit separation, parting material is inoculated in adventitious bud proliferation medium and breeds, adventitious bud proliferation medium is MS+6-BA1.0 ~ 3.0mg/L+NAA0.05 ~ 0.2mg/L+PVP0.1 ~ 0.5g/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

5), adventitious bud rooting induction: by Seedling height to the indefinite bud of growing thickly of 3 ~ 4cm from the single separation of base portion, the induction of adventive root is carried out in access root media, root media is MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L or 1/2MS+IBA0.01 ~ 0.5mg/L+PVP0.1 ~ 0.5g/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

6), plantlet in vitro domestication and transplanting: after indefinite bud base portion of growing thickly grows the adventive root of 4 ~ 62 ~ 3cm, seedling of taking root moves to greenhouse, scattered light is opened bottle cap again and is cultivated 1 ~ 2d after cultivating 3 ~ 5d, Calotropis gigantea plantlet in vitro is carefully connected from blake bottle root to take out, clean the medium on plantlet in vitro surface with running water, then after plantlet in vitro surface moisture is slightly dried in ventilating and cooling place by Calotropis gigantea plantlet in vitro, plant in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse;

Seed germination, adventitious bud inducing and propagation, adventitious bud rooting induction minimal medium are MS medium, and dosage of sucrose is 25 ~ 40g/L, and coagulating agent is agar powder, and consumption is 8 ~ 9g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing.