CN102144550B - Tissue culture method for flower buds of hemerocallis middendorfii 'prunus lannesiana wils' - Google Patents
Tissue culture method for flower buds of hemerocallis middendorfii 'prunus lannesiana wils' Download PDFInfo
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Abstract
The invention discloses a tissue culture method for the flower buds of the hemerocallis middendorfii 'prunus lannesiana wils', which comprises the steps of: soaking the flower buds by cleanser essence, 75% alcohol and a 0.1% HgCl2 solution sequentially to obtain the sterile flower buds, wherein the sterilization effect is better, the follow-up culture pollution is less, and the death rate of the flower buds is lower; culturing in an inducing way by an inducing culture medium to form into the callus so as to obtain the callus flower buds; and culturing by a differential culture medium to form into the adventitious buds so as to obtain the tissue culture seedlings of the 'prunus lannesiana wils'. The method adopts the hierarchical culture of the callus buds and the adventitious buds, wherein the adventitious buds are faster in propagation and better in quality, one flower bud of the hemerocallis middendorfii 'prunus lannesiana wils' can propagate 20 thousands available root growing tissue culture seedlings, the adventitious buds can be propagated at a time every one moth, and the propagation coefficient is always kept at 4.2-5.2, so that the tissue culture method is short in culture period, and lays a foundation for the high-propagation rate factorial production of the hemerocallis middendorfii 'prunus lannesiana wils' on a large scale.
Description
Technical field
The present invention relates to hemerocailis middendorffi tissue cultivating technology, be specifically related to the tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud.
Background technology
Hemerocailis middendorffi (Hemerocallis middendorfii) is Liliaceae hemerocallis perennial root herbaceous plant, and color is magnificent, and leaf is like fragrant thoroughwort, and flower is as lily, and the florescence is longer.The hemerocailis middendorffi designs and varieties are various, and the colors such as yellowish, golden yellow, light yellow, pale red, bright red, dark red are arranged, and the alternate secondary color alternate with reddish yellow of pink colour more arranged.In recent years, China meets the need of market from external introduction hemerocailis middendorffi new varieties successively, hemerocailis middendorffi wherein " Yu Yihuang " is the new varieties of just introducing, flower shape is larger, and pattern is eye-catching orange-yellow, at the beginning of 6 months, blooms and lasts till the first tenday period of a month in October, florescence reaches 4 months, the strong adaptability management is more extensive, and " Yu Yihuang " is evergreen in the Jiangsu and Zhejiang Provinces area four seasons simultaneously, rather rare in hemerocailis middendorffi; It is not only outstanding ground cover plant, but also can be applicable to courtyard greening, and potted plant viewing and admiring, in the design of flower border and artistic flower arrangement, has wide DEVELOPMENT PROSPECT." Yu Yihuang " kind Natural seed setting rate is low, and seed germination rate is low, adopts at present the vegetative propagation modes such as plant division, and reproduction rate is low, and a general year growth coefficient is 4-5, is difficult to the business-like demand of satisfying the market.Tissue culture technique is the effective way of a large amount of Fast-propagation elite germplasms, is also the important foundation that plant carries out genetic improvement simultaneously; As elementary as Guo Da in nineteen ninety, the scape of hemerocailis middendorffi Hybrid of just take is explant, with MS+IBA0.4mgL-1+BA4.0mgL-1 evoked callus, and under different exogenous hormones the propagation situation of indefinite bud relatively, obtain best proliferated culture medium MS+NAA1.0mgL-1+BA5.0mgL-1, successfully obtain the seedling of taking root.Wang Hanhai etc. also be take stem apex as explant, successfully obtain hemerocailis middendorffi " golden doll " kind group training seedling.But the group training research to " Yu Yihuang " there is not yet report; therefore explore " Yu Yihuang " tissue culture technology, realize its biological control, not only can enrich the garden plants kind; meet the need of market, also the tissue culture technology research of other plant of Liliaceae had to important reference.
Summary of the invention
Technical problem to be solved by this invention is to provide the tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud, and the method has that sterilization effect is better, and lethality is lower, and the short and quality of indefinite bud cultivation period is advantage preferably.
The present invention solves the problems of the technologies described above adopted technical scheme: the tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud comprises the steps:
A, in fine day, adopt lower formation one week noon with interior hemerocailis middendorffi " Yu Yihuang " bud, first with liquid detergent, soak 1h, rinse 4～5 times with flowing water afterwards, then the alcohol immersion that is 75% by mass percentage concentration 30 seconds, the HgCl that is then 0.1% by mass percentage concentration
2solution soaks 10 minutes, and aseptic water washing 5～6 times, obtain aseptic bud;
Ovary is exposed at b, the top of excising above-mentioned aseptic bud, then half-and-half cut, be inoculated on the inducing culture that contains 6-benzyl purine (6-BA), methyl α-naphthyl acetate (NAA) and sucrose, induce and cultivate 55～66 days, callus forms, and obtains the callus bud, in this inducing culture, the concentration of described 6-benzyl purine is 5.0 mg/L, and described concentration of NAA is 5.0 mg/L, and the mass percentage concentration of described sucrose is 3.0%;
C, above-mentioned callus bud is inoculated on the differential medium that contains 6-benzyl purine, methyl α-naphthyl acetate and sucrose, differentiation is cultured to adventitious bud formation, obtain " Yu Yihuang " group training seedling, in this differential medium, the concentration of described 6-benzyl purine is 1.5mg/L, described concentration of NAA is 0.3 mg/L, and the mass percentage concentration of described sucrose is 3.0%;
The cultivation temperature that the differentiation of inducing cultivation and step c of d, above-mentioned steps b is cultivated is all 24～26 ℃, and illumination is all 2000 Lux, and every day, light application time was all 16 h.
The basic culture solution that above-mentioned inducing culture and differentiation are cultivated is MS(Murashige and Skoog).
Compared with prior art, the invention has the advantages that the tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud, bud is successively by liquid detergent, 75% ethanol and 0.1% HgCl
2solution soaks and obtains aseptic bud, and sterilization effect is better, and follow-up cultivation is of reduced contamination, and the bud lethality is lower, then induces with inducing culture and cultivate the formation callus, obtains the callus bud; Then be cultured to adventitious bud formation with differential medium, obtain " Yu Yihuang " group training seedling; Callus and indefinite bud breeding graded, adventitious bud proliferation is very fast, and quality is better, a hemerocailis middendorffi " Yu Yihuang " bud in 1 year, can breed 20,000 young plants can with the group training seedling of taking root, indefinite bud can be bred once in every 1 month, and growth coefficient remains between 4.2～5.2, so cultivation period is short, extensive for realizing hemerocailis middendorffi " Yu Yihuang ", the batch production production of high reproductive rate lays the foundation.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
The tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud, at the noon of fine day, with scissors, hemerocailis middendorffi " Yu Yihuang " being formed to one week carefully cuts and takes back with interior bud, soak 1h with liquid detergent, flowing water rinses 4～5 times again, move to superclean bench after minute installing and adopt mass concentration 75% alcohol immersion 30 seconds, the HgCl that is then 0.1% by mass percentage concentration
2solution soaks 10 minutes, and aseptic water washing 5～6 times, obtain aseptic bud, processes like this sterilization effect better, and lethality is also lower; Explant using aseptic bud as evoked callus, ovary is exposed at the top of excision bud, then half-and-half cut, be inoculated on the inducing culture that contains 6-BA, NAA and sucrose, in cultivation temperature, it is 24～26 ℃, illumination is 2000 Lux, every day, light application time was under 16 h conditions, to induce to cultivate 55～66 days, formed callus more than 70%, obtain the callus bud, in this inducing culture, the concentration of 6-benzyl purine is 5.0 mg/L, concentration of NAA is 5.0 mg/L, and the mass percentage concentration of sucrose is 3.0%; The callus bud that forms callus is inoculated on the differential medium that contains 6-BA, NAA and sucrose, in cultivation temperature, it is 24～26 ℃, illumination is 2000 Lux, every day, light application time was that under 16 h conditions, differentiation is cultured to adventitious bud formation, obtain " Yu Yihuang " group training seedling, in this differential medium, the concentration of 6-benzyl purine is 1.5mg/L, concentration of NAA is 0.3 mg/L, and the mass percentage concentration of sucrose is 3.0%; The induction duration of above callus is 2 months, and the formation cycle is 1 month, and be 1 month proliferating cycle, moves in circles, as long as obtain indefinite bud, every 1 month propagation once, an average bud can obtain group training seedling 20000 strains of taking root in 1 year.
According to our design and experiment, draw, under certain condition of mercuric chloride processing time, the lethality of explant increased and raises with processing time of ethanol, and the pollution rate of explant also reduces with the processing time increase of mercuric chloride and ethanol simultaneously; The time that the pollution rate of explant is processed along with mercuric chloride in the situation of Ethanol Treatment time consistency increases and reduces, but lethality also synchronously rises; Therefore in conjunction with sterilizing efficiency and lethality, we choose with 75% alcohol immersion 30 seconds and 0.1% HgCl
2solution soaks the sterilization method of 10 minutes, suitable to hemerocailis middendorffi " Yu Yihuang " bud.
First, with the NAA of higher concentration and inducing of 6-BA, can make bud explant callusization better, and only occur expanding can not callus phenomenon less; Use the differentiation of NAA and the 6-BA of low concentration, callus better differentiates indefinite bud again, and the blade of indefinite bud can be curling deformity even, and the generation of the phenomenon such as brown stain, so differentiation adventitious buds is better, and better quality.
Claims (1)
1. the tissue culture method of a hemerocailis middendorffi " Yu Yihuang " bud, is characterized in that comprising the steps:
A, adopt the bud of lower hemerocailis middendorffi " Yu Yihuang " noon in fine day, wherein the formation time of bud is in one week, first with liquid detergent, soaks 1h, rear with flowing water flushing 4～5 times, the alcohol immersion that is 75% by mass percentage concentration again 30 seconds, the HgCl that is then 0.1% by mass percentage concentration
2solution soaks 10 minutes, and aseptic water washing 5～6 times, obtain aseptic bud;
Ovary is exposed at b, the top of excising above-mentioned aseptic bud, then half-and-half cut, be inoculated on the inducing culture that contains 6-benzyl purine, methyl α-naphthyl acetate and sucrose, induce and cultivate 55～66 days, callus forms, and obtains the callus bud, in this inducing culture, the concentration of described 6-benzyl purine is 5.0mg/L, and described concentration of NAA is 5.0mg/L, and the mass percentage concentration of described sucrose is 3.0%;
C, above-mentioned callus bud is inoculated on the differential medium that contains 6-benzyl purine, methyl α-naphthyl acetate and sucrose, differentiation is cultured to adventitious bud formation, obtain " Yu Yihuang " group training seedling, in this differential medium, the concentration of described 6-benzyl purine is 1.5mg/L, described concentration of NAA is 0.3mg/L, and the mass percentage concentration of described sucrose is 3.0%;
The cultivation temperature that the differentiation of inducing cultivation and step c of d, above-mentioned steps b is cultivated is all 24～26 ℃, and illumination is all 2000Lux, and every day, light application time was all 16h.
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| CN112167048A (en) * | 2020-08-13 | 2021-01-05 | 云南吉成园林科技股份有限公司 | Breeding and breeding method for hemerocallis fulva |
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