CN102144550A - 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 the perennial perennial root herbaceous plant of Liliaceae hemerocallis, 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 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 bigger, and pattern is eye-catching orange-yellow, and blooming at the beginning of 6 months lasts till the first tenday period of a month in October, florescence reaches 4 months, the strong management of adaptability is more extensive, and " Yu Yihuang " is evergreen in the Jiangsu and Zhejiang Provinces area four seasons simultaneously, and is 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 design of flower border and the artistic flower arrangement, has the wide development prospect." Yu Yihuang " kind nature ripening rate is low, and the seed germination rate is low, adopts vegetative propagation modes such as plant division at present, and reproduction rate is low, and a general year growth coefficient is 4-5, is difficult to satisfy the demand of market businessization.Tissue culture technique is the effective way of a large amount of elite germplasms of breeding fast, also is the important foundation that plant carries out genetic improvement simultaneously; Elementary as Guo Da in nineteen ninety, just the scape with the hemerocailis middendorffi Hybrid is an 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 to take root seedling.Wang Hanhai etc. also are explant with the stem apex, successfully obtain hemerocailis middendorffi " golden doll " kind tissue cultivating seedling.But the group training research to " Yu Yihuang " does not appear in the newspapers as yet; therefore explore " Yu Yihuang " tissue culture technology, realize its scale breeding, not only can enrich the garden plants kind; meet the need of market, also the tissue culture technology research to other plant of Liliaceae has important reference meanings.
Summary of the invention
Technical problem to be solved by this invention provides the tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud, and this 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 the technical scheme that is adopted: the tissue culture method of a kind of hemerocailis middendorffi " Yu Yihuang " bud comprises the steps:
A, adopt down fine noon and to form a week with interior hemerocailis middendorffi " Yu Yihuang " bud, soak 1h with liquid detergent earlier, the back is washed 4～5 times with flowing water, is 75% alcohol immersion 30 seconds with mass percentage concentration again, is 0.1% HgCl then with mass percentage concentration
2Solution soaking 10 minutes, aseptic water washing 5～6 times obtains aseptic bud;
Ovary is exposed at the top of b, the above-mentioned aseptic bud of excision, half-and-half cut then, be inoculated on the inducing culture that contains 6-benzyl purine (6-BA), methyl (NAA) and sucrose, inducing culture 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 methyl concentration 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 and sucrose, differentiation culture to indefinite bud forms, obtain " Yu Yihuang " tissue cultivating seedling, in this differential medium, the concentration of described 6-benzyl purine is 1.5mg/L, described methyl concentration is 0.3 mg/L, and the mass percentage concentration of described sucrose is 3.0%;
The cultivation temperature of the differentiation culture of the inducing culture of d, above-mentioned steps b and step c all is 24～26 ℃, and illumination all is 2000 Lux, and every day, light application time all was 16 h.
The basic culture solution of above-mentioned inducing culture and differentiation culture 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 soaking obtains aseptic bud, and sterilization effect is better, and follow-up cultivation is of reduced contamination, and the bud lethality is lower, forms callus with the inducing culture inducing culture again, obtains the callus bud; Be cultured to indefinite bud with differential medium then and form, obtain " Yu Yihuang " tissue cultivating seedling; Callus and indefinite bud breeding graded, adventitious bud proliferation is very fast, and quality is better, a hemerocailis middendorffi " Yu Yihuang " bud can breed in 1 year 20000 young plants can with the tissue cultivating seedling of taking root, indefinite bud can be bred once in per 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 " is formed carefully to cut with interior bud in a week and take back, soak 1h with liquid detergent, the flowing water flushing is 4～5 times again, moving to superclean bench after branch installs and adopted mass concentration 75% alcohol immersion 30 seconds, is 0.1% HgCl then with mass percentage concentration
2Solution soaking 10 minutes, aseptic water washing 5～6 times obtains aseptic bud, and it is preferable to handle sterilization effect like this, and lethality is also lower; With the explant of aseptic bud as evoked callus, ovary is exposed at the top of excision bud, half-and-half cut then, be inoculated on the inducing culture that contains 6-BA, NAA and sucrose, in cultivation temperature is 24～26 ℃, illumination is 2000 Lux, every day, light application time was an inducing culture 55～66 days under the 16 h conditions, formed callus more than 70%, obtain the callus bud, in this inducing culture, the concentration of 6-benzyl purine is 5.0 mg/L, methyl concentration 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 is 24～26 ℃, illumination is 2000 Lux, every day, light application time was that differentiation culture to indefinite bud forms under the 16 h conditions, obtain " Yu Yihuang " tissue cultivating seedling, in this differential medium, the concentration of 6-benzyl purine is 1.5mg/L, methyl concentration 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, move in circles, as long as obtain indefinite bud, every 1 month propagation once, tissue cultivating seedling 20000 strains that an average bud can obtain to take root in a year.
Design and experiment according to us draw, and under certain condition of mercuric chloride processing time, death rate of explants increases with processing time of ethanol and raises, 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 handled along with mercuric chloride under the situation of Ethanol Treatment time unanimity increases and reduces, but lethality also rises synchronously; Therefore we choose with 75% alcohol immersion 30 seconds and 0.1% HgCl in conjunction with sterilizing efficiency and lethality
210 minutes sterilization method of solution soaking, suitable to hemerocailis middendorffi " Yu Yihuang " bud.
Earlier, can make bud explant callusization better with the NAA of higher concentration and inducing of 6-BA, and only occur expanding can not callus phenomenon less; Use the differentiation of the NAA and the 6-BA of low concentration again, callus better differentiates indefinite bud, and the blade of indefinite bud can not curl even deformity, and the generation of 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 down fine noon and to form a week with interior hemerocailis middendorffi " Yu Yihuang " bud, soak 1h with liquid detergent earlier, the back is washed 4～5 times with flowing water, is 75% alcohol immersion 30 seconds with mass percentage concentration again, is 0.1% HgCl then with mass percentage concentration
2Solution soaking 10 minutes, aseptic water washing 5～6 times obtains aseptic bud;
Ovary is exposed at the top of b, the above-mentioned aseptic bud of excision, half-and-half cut then, be inoculated on the inducing culture that contains 6-benzyl purine, methyl and sucrose, inducing culture 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 methyl concentration 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 and sucrose, differentiation culture to indefinite bud forms, obtain " Yu Yihuang " tissue cultivating seedling, in this differential medium, the concentration of described 6-benzyl purine is 1.5mg/L, described methyl concentration is 0.3 mg/L, and the mass percentage concentration of described sucrose is 3.0%;
The cultivation temperature of the inducing culture of the inducing culture of d, above-mentioned steps b and step c all is 24～26 ℃, and illumination all is 2000 Lux, and every day, light application time all was 16 h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461135A (en) * | 2013-09-18 | 2013-12-25 | 宁波市农业科学研究院 | Method for propagating hemerocallis hybridus |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869062A (en) * | 2009-04-24 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of Hemerocallis dumortieri |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869062A (en) * | 2009-04-24 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of Hemerocallis dumortieri |
Non-Patent Citations (1)
| Title |
|---|
| 孔刚等: "大花萱草的组织培养", 《国土与自然资源研究》, no. 03, 20 September 2001 (2001-09-20) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461135A (en) * | 2013-09-18 | 2013-12-25 | 宁波市农业科学研究院 | Method for propagating hemerocallis hybridus |
| CN103461135B (en) * | 2013-09-18 | 2015-10-07 | 宁波市农业科学研究院 | A kind of propagation method of hemerocailis middendorffi |
| 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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