CN105123517A - Culture medium for converting Chinese herbaceous peony blades into buds directly through tissue culture and culture method - Google Patents

Abstract

The invention belongs to the field of tissue culture of plants, and particularly relates to a culture medium for converting Chinese herbaceous peony blades into buds directly through tissue culture and a culture method. The method comprises the steps of: selecting young tender Chinese herbaceous peony blades in late March; and after sterilization, inoculating the selected young tender Chinese herbaceous peony blades on the culture medium at the temperature of 25+/-2 DEG C, and performing dark culture for 2 months to obtain adventitious buds. The culture medium is an improved MS basic culture medium, into which 0.1-3.0mg.L-1 of TDZ, 5-15% of coconut milk, 30g.L-1 of additional cane sugar, 600mg.L-1 of casein hydrolysate and 6.4g.L-1 of agar are added, and the pH of the culture medium is 5.8. Through the adoption of the method, the highest bud ratio of the Chinese herbaceous peony blades can reach 85%, problems can be effectively solved, human resources and material resources are saved, the regeneration time of Chinese herbaceous peonies is shortened, technical support is provided for the rapid propagation of excellent varieties of the Chinese herbaceous peonies, and market prospects are widened.

Description

Chinese herbaceous peony leaf tissue cultivates the medium and cultural method that directly become bud

Technical field

The invention belongs to the field of tissue culture of plant, be specifically related to a kind of Chinese herbaceous peony leaf tissue and cultivate the medium and cultural method that directly become bud.

Background technology

Chinese herbaceous peony (PaeonialactifloraPall.) is traditional famous flower of China, ranks " flower phase ", is only second to " flower king " tree peony, has the cultivation history of more than 4000 year in China.The flower of Chinese herbaceous peony is very large, and pattern is gorgeous, and flower pattern is attractive in appearance, and the fragrance of a flower is strong fragrant, dark liking by broad masses of the people, and some have the kind of novel proterties, and market prospects are particularly wide.As common 3 ~ 5 years, raw Chinese herbaceous peony cultivar price was generally 20 ~ 30 yuan, and comparatively novel kind such as ' gold wheel ' price is about 10 times of common variety, and demand is huge.And it is numerous how fast and efficiently these novel kinds to be carried out expansion, be put a difficult problem in the urgent need to address in face of grower.

The most frequently used propagation method of current Chinese herbaceous peony is division propagation and seed propagation, although the former can keep the proterties of original kind, but need plant strain growth 3 ~ 5 years ability to divide once, and the cycle is longer, reproduction coefficient is lower, the maternal plant ornamental value in next year after plant division obviously declines; The latter can not keep the proterties of original kind, needs again the virgin phase through 4 ~ 5 just can bloom, and therefore, both are all not suitable for commercially producing on a large scale.Plant Tissue Breeding is the one effective method the most of micropropagation of plants, and compared with Traditional breeding processes, tissue cultures can keep original kind and merit, expand its reproduction coefficient, again not by the impact in season, can operate in the anniversary, improve the efficiency of breeding significantly.CN102428872B discloses a kind of method being obtained complete Chinese herbaceous peony plant by Seed embryo culture, but can not keep maternal proterties completely due to embryo, and the expansion that therefore can not be used for improved seeds is numerous.Paper ' Herbaceous peony ' rich and honour greatly ' inducing clumping bud and the rooting technique ' (Wu Hongjuan that the people such as Wu Hongjuan deliver, Shen Miaomiao, Yu Xiaonan. Herbaceous peony ' rich and honour greatly ' inducing clumping bud and rooting technique. Journal of northeast Forestry university, 2011, 39 (9): 20-22.), ' Chinese herbaceous peony different cultivars Primary culture compares and the research of ' cinnabar is sentenced ' inducing clumping bud ' (Wu Hongjuan, Yu Xiaonan. Chinese herbaceous peony different cultivars Primary culture compares and the research of ' cinnabar is sentenced ' inducing clumping bud. Agricultural University of the Inner Mongol's journal, 2010, 31 (4): 29-33.) report with the method for underground sleeping bud for explant induction Multiple Buds, the method is for the numerous existence of expansion 3 deficiencies of improved seeds: the 1. underground sleeping bud limited amount that comprises of a plant, the reproductive efficiency being explant with it is not high, 2. every strain plantlet in vitro can only produce 3 ~ 5 Multiple Buds, and reproduction coefficient is not high, 3. whole process must experience the process of a underground bud Primary culture, and the human and material resources of at substantial, raise the cost.Paper ' inductive technology of Chinese herbaceous peony the indefinite bud ' (Hu Yingquan that the people such as Hu Yingquan deliver; Feng Haihua, Shi Baoling. the inductive technology of Chinese herbaceous peony indefinite bud, Shanxi Forestry science and technology; 2003; supplementary issue: 23-24,33.) tissue cultures is carried out to the tender stem of Chinese herbaceous peony, MS+BA3.5mg/L+ sucrose 30g/L+ agar 6g/L medium a step can become bud; shorten into the bud time; but these paper data are comparatively ambiguous, and the time of statistics also hands over, the inductivity of final bud is the highest only has 74%.The paper that the people such as Wang Jifeng deliver ' 5 Cultivars of Chinese Herbaceous Peony callus induction and differentiation researchs ' (Wang Jifeng, Li Qing, the Meng can .5 Cultivars of Chinese Herbaceous Peony callus induction and differentiation research. Beijing Forestry University's journal, 2011,32 (3): 213-216.) successfully induce callus in, and differentiate indefinite bud, but its callus brownization is serious, vitrifying, frangible, inductivity is low, only has 7.95%.Paper ' research of Chinese herbaceous peony callus induction and the differentiation adventitious buds ' (Sun Xiaomei that the people such as Sun Xiaomei deliver, Cheng Wan, Liu Ping, Zhou Wenqiang, wangdan, Yang Hongguang. the research of Chinese herbaceous peony callus induction and differentiation adventitious buds. Agricultural University Of Shenyang's journal, 2014-02,45 (1): 24-27.) Chinese herbaceous peony cotyledon, stem, blade are cultivated, the differentiation adventitious buds explant being optimum with base portion stem, but differentiation rate only has 35%, and need through the callus of induce stage.In sum, in Chinese herbaceous peony tissue culture procedures, before successfully differentiate indefinite bud per capita, but they are explant mainly with embryo, underground sleeping bud, stem section etc., not only differentiation rate is low, and subculture number many (implementing in two steps), and experimentation is loaded down with trivial details, cycle is longer, can not obtain desirable effect.

Summary of the invention

The present invention, in order to solve the key technical problem in Chinese herbaceous peony group training process, provides and a kind ofly becomes medium and the cultural method of bud with Chinese herbaceous peony blade for explant one step, provide technical support for Chinese herbaceous peony improved seeds are numerous soon.

The Chinese herbaceous peony leaf tissue that the present invention adopts cultivates the medium directly becoming bud, it is characterized in that, improvement MS minimal medium, adds TDZ0.1 ~ 3.0mgL ^-1+ Coconut Juice 5 ~ 15%, additional saccharose 30gL ^-1, caseinhydrolysate 600mgL ^-1, agar 6.4gL ^-1, pH is 5.8.

The invention also discloses a kind of Chinese herbaceous peony leaf tissue and cultivate the cultural method directly becoming bud, it is characterized in that, comprise the following steps: late March chooses the tender Chinese herbaceous peony blade of children; Be inoculated in temperature 25 ± 2 DEG C on medium after sterilizing, light culture 2 months, obtain indefinite bud; Described medium is improvement MS minimal medium, adds TDZ0.1 ~ 3.0mgL ^-1+ Coconut Juice 5 ~ 15%, additional saccharose 30gL ^-1, caseinhydrolysate 600mgL ^-1, agar 6.4gL ^-1, pH is 5.8.

The sterilizing methods that the sterilizing methods that described blade sterilization treatment adopts alcohol to combine with mercuric chloride adopts alcohol to combine with mercuric chloride, concrete steps are as follows:

1. first young leaflet tablet is rinsed 10min in flowing water, remove exterior earth, then clean with liquid detergent, running water is clean;

2. be placed on superclean bench by young leaflet tablet, be positioned in beaker, with 75% alcohol disinfecting 30s, sterile water rinses 5 ~ 6 times repeatedly;

3. again with 0.1% mercuric chloride sterilization 3min, sterile water rinses 5 ~ 6 times repeatedly;

4. explant is placed in suck dry moisture on aseptic filter paper, cuts marginal portion and the master pulse of blade, and the size being cut into 1cm × 1cm is inoculated on inducing culture, for avoiding cross-infection, and every bottle graft kind 3 blades.

Beneficial effect of the present invention is embodied in:

(1) in the present invention, Chinese herbaceous peony blade bud ratio reaches as high as 85%, overcomes the technology barrier of Chinese herbaceous peony blade through Calli Differentiation indefinite bud difficulty.

(2) in the present invention, Chinese herbaceous peony leaf tissue is cultivated and is become bud without the direct step of callus phase, avoids because of repeatedly subculture and the problem of labor intensive, material resources, shortens the time of Chinese herbaceous peony regeneration, decrease the contamination hazard in switching process simultaneously.

(3) blade often opening inoculation in the present invention can form 3-8 indefinite bud, improves reproduction coefficient.

Accompanying drawing explanation

Fig. 1 is the Chinese herbaceous peony blade be inoculated on medium.

Fig. 2 is adventitious bud formation process.

Embodiment

Embodiment 1

With Chinese herbaceous peony holder osmanthus type kind ' purple phoenix feather ' of planting in Yangzhou University's gardening and plant protection institute Chinese herbaceous peony Germplasm Resources for try material to carry out this research work:

(1) draw materials the time: on March 28th, 2014;

(2) selection: choose the blade that children is tender;

(3) sterilizing of blade and inoculation:

After tender leaf is adopted, adopt the sterilizing methods that alcohol combines with mercuric chloride, concrete steps are as follows:

1. first young leaflet tablet is rinsed 10min in flowing water, remove exterior earth, then use liquid detergent (Shanghai Whitecat Shareholding Co., Ltd.) to clean, running water is clean;

2. be placed on superclean bench by young leaflet tablet, be positioned in beaker, with 75% alcohol disinfecting 30s, sterile water rinses 5 ~ 6 times repeatedly;

3. sterilize 3min to use 0.1% mercuric chloride (chemical plant, Chinese and Western, Shanghai) again, and sterile water rinses 5 ~ 6 times repeatedly;

4. explant is placed in suck dry moisture on aseptic filter paper, cuts marginal portion and the master pulse of blade, and the size being cut into 1cm × 1cm is inoculated on inducing culture, and for avoiding cross-infection, every bottle graft kind 3 blades, inoculate 200 bottles altogether.

(4) culture medium prescription and cultural method:

1. culture medium prescription: improvement MS minimal medium, adds TDZ1.0mgL ^-1+ Coconut Juice 5%, additional saccharose 30gL ^-1, hydrolysis network protein 60 0mgL ^-1, agar 6.4gL ^-1, pH is 5.8.TDZ is purchased from Beijing Suo Laibao Science and Technology Ltd., sucrose is purchased from Shanghai Experimental Reagent Co., Ltd., caseinhydrolysate is purchased from Sangon Biotech (Shanghai) Co., Ltd., agar is purchased from Beijing Suo Laibao Science and Technology Ltd., Coconut Juice is purchased from Shanghai Jia Guo Food Co., Ltd.

Improveing MS minimal medium formula in the present invention is:

Macroelement

Potassium nitrate KNO ₃250mg/L

Ammonium nitrate NH ₄nO ₃500mg/L

Potassium dihydrogen phosphate KH ₂pO ₄550mg/L

Magnesium sulfate MgSO ₄7H ₂o250mg/L

Nitrate of lime Ca (NO ₃) ₂4H2O500mg/L

Trace element

Potassium iodide KI0.83mg/L

Boric acid H ₃bO ₃6.2mg/L

Manganese sulphate MnSO ₄4H ₂o22.3mg/L

Zinc sulphate ZnSO ₄7H ₂o8.6mg/L

Sodium molybdate Na ₂moO ₄2H ₂o0.25mg/L

Copper sulphate CuSO ₄5H ₂o0.025mg/L

Cobalt chloride CoCl ₂6H ₂o0.025mg/L

Molysite

Disodium ethylene diamine tetraacetate Na ₂eDTA37.3mg/L

Ferrous sulfate FeSO ₄7H ₂o27.8mg/L

Organic principle

Inositol 100mg/L

Glycine 2mg/L

Thiamine hydrochloride VB ₁0.1mg/L

Puridoxine hydrochloride VB ₆0.5mg/L

Nicotinic acid VB ₅0.5mg/L

Sucrose 30g/L

Agar 6.4g/L.

2. condition of culture: temperature 25 ± 2 DEG C, light culture 2 months.

(5) statistical indicator and method: explant starts statistics and sprouts and brownization situation after 60d is cultivated in group training room.

Bud ratio=sprout organize number/inoculation blade amt × 100%;

Melting brown rate=brownization organize number/inoculation blade amt × 100%.

(6) result: the blade inoculation of 1cm × 1cm size is as shown in Figure 1 carried out light culture in Bud polarization medium, within 1.5 ~ 2 months, can observe at the beginning of the indefinite bud formed as shown in Figure 2, blade bud ratio can reach 84.60%, often open blade and can form 3-8 indefinite bud, melting brown rate is only 1.93%.

Claims (3)

1. Chinese herbaceous peony leaf tissue cultivates the medium directly becoming bud, it is characterized in that, improvement MS minimal medium, adds TDZ0.1 ~ 3.0mgL ^-1+ Coconut Juice 5 ~ 15%, additional saccharose 30gL ^-1, caseinhydrolysate 600mgL ^-1, agar 6.4gL ^-1, pH is 5.8.

2. Chinese herbaceous peony leaf tissue cultivates the cultural method directly becoming bud, it is characterized in that, comprises the following steps: late March chooses the tender Chinese herbaceous peony blade of children; Be inoculated in temperature 25 ± 2 DEG C on medium after sterilizing, light culture 2 months, obtain indefinite bud; Described medium is improvement MS minimal medium, adds TDZ0.1 ~ 3.0mgL ^-1+ Coconut Juice 5 ~ 15%, additional saccharose 30gL ^-1, caseinhydrolysate 600mgL ^-1, agar 6.4gL ^-1, pH is 5.8.

3. method according to claim 2, is characterized in that, the sterilizing methods that described blade sterilization treatment adopts alcohol to combine with mercuric chloride, and concrete steps are as follows:

1. first young leaflet tablet is rinsed 10min in flowing water, remove exterior earth, then clean with liquid detergent, running water is clean;

2. be placed on superclean bench by young leaflet tablet, be positioned in beaker, with 75% alcohol disinfecting 30s, sterile water rinses 5 ~ 6 times repeatedly;

3. again with 0.1% mercuric chloride sterilization 3min, sterile water rinses 5 ~ 6 times repeatedly;

4. explant is placed in suck dry moisture on aseptic filter paper, cuts marginal portion and the master pulse of blade, and the size being cut into 1cm × 1cm is inoculated on medium, for avoiding cross-infection, and every bottle graft kind 3 blades.