CN106106155B - A kind of method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology - Google Patents

A kind of method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology Download PDF

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Publication number
CN106106155B
CN106106155B CN201610493055.XA CN201610493055A CN106106155B CN 106106155 B CN106106155 B CN 106106155B CN 201610493055 A CN201610493055 A CN 201610493055A CN 106106155 B CN106106155 B CN 106106155B
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seedling
bletilla striata
bulb
micro
culture
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CN106106155A (en
Inventor
刘勇
葛红心
李厚华
王胜宝
郝兴顺
桓德强
吴玉红
陈进
魏玲
魏芳勤
王艳龙
陈永刚
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Hanzhoung Institute Of Agricultural Sciences (shaanxi Rice Research Institute)
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Hanzhoung Institute Of Agricultural Sciences (shaanxi Rice Research Institute)
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00Cultivation of specific crops or plants not otherwise provided for

Abstract

The invention discloses a kind of method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology.This method is first to carry out aseptic seeding with bletilla striata seeds, culture obtains the protocorm of 1~2 leaflet of differentiation, it is transferred to again on the culture medium of " activated carbon of MS+15% potato juices+0.1% " and cultivates bottle seedling, and produce micro- bulb by changing condition of culture induction, then, in tissue culture bottle rudiment micro- bulb sowing and breeding bletilla striata seedling.Compared with prior art, the present invention has that tissue culture program is simple, and cost is cheap, and planting percent is high, is bred as seedling seedling well developed root system, robust growth, seedling raise period is short and transplanting crop field after the advantages that survival rate is high, growth potential is strong.

Description

A kind of method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology
Technical field
The present invention relates to plant seedling raising technology field, is specifically related to a kind of numerous using the micro- bulb of tissue culture technology induction Educate the method for bletilla striata seedling.
Background technology
The bletilla striata is also known as bletilla, purple orchid, Gan Gen, Lian Jicao, is orchid family bletilla striata category perennial herb plant, in being distributed in The states such as state to Korea, Japan, China are distributed in the ground such as East China, south China and Henan, Shaanxi, Sichuan, Yunnan.The stem tuber of the bletilla striata is China's traditional Chinese medicine, early in《Sheng Nong's herbal classic》In just it is on the books,《Chinese Pharmacopoeia》It is middle record with tonifying lung, detumescence, myogenic, Hemostasis, sore and other effects, hinder the diseases such as hemoptysis, metal-inflicted wound bleeding, canker sores, soup fire burn, rhagadia manus et pedis for treating lung.This Outside, bletilla striata pattern is gorgeous, and a kind of garden landscape plant, therefore market is very vigorous to the demand of the bletilla striata well.In recent years To increase with market demand, bletilla striata price also rises year by year.Due to unrestrictedly artificial excavation for a long time, wild resource reserves Increasingly reduce, endangered, the bletilla striata is classified as one of Wild Medicinal of focused protection by country.To protect the wild money of the bletilla striata Source, artificial breeding and the cultivation bletilla striata are imperative.
The seed of the bletilla striata is very tiny and without endosperm, thus under natural situation be difficult sprout and growth, seedling it is numerous More difficulty is educated, traditional cultivation carries out division propagation mainly by the mode of segmentation pseudobulb.General 1 pseudobulb is only capable of shape in 1 year Into 1~3 new pseudobulb, therefore, division propagation cycle length, reproductive efficiency is low, and consumes kind of amount greatly, it is difficult to meet that large area is planted The needs of training.In recent years, tissue culture technique is widely used in bletilla striata seedling breeding, typically uses bletilla striata maturation capsule as material Material, carries out aseptic seeding on different culture media, and seed is white using method for tissue culture progress clonal reproduction, realization after sprouting The large-scale production of splendid achnatherum seedling.Chinese scholar employs " aseptic seeding → protocorm in terms of the bletilla striata is bred using tissue culture technology Differentiation → root induction → acclimatization and transplantses of → induction Protocorm Multiplication → induced bud ", " aseptic seeding → protocorm → induced bud Differentiation → root induction → acclimatization and transplantses ", " differentiation → root induction of aseptic seeding → protocorm → induced bud → hardening moves The technology paths such as cultivation ".These technical schemes are typically necessary after aseptic seeding again by 2~3 switchings, in different cultures Complete the tissue culture process such as break up, breed, taking root on base, then again by hardening, transplant and can just obtain bletilla striata seedling.And bletilla striata group Survival rate is not high after training transplantation of seedlings, and root system is thin and delicate after transplant survival, and growth is slow, could be transplanted after needing precision management 6~12 months Crop field.Therefore, tissue culture program step is cumbersome, and transplanting survival rate is not high, and transplanted seedling growth is limitation bletilla striata tissue culture raising technology slowly The restraining factors of scale application.
The content of the invention
To solve the above problems, the present invention provides following technical scheme:
A kind of method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology, it is characterised in that comprise the following steps:
A, by bletilla striata seeds aseptic seeding on MS culture mediums, in 25 DEG C ± 1 DEG C, intensity of illumination 1000Lx of temperature, illumination 50d is cultivated under the conditions of time 12h/d, obtains the protocorm with 1~2 leaflet;
B, the obtained protocorms of step A are transferred on the culture medium of " activated carbon of MS+15% potato juices+0.1% ", 25 DEG C ± 1 DEG C of temperature, 45d is cultivated under the conditions of intensity of illumination 2000Lx, light application time 12h/d, obtain white with 3~4 leaves Splendid achnatherum tissue-culture container seedling;
C, by the bletilla striata tissue-culture container seedling cultivated through step B in 15 DEG C ± 1 DEG C, intensity of illumination 2000Lx of temperature, light application time 40d is cultivated under the conditions of 10h/d, induction bletilla striata tissue-culture container seedling forms micro- bulb;
D, by the bletilla striata tissue-culture container seedling cultivated through step C in 25 DEG C ± 1 DEG C, intensity of illumination 1000Lx of temperature, light application time 60~80d is cultivated under the conditions of 12h/d, micro- bulb expands and sprouts sprouting, tissue-cultured seedling is taken out from tissue culture bottle, peels off old leaf, Wash culture medium off, obtain micro- bulb of rudiment;
E, selecting step D obtains more than diameter 3mm in micro- bulb, and bud is grown 0.3~1.0cm person and is seeded in equipped with seedling medium Seedling culture hole plate in, depth of planting 1cm pours permeable after broadcasting, be placed in vinyl house and carry out seedling management, canopy temperature control system is white It 25~30 DEG C, at 15~20 DEG C of night, compound seedling medium water content is maintained at 70%~75% before emergence, after emergence water management see It is dry see it is wet.After planting 70d, bletilla striata seedling grow 3~5 leaves, transplant crop field during more than height of seedling 6cm.
A kind of MS culture mediums described in above-mentioned steps A and B, it is characterised in that cane sugar content 30g/L, agar content are 6.5g/L, pH value 5.8.
A kind of seedling medium described in above-mentioned steps E, it is characterised in that by rural area soil, decomposed sawdust and decomposed chicken Excrement by volume 50:40:10 mix.
The beneficial effects of the invention are as follows:1. tissue culture program is simple, it is only necessary to obtains having 1~2 leaflet in aseptic seeding Protocorm after transfer once.2. inducing bletilla striata bottle seedling to form micro- bulb by changing condition of culture, simple to operate, cost is low It is honest and clean.It is 3. short with micro- bulb sowing and breeding bletilla striata seedling planting percent height of rudiment, seedling well developed root system, robust growth, seedling raise period.With Prior art is compared, and the present invention has that tissue culture program is simple, use manpower and material resources sparingly, cost is cheap, workable, breeding cycle The advantages that survival rate is high, growth potential is strong behind short, incubation seedling stalwartness and transplanting crop field.
Brief description of the drawings
Fig. 1 is the protocorm bottle seedling for starting to break up blade.
Fig. 2 is micro- bulb of rudiment.
Fig. 3 is micro- bulb tree seedling after emergence.
Fig. 4 is that micro- bulb sows the sturdy root system of bletilla striata seedling.
Embodiment
With reference to embodiment and embodiment, the invention will be further described.
Embodiment:
Step 1:By the bletilla striata fruit folder of maturation with being transferred to 0.2% after 70% ethanol postincubation 1min under superclean bench Mercuric chloride solution soaks 8min, and appropriate stir makes fruit clamp surface be totally submerged in solution several times in immersion process;Fruit folder is pulled out, with nothing Bacterium water rinses 4 times, then blots fruit clamp surface moisture with sterilized filter paper;Dug with the tweezers and scalpel handled through flame disinfection Fruit folder is driven, by bletilla striata seeds aseptic seeding in the tissue culture bottle equipped with MS culture mediums;In 25 DEG C ± 1 DEG C of temperature, intensity of illumination 50d is cultivated under the conditions of 1000Lx, light application time 12h/d, obtains the protocorm with 1~2 leaflet.
Step 2:The protocorm with 1~2 leaflet obtained through step 1 culture is transferred to containing 15% potato juice On the MS culture mediums of 0.1% activated carbon, 200ml tissue culture bottles are inoculated with 20 protocorms.In 25 DEG C ± 1 DEG C of temperature, intensity of illumination 45d is cultivated under the conditions of 2000Lx, light application time 12h/d, obtains the bletilla striata tissue-culture container seedling with 3~4 leaves.
Step 3:By the bletilla striata tissue-culture container seedling cultivated through step 2 in 15 DEG C ± 1 DEG C, intensity of illumination 2000Lx of temperature, illumination 40d is cultivated under the conditions of time 10h/d, induction bletilla striata tissue-culture container seedling forms micro- bulb.
Step 4:By the bletilla striata tissue-culture container seedling cultivated through step 3 in 25 DEG C ± 1 DEG C, intensity of illumination 1000Lx of temperature, illumination 60~80d is cultivated under the conditions of time 12h/d, micro- bulb is expanded and sprouts sprouting, and tissue-cultured seedling is taken out from tissue culture bottle, is peelled off old Leaf, wash culture medium off, obtain micro- bulb of rudiment.
Step 5:More than diameter 3mm in micro- bulb that selecting step 4 obtains, bud grow 0.3~1.0cm person and are seeded in be equipped with and educate In the seedling culture hole plate of seedling matrix, depth of planting 1cm pours permeable after broadcasting, be placed in vinyl house and carry out seedling management, canopy temperature control Make 25~30 DEG C of daytime, at 15~20 DEG C of night, compound seedling medium water content is maintained at 70%~75% before emergence, moisture after emergence Management see it is dry see it is wet.After planting 70d, bletilla striata seedling grow 3~5 leaves, transplant crop field during more than height of seedling 6cm.
MS culture mediums described in above-mentioned steps 1 and 2, its cane sugar content are 30g/L, agar content 6.5g/L, and pH value is 5.8。
Seedling medium described in above-mentioned steps 5 is by rural area soil, decomposed sawdust and decomposed chicken manure by volume 50:40: 10 mix.
Embodiment 1
The bletilla striata seeds of selection are the pale reddish brown bletilla striata fruit folder of family's kind in the harvesting of Hanzhong City Liuba County.According to described in embodiment After method carries out step 1 aseptic seeding and Protocorm, the culture of step 2 tissue-cultured seedling and the micro- bulb of step 3 induced synthesis, through adjusting It is 84.3% to look into the micro- bulb inductivity of statistics.Continue to cultivate through step 4 again, counted when cultivating 70d, micro- bulb average diameter 4.6mm, the long 0.59cm of average bud;Counted when cultivating 80d, micro- bulb average diameter 4.8mm, the long 0.64cm of average bud.Selection is straight More than footpath 3mm, micro- bulb that bud grows 0.3~1.0cm are sowed and managed according to the method described in embodiment step 5, on the same day Bletilla tissue culture seedlings are transplanted as control, investigation statisticses planting percent and seedling quality situation are shown in Table 1 when broadcasting rear 70d, as can be seen from the table The indexs such as micro- bulb tree seedling (processing 1) planting percent, the number of blade, height of seedling, radical and root be thick are significantly better than same kind tissue-cultured seedling Directly transplanting (processing 3).
Table 1:The micro- bulb tree seedling of the bletilla striata is compared with tissue culture transplanted seedling quality
Embodiment 2
The difference from Example 1 of embodiment 2 is:The bletilla striata seeds of selection are in the wild of Hanzhong City Lueyang County harvesting Chrysanthemum bletilla striata fruit is pressed from both sides.It is 89.5% through the micro- bulb inductivity of investigation statisticses after the micro- bulb of step 3 induced synthesis.Again through step 4 after Continuous culture, is counted, micro- bulb average diameter 4.3mm when cultivating 60d, the long 0.45cm of average bud.Select more than diameter 3mm, bud length 0.3~1.0cm micro- bulb is sowed according to the method described in step 5 in embodiment and management, transplants bletilla tissue culture seedlings on the same day As control, investigation statisticses planting percent and seedling quality situation are shown in Table 1 when broadcasting rear 70d, as can be seen from the table micro- bulb tree seedling (place 3) planting percent, the number of blade, height of seedling, radical and root be thick etc. that index is significantly better than tissue culture seedling direct transplantation (processing 4) for reason.

Claims (3)

  1. A kind of 1. method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology, it is characterised in that comprise the following steps:
    A, by bletilla striata seeds aseptic seeding on MS culture mediums, in 24~26 DEG C, intensity of illumination 1000Lx of temperature, light application time 50d is cultivated under the conditions of 12h/d, obtains the protocorm with 1~2 leaflet;
    B, the obtained protocorms of step A are transferred on the culture medium of " activated carbon of MS+15% potato juices+0.1% ", in temperature 24 45d is cultivated under the conditions of~26 DEG C, intensity of illumination 2000Lx, light application time 12h/d, obtains the bletilla striata tissue culture bottle with 3~4 leaves Seedling;
    C, by the bletilla striata tissue-culture container seedling cultivated through step B in 14~16 DEG C of temperature, intensity of illumination 2000Lx, light application time 10h/d Under the conditions of cultivate 40d, induction bletilla striata tissue-culture container seedling forms micro- bulb;
    D, by the bletilla striata tissue-culture container seedling cultivated through step C in 24~26 DEG C of temperature, intensity of illumination 1000Lx, light application time 12h/d Under the conditions of cultivate 60~80d, micro- bulb expands and sprouts sprouting, tissue-cultured seedling is taken out from tissue culture bottle, peels off old leaf, washes training off Base is supported, obtains micro- bulb of rudiment;
    E, selecting step D obtains more than diameter 3mm in micro- bulb, and bud grows 0.3~1.0cm person and is seeded in educating equipped with seedling medium In seedling hole tray, depth of planting 1cm pours permeable after broadcasting, be placed in vinyl house and carry out seedling management, canopy temperature control system is on daytime 25 ~30 DEG C, at 15~20 DEG C of night, compound seedling medium water content is maintained at 70%~75% before emergence, and water management, which is shown in, after emergence dry sees Wet, after planting 70d, bletilla striata seedling grows 3~5 leaves, transplants crop field during more than height of seedling 6cm.
  2. A kind of 2. method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology described in claim 1, it is characterised in that MS culture mediums cane sugar content described in step A and B is 30g/L, agar content 6.5g/L, pH value 5.8.
  3. A kind of 3. method for inducing micro- bulb to breed bletilla striata seedling using tissue culture technology described in claim 1, it is characterised in that Seedling medium described in step E is by rural area soil, decomposed sawdust and decomposed chicken manure by volume 50:40:10 mix.
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