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 PDFInfo
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- 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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- 241001313857 Bletilla striata Species 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000005516 engineering process Methods 0.000 title claims abstract description 13
- 230000001939 inductive effect Effects 0.000 title claims abstract description 8
- 238000010899 nucleation Methods 0.000 claims abstract description 11
- 230000006698 induction Effects 0.000 claims abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 4
- 238000005286 illumination Methods 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 3
- 241000287828 Gallus gallus Species 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 229960004793 sucrose Drugs 0.000 claims description 3
- 238000012549 training Methods 0.000 claims description 3
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 3
- 229920002554 vinyl polymer Polymers 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 210000003608 fece Anatomy 0.000 claims description 2
- 239000010871 livestock manure Substances 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 5
- 230000004069 differentiation Effects 0.000 abstract description 4
- 238000009331 sowing Methods 0.000 abstract description 2
- 235000013399 edible fruits Nutrition 0.000 description 7
- 241001313855 Bletilla Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 241001523383 Achnatherum Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000905957 Channa melasoma Species 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 240000005250 Chrysanthemum indicum Species 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
- 240000000233 Melia azedarach Species 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 206010051373 Wound haemorrhage Diseases 0.000 description 1
- 208000002399 aphthous stomatitis Diseases 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 238000011534 incubation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 230000001114 myogenic effect Effects 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Cultivation Of Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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
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)
- 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.
- 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.
- 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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