CN109169275A - A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method - Google Patents

A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method Download PDF

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Publication number
CN109169275A
CN109169275A CN201811066484.4A CN201811066484A CN109169275A CN 109169275 A CN109169275 A CN 109169275A CN 201811066484 A CN201811066484 A CN 201811066484A CN 109169275 A CN109169275 A CN 109169275A
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reddish brown
bletilla striata
pale reddish
brown trident
culture
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CN201811066484.4A
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史永弟
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Gansu Yuan Yi Biotechnology Co Ltd
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Gansu Yuan Yi Biotechnology Co Ltd
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Priority to CN201811066484.4A priority Critical patent/CN109169275A/en
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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
    • A01H4/001Culture apparatus for tissue culture
    • 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
    • A01H4/008Methods for regeneration to complete plants

Abstract

The invention discloses a kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and methods, comprising the following steps: after pale reddish brown trident bletilla striata capsule is cleaned, sterilizes, sterilizes by S1., seed in pale reddish brown trident bletilla striata capsule is spread on MS basal medium;S2. after the expansion of pale reddish brown trident bletilla striata cotyledon, single plant switching is divided to induce Multiple Buds on inducing clumping bud culture medium;S3. pale reddish brown trident bletilla striata Multiple Buds are separated into single plant on superclean bench, switching carries out Multiplying culture on proliferated culture medium;S4. the pale reddish brown trident bletilla striata seedling switching that growth length is 2-3cm is subjected to rooting induction on root media.By carrying out disinfection using pale reddish brown trident bletilla striata capsule as explant, the direct injury in disinfecting process to seed can be reduced;Reasonable culture medium prescription improves germination percentage and survival rate, accelerates breeding, not only accelerates the speed of production of scale, also improve the pale reddish brown trident bletilla striata quality of production, solve the pale reddish brown trident bletilla striata market status that supply falls short of demand.

Description

A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method
Technical field
The present invention relates to field of plant tissue culture technique, it particularly relates to a kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation Culture medium and method.
Background technique
The bletilla striata is orchid family bletilla striata category perennial herb bulbous plant, alias galanga, Bai Gen, bletilla, purple orchid, Lian Jicao, sweet Root etc., dry root is used as medicine, and property is bitter, sweet, puckery, slightly cold, return lung, liver, stomach meridian.The major functions: astringing to arrest bleeding, detumescence and promoting granulation;For hemoptysis It spits blood, traumatic hemorrhage, sore swollen toxin, chapped skin;Pulmonary tuberculosis hemoptysis, ulcer haemorrhage etc..In addition, there are also very high sights for the bletilla striata Reward value and economic value.
Bletilla striata seeds germination rate is low with survival rate, slow growth, in addition widely used, demand is big, at present artificial breeding Predominantly stem tuber rhizome cutting propagation, breeding coefficient is low to be not easy to be mass produced, and therefore, it is difficult to meet the needs of market.
For the problems in the relevant technologies, currently no effective solution has been proposed.
Summary of the invention
For above-mentioned technical problem in the related technology, the present invention proposes a kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium And method, it is able to solve above-mentioned technical problem.
To realize the above-mentioned technical purpose, the technical scheme of the present invention is realized as follows:
A kind of pale reddish brown trident bletilla striata tissue culture and rapid propagation method, comprising the following steps:
S1. seed is sprouted: after being cleaned by pale reddish brown trident bletilla striata capsule, sterilize, sterilize, by pale reddish brown trident bletilla striata capsule Interior seed is spread on MS basal medium, promotes to germinate;
S2. inducing clumping bud culture: after the expansion of pale reddish brown trident bletilla striata cotyledon, single plant switching is divided to train in inducing clumping bud It supports and induces Multiple Buds on base;
S3. Multiplying culture: pale reddish brown trident bletilla striata Multiple Buds are separated into single plant on superclean bench, switching is trained in proliferation It supports and carries out Multiplying culture on base, cultivate 30-40 days;
S4. culture of rootage: the pale reddish brown trident bletilla striata seedling that growth length is 2-3cm is transferred and is given birth on root media Root induction.
Further, the formula of inducing clumping bud culture medium are as follows: MS basal medium, 0.1mg/L Thidiazuron, 0.1mg/L Methyl α-naphthyl acetate.
Further, the formula of the proliferated culture medium are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate.
Further, the formula of the root media are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate, 0.2g/L active carbon.
Further, sucrose 30g, agar 5.8g are added in every liter of culture medium, pH value is adjusted to 5.80-5.90, goes out at 121 DEG C 12h is cultivated under bacterium 20min, illumination 2000lx, cultivation temperature is 24 ± 2 DEG C, wherein the culture medium includes inducing clumping bud training Support base, proliferated culture medium, root media.
Further, cleaning, the specific steps for sterilizing, sterilizing described in step S1 are as follows: be chosen as ripe full and complete without small holes caused by worms Whole pale reddish brown trident bletilla striata capsule, after dish washing liquid cleans up, 75% alcohol disinfecting 30s, sterile distilled water flushing 3 times, 0.1% Mercuric chloride shakes 8-18min, and sterile distilled water rinses 4-6 times, 5min/ times, blots the excessive moisture on capsule skin with sterilizing paper.
Further, it after carrying out rooting induction on root media described in step S4, after culture 20 days, transfers in sugar-free After progress sugar-free is taken root or cultivated 40 days in box, it is placed on greenhouse and first carries out covered experienced seedling 15 days, then experienced seedling at least 15 of uncapping It transplants after it to hole tray.
Further, step S4 is replaced with following steps: after the proliferation seedling size fractionation that step S3 is obtained, being transferred to nothing In sugar culture box, 5 natural ventilation holes, 1 natural permeable hole of preserving container itself are sealed with preservative film after being inoculated with Gas exchanges are carried out, remaining 5 natural ventilation hole is opened one by one after the long about 5-8cm of the big bletilla striata seedling of pale reddish brown trident and carries out gases CO is led in exchange in culture vessel after 7 days2Gas, after 25-35 days, transplantation of seedlings out.
A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium, including inducing clumping bud culture medium, proliferated culture medium, training of taking root Support base;Wherein,
The formula of inducing clumping bud culture medium are as follows: MS basal medium, 0.1mg/L Thidiazuron, 0.1mg/L methyl α-naphthyl acetate;
The formula of proliferated culture medium are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate;
The formula of root media are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate, 0.2g/L active carbon.
Beneficial effects of the present invention: by carrying out disinfection using pale reddish brown trident bletilla striata capsule as explant, disinfection can be reduced In the process to the direct injury of seed, pollution rate raising survival rate can be reduced by adjusting sterilization time;Reasonable culture medium prescription, mentions High-incidence bud rate and survival rate accelerate breeding, not only accelerate the speed of production of scale, also improve pale reddish brown trident bletilla striata production Quality solves the pale reddish brown trident bletilla striata market status that supply falls short of demand.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's every other embodiment obtained, shall fall within the protection scope of the present invention.
A kind of pale reddish brown trident bletilla striata tissue culture and rapid propagation method, comprising the following steps:
S1. seed is sprouted: selecting mature and plump and completely the big bletilla striata capsule of pale reddish brown trident, dish washing liquid cleaning are dry without small holes caused by worms After net, 75% alcohol disinfecting 30s, sterile distilled water is rinsed 3 times, and 0.1% mercuric chloride shakes 8-18min, and sterile distilled water rinses 4-6 It is secondary, 5min/ times, the excessive moisture on capsule skin is blotted with sterilizing paper, scissors presss from both sides capsule with tweezers after one end clip of capsule Seed is shaken off to support in MS and cultivates 20-30d on base and culture solution.To pale reddish brown trident bletilla striata seeds germination (culture medium is emerald green) with Afterwards, the plumule absorption of growing space deficiency, part top continues to cultivate less than bottom nutrition so being transferred on MS culture medium 20-30 days.In every liter of culture medium plus sucrose 30g, agar 5.8g, pH value are adjusted between 5.80-5.90, sterilize at 121 DEG C 20min (following culture medium is herewith handled) cultivates (12h, 2000lx) under illumination, and cultivation temperature is 24 ± 2 DEG C (similarly hereinafter).With Pale reddish brown trident bletilla striata capsule carries out disinfection as explant, can reduce the direct injury in disinfecting process to seed, adjustment sterilizing Time can reduce pollution rate and improve survival rate.
S2. inducing clumping bud culture: after the expansion of pale reddish brown trident bletilla striata cotyledon, i.e. when long 0.3-0.8cm, single plant is divided to turn It connects on inducing clumping bud culture medium, connects 10 bottles, every bottle connects 14 plants, 3 repetitions, the formula of inducing clumping bud culture medium are as follows: MS Basal medium, 0.1mg/L Thidiazuron, 0.1mg/L methyl α-naphthyl acetate, culture obviously induce Multiple Buds after 15 days, grow thickly after 30 days Bud is up to 4 times or more;
S3. Multiplying culture: pale reddish brown trident bletilla striata Multiple Buds are separated into single plant on superclean bench, switching is trained in proliferation It supports and carries out Multiplying culture, the formula of proliferated culture medium on base are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate connects 10 bottles, and every bottle connects 10 plants, and 3 repetitions are cultivated 30-40 days, and proliferation times are high, the period is short, proliferation seedling Growing way is good;
S4. culture of rootage: culture of rootage is divided into two ways, and one is pale reddish brown trident that growth length is 2-3cm is white Splendid achnatherum seedling is transferred on the root media for having added active carbon, and proliferation while carries out rooting induction, the formula of root media are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate, 0.2g/L active carbon.It, can after cultivating 20d Switching carries out sugar-free in no sugar bowl and takes root, and after can also cultivating 40 days, is placed on greenhouse and first carries out covered experienced seedling 15 days, then It transplants after uncapping experienced seedling 15 days or more to hole tray.
Another kind is carbonless base paper rooting method, after the proliferation seedling size fractionation that step S3 is obtained, is transferred to sugar-free training It supports in box, seals 5 natural ventilation holes with preservative film after being inoculated with, 1 natural permeable hole of preserving container itself carries out Gas exchanges open remaining 5 natural ventilation hole after the long about 5-8cm of the big bletilla striata seedling of pale reddish brown trident one by one and carry out gas friendships It changes, leads to CO after 7 days in culture vessel2Gas, after 25-35 days, transplantation of seedlings out.Without practicing seedling for a long time before transplanting, it is within 1-3 days It can.
Root media is taken root with carbonless base paper to be combined, and the number of days for experienced seedling of taking root, and sugar-free tissue culture half are substantially reduced The perfect blade function of closed culture environment, improves the photosynthetic capacity of pale reddish brown trident bletilla tissue culture seedlings, then improves Transplanting survival rate.
MS basal medium, contained component and content such as table 1:
1 MS basal medium of table
The pale reddish brown trident bletilla striata is cultivated according to the method for embodiment, changes the time that wherein 0.1% mercuric chloride shakes sterilizing, time Be respectively set to 8min, 12min, 15min and 18min, detect different sterilization times to pale reddish brown trident bletilla striata seeds pollution rate with The influence of survival rate, the results are shown in Table 2.
Influence of the different disinfecting times of table 2 to seed contamination rate and survival rate
It can analyze to obtain from table 2, arrive mercuric chloride sterilization time 18 minutes, survival rate starts to have a declining tendency, pollution rate It keeps with the effect of sterilizing 15 minutes always, so mercuric chloride sterilization time is maintained at 8-18 minutes, can both keeping high survival Rate can keep minimum pollution rate again.
The pale reddish brown trident bletilla striata is cultivated according to the method for embodiment, changes the formula of wherein inducing clumping bud culture medium, simultaneously Do 8 groups of experiments, it is as shown in table 3 that 8 groups of experiments are respectively adopted different inducing clumping bud culture medium prescriptions, compares 8 groups of experiment cultures Multiple Buds quantity and proliferation rate afterwards, the results are shown in Table 4.
The corresponding inducing clumping bud culture medium prescription of 38 groups of experiments of table
Influence of the 4 different culture medium formula of table to inducing clumping bud
Wherein, the "+" in " foundation portion black ++ " represents nigrescence degree, and "+", and to represent foundation portion nigrescence situation tighter Weight.
From table 4 analyze it is found that using inducing clumping bud culture medium formula are as follows: MS basal medium, 0.1mg/L thiophene benzene Grand, the pale reddish brown trident bletilla striata of 0.1mg/L methyl α-naphthyl acetate culture is always best from several and proliferation rate of sprouting, and plant in it is dark green, grow thickly Bud is healthy and strong, has root, and foundation portion is without nigrescence phenomenon.
By in step S1, seed is sprinkling upon respectively in MS basal medium and MS nutrient solution, compare the survival rate of seed with it is abnormal The probability of shape seedling, experimental result show, seed is sprinkling upon MS basal medium and is remarkably improved the survival rate of seed while reducing The probability of lopsided seedling.
In conclusion by means of above-mentioned technical proposal of the invention, by using pale reddish brown trident bletilla striata capsule as explant It carries out disinfection, the direct injury in disinfecting process to seed can be reduced, adjustment sterilization time can reduce pollution rate and improve survival rate; Reasonable culture medium prescription improves germination percentage and survival rate, accelerates breeding, not only accelerates the speed of production of scale, also mention The high pale reddish brown trident bletilla striata quality of production, solves the pale reddish brown trident bletilla striata market status that supply falls short of demand.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims (9)

1. a kind of pale reddish brown trident bletilla striata tissue culture and rapid propagation method, which comprises the following steps:
S1. seed is sprouted: after being cleaned by pale reddish brown trident bletilla striata capsule, sterilize, sterilize, by pale reddish brown trident bletilla striata capsule kinds Son is spread on MS basal medium, promotes to germinate;
S2. inducing clumping bud culture: after the expansion of pale reddish brown trident bletilla striata cotyledon, divide single plant switching in inducing clumping bud culture Multiple Buds are induced on base;
S3. Multiplying culture: pale reddish brown trident bletilla striata Multiple Buds are separated into single plant on superclean bench, are transferred in proliferated culture medium Upper carry out Multiplying culture is cultivated 30-40 days;
S4. culture of rootage: the pale reddish brown trident bletilla striata seedling switching that growth length is 2-3cm take root on root media luring It leads.
2. pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that inducing clumping bud culture medium Formula are as follows: MS basal medium, 0.1mg/L Thidiazuron, 0.1mg/L methyl α-naphthyl acetate.
3. pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that the proliferated culture medium is matched Side are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate.
4. pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that the root media is matched Side are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate, 0.2g/L active carbon.
5. -4 any pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that add sugarcane in every liter of culture medium Sugared 30g, agar 5.8g, pH value are adjusted to 5.80-5.90, and sterilize 20min at 121 DEG C, and 12h is cultivated under illumination 2000lx, training Supporting temperature is 24 ± 2 DEG C, wherein the culture medium includes inducing clumping bud culture medium, proliferated culture medium, root media.
6. pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that clean, disappear described in step S1 Poison, sterilization specific steps are as follows: be chosen as that ripe full and completely pale reddish brown trident bletilla striata capsule, dish washing liquid clean up without small holes caused by worms Afterwards, 75% alcohol disinfecting 30s, sterile distilled water rinse 3 times, and 0.1% mercuric chloride shakes 8-18min, and sterile distilled water rinses 4-6 times, 5min/ times, the excessive moisture on capsule skin is blotted with sterilizing paper.
7. pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that in training of taking root described in step S4 It supports after carrying out rooting induction on base, after culture 20 days, switching carries out being placed on after sugar-free takes root or cultivate 40 days in no sugar bowl Greenhouse first carries out covered experienced seedling 15 days, then transplants after uncapping experienced seedling at least 15 days to hole tray.
8. pale reddish brown trident bletilla striata tissue culture and rapid propagation method according to claim 1, which is characterized in that replace step with following steps S4: after the proliferation seedling size fractionation that step S3 is obtained, being transferred in carbonless base paper box, with preservative film sealing 5 after being inoculated with 1 natural permeable hole in a natural ventilation hole, preserving container itself carries out gas exchanges, is about to the big bletilla striata seedling of pale reddish brown trident Gas exchanges are carried out to open remaining 5 natural ventilation hole after 5-8cm one by one, lead to CO after 7 days in culture vessel2Gas, 25- After 35 days, transplantation of seedlings out.
9. a kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium, which is characterized in that including inducing clumping bud culture medium, Multiplying culture Base, root media;Wherein,
The formula of inducing clumping bud culture medium are as follows: MS basal medium, 0.1mg/L Thidiazuron, 0.1mg/L methyl α-naphthyl acetate;
The formula of proliferated culture medium are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate;
The formula of root media are as follows: MS basal medium, 1.2mg/L6- benzyl aminoadenine, 0.5mg/L methyl α-naphthyl acetate, 0.2g/L active carbon.
CN201811066484.4A 2018-09-13 2018-09-13 A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method Pending CN109169275A (en)

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