CN105104196A - Rapid propagation method for hedyotis corymbosa tissue culture seedlings - Google Patents
Rapid propagation method for hedyotis corymbosa tissue culture seedlings Download PDFInfo
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- CN105104196A CN105104196A CN201510518049.0A CN201510518049A CN105104196A CN 105104196 A CN105104196 A CN 105104196A CN 201510518049 A CN201510518049 A CN 201510518049A CN 105104196 A CN105104196 A CN 105104196A
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Abstract
A rapid propagation method for hedyotis corymbosa tissue culture seedlings includes the following steps that 1, a non-cracking fruit of hedyotis corymbosa is used as an explant to be disinfected; 2, the disinfected explant is stripped off on a clean bench, seeds are inoculated to an MS culture medium and induced to sprout to obtain sterile test-tube seedlings; 3, the sterile test-tube seedlings are placed in an MS propagation culture medium and cultured for rapid propagation to obtain cluster buds; 4, the cluster buds are placed in an MS rooting culture medium and cultured to obtain complete seedlings with roots; 5, the complete seedlings with the roots are hardened and then transplanted in a seedbed to grow for a month, and then the complete seedlings are transplanted to a land. The propagation coefficient of the hedyotis corymbosa cluster buds obtained through the culture method reaches 20-30 times, the rooting rate of the obtained tissue culture seedlings reaches 98.5%, the survival rate of seedbed transplant reaches 95%, and the large-scale seedling culture for hedyotis corymbosa is effectively achieved.
Description
Technical field
The present invention relates to a kind of method for propagation, particularly a kind of method for quickly breeding of Herba Hedyotidis Corymbosae plantlet in vitro.
Background technology
Herba Hedyotidis Corymbosae, Classification system OldenlandiacorymbosaL., have another name called HERBA HEDYOTIS DIFFUSAE, corymbose hedyotis herb, for Rubiaceae plants of Hedyotis, be distributed in south China each province, output is larger, its dry all herbal medicine, containing multiple triterpene compound in medicinal material, wherein malol content is the highest, is its main active.Pharmacological research finds, malol has antitumor, anti-hepatitis, the multiple pharmacological effect such as antiviral, antibacterial, has important development and application values.Current Herba Hedyotidis Corymbosae medicinal material and seedling are mainly derived from wild resource, also do not have the report of artificial breeding and artificial planting technique.Serious artificial destruction is suffered in order to prevent Herba Hedyotidis Corymbosae wild resource, ensure the sustainable use of Herba Hedyotidis Corymbosae resource, need to carry out seedling breeding technical research to Herba Hedyotidis Corymbosae, with ensure excavate on a large scale after sufficient seedling can be provided to meet the needs of resource updates.By the tissue culture technique of biotechnology, Herba Hedyotidis Corymbosae seedling can be produced effectively rapidly, realize the factorial seedling growth of Herba Hedyotidis Corymbosae high quality seedling, with the needs in satisfied production.
Summary of the invention
The object of this invention is to provide a kind of method for quickly breeding of Herba Hedyotidis Corymbosae plantlet in vitro, it can improve reproduction speed and the quality of Herba Hedyotidis Corymbosae seedling, realizes the factorial seedling growth of Herba Hedyotidis Corymbosae high quality seedling, with the needs in satisfied production.
The present invention achieves the above object by the following technical programs: a kind of method for quickly breeding of Herba Hedyotidis Corymbosae plantlet in vitro, comprises the following steps:
(1) selection of explant and sterilization: get the non-dehiscent fruit of maturation of Herba Hedyotidis Corymbosae as explant, use 2% (v/v) liquid detergent aqueous solution soaking 5min, wire tap water 15-30min successively, with the addition of 2-3 and drip 100 milliliter of 0.1% (v/v) mercuric chloride sterilization 8-10min of Tween-20, aseptic water washing 3-5 time, finally remove surface moisture with sterilized filter paper, obtain explant, wherein sterile water is through autoclaved distilled water
(2) seed germination obtains in vitro cuttings: the explant strip off on superclean bench step (1) obtained, seed is inoculated in MS medium, be 23-27 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 30 days under the condition of 8-10 hour/day, obtains in vitro cuttings, wherein add benzyladenine 6-BA, 30g/L sucrose of 0.2mg/L and the agar of 3.4g/L in MS medium after seed sprouting, the pH value of medium is 5.8
(3) test-tube plantlet Multiple Buds Fast-propagation is cultivated: the in vitro cuttings obtained in step (2) is placed in MS propagating culture medium, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate under the condition of 8-10 hour/day to obtain test-tube plantlet Multiple Buds in 30 days, wherein add kinetin KT, 30g/L sucrose of heteroauxin IAA, 0.1-1.0mg/L and the agar of 3.4g/L of benzyladenine 6-BA, 0.1-0.3mg/L of 0.5-3.0mg/L in MS propagating culture medium, the pH value of medium is 5.8
(4) Multiple Buds culture of rootage: the stem section Multiple Buds obtained in step (3) being cut into band terminal bud or leaf bud, be placed in 1/2MS root media, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate the whole plant obtaining for 30 days being with root under the condition of 12-14 hour/day, wherein add root-inducing powder ABT, 30g/L sucrose of heteroauxin IAA, 0.1-0.3mg/L and the agar of 3.4g/L of 0.1-1.0mg/L in 1/2MS root media, the pH value of medium is 5.8
(5) hardening and transplanting: after step (4) obtains the whole plant being with root, bottle cap is opened in the indoor being 25 DEG C in room temperature, a small amount of running water is added in bottle, hardening 2-4 days, after surface horny is formed, seedling is taken out, clean root medium, be transplanted to immediately in seedbed, grow one month in seedbed after, transplant land for growing field crops.
Transplant in the rear week, every day early 8 to spraying evening 6 3-5 time, each 10min, after this every day, early 8 points, evening 6 respectively sprayed 1 time, each 10min; Temperature condition during transplanting is 20-28 DEG C, relative moisture 75-80%, and sunshade rate is 80%.
Outstanding advantages of the present invention is:
(1) adopt tissue culture technique to breed Herba Hedyotidis Corymbosae seedling and have that reproduction speed is fast, seedling quality is homogeneous; and not by the outstanding advantages of time, space, restriction in season, shorten the seedling breeding cycle, improve seedling quality; accomplish scale production, meet the needs on producing.
(2) the Herba Hedyotidis Corymbosae adventitious buds proliferation coefficient adopting cultural method of the present invention to obtain reaches 20-30 doubly, Multiple Buds is healthy and strong, easily take root after being inoculated into root media, rooting rate can reach more than 98.5%, transplants seedbed survival rate to 95% after hardening.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further illustrated.
Embodiment 1
An example of the quick breeding method for tissue culture of Herba Hedyotidis Corymbosae of the present invention, comprises the following steps:
(1) selection of explant and sterilization: get the non-dehiscent fruit of maturation of Herba Hedyotidis Corymbosae as explant, use 2% (v/v) liquid detergent aqueous solution soaking 5min, wire tap water 15-30min successively, with the addition of 2-3 and drip 100 milliliter of 0.1% (v/v) mercuric chloride sterilization 8-10min of Tween-20, aseptic water washing 3-5 time, finally remove surface moisture with sterilized filter paper, obtain explant, wherein sterile water is through autoclaved distilled water
(2) seed germination obtains in vitro cuttings: the explant strip off on superclean bench step (1) obtained, seed is inoculated in MS medium, be 23-27 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 30 days under the condition of 8-10 hour/day, obtains in vitro cuttings, wherein add benzyladenine 6-BA, 30g/L sucrose of 0.2mg/L and the agar of 3.4g/L in MS medium after seed sprouting, the pH value of medium is 5.8
(3) test-tube plantlet Multiple Buds Fast-propagation is cultivated: the in vitro cuttings obtained in step (2) is placed in MS propagating culture medium, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate under the condition of 8-10 hour/day to obtain test-tube plantlet Multiple Buds in 30 days, wherein add kinetin KT, 30g/L sucrose of heteroauxin IAA, 1.0mg/L and the agar of 3.4g/L of benzyladenine 6-BA, 0.3mg/L of 0.5mg/L in MS propagating culture medium, the pH value of medium is 5.8, adventitious buds proliferation coefficient is 23.5
(4) Multiple Buds culture of rootage: the stem section Multiple Buds obtained in step (3) being cut into band terminal bud or leaf bud, be placed in 1/2MS root media, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate the whole plant obtaining for 30 days being with root under the condition of 12-14 hour/day, wherein add root-inducing powder ABT, 30g/L sucrose of heteroauxin IAA, 0.3mg/L and the agar of 3.4g/L of 0.1mg/L in 1/2MS root media, the pH value of medium is 5.8, rooting rate is 93.0%
(5) hardening and transplanting: after step (4) obtains the whole plant being with root, bottle cap is opened in the indoor being 25 DEG C in room temperature, a small amount of running water is added in bottle, hardening 2-4 days, after surface horny is formed, seedling is taken out, clean root medium, be transplanted to immediately in seedbed, transplant land for growing field crops grow one month in seedbed after, transplanting survival rate is 95.5%.
Embodiment 2
Another example of the quick breeding method for tissue culture of Herba Hedyotidis Corymbosae of the present invention, comprises the following steps:
(1) selection of explant and sterilization: get the non-dehiscent fruit of maturation of Herba Hedyotidis Corymbosae as explant, use 2% (v/v) liquid detergent aqueous solution soaking 5min, wire tap water 15-30min successively, with the addition of 2-3 and drip 100 milliliter of 0.1% (v/v) mercuric chloride sterilization 8-10min of Tween-20, aseptic water washing 3-5 time, finally remove surface moisture with sterilized filter paper, obtain explant, wherein sterile water is through autoclaved distilled water
(2) seed germination obtains in vitro cuttings: the explant strip off on superclean bench step (1) obtained, seed is inoculated in MS medium, be 23-27 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 30 days under the condition of 8-10 hour/day, obtains in vitro cuttings, wherein add benzyladenine 6-BA, 30g/L sucrose of 0.2mg/L and the agar of 3.4g/L in MS medium after seed sprouting, the pH value of medium is 5.8
(3) test-tube plantlet Multiple Buds Fast-propagation is cultivated: the in vitro cuttings obtained in step (2) is placed in MS propagating culture medium, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate under the condition of 8-10 hour/day to obtain test-tube plantlet Multiple Buds in 30 days, wherein add kinetin KT, 30g/L sucrose of heteroauxin IAA, 0.1mg/L and the agar of 3.4g/L of benzyladenine 6-BA, 0.1mg/L of 3.0mg/L in MS propagating culture medium, the pH value of medium is 5.8, adventitious buds proliferation coefficient is 27.8
(4) Multiple Buds culture of rootage: the stem section Multiple Buds obtained in step (3) being cut into band terminal bud or leaf bud, be placed in 1/2MS root media, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate the whole plant obtaining for 30 days being with root under the condition of 12-14 hour/day, wherein add root-inducing powder ABT, 30g/L sucrose of heteroauxin IAA, 0.1mg/L and the agar of 3.4g/L of 1.0mg/L in 1/2MS root media, the pH value of medium is 5.8, rooting rate is 98.5%
(5) hardening and transplanting: after step (4) obtains the whole plant being with root, bottle cap is opened in the indoor being 25 DEG C in room temperature, a small amount of running water is added in bottle, hardening 2-4 days, after surface horny is formed, seedling is taken out, clean root medium, be transplanted to immediately in seedbed, transplant land for growing field crops grow one month in seedbed after, transplanting survival rate is 97.5%.
Embodiment 3
Another example of the quick breeding method for tissue culture of Herba Hedyotidis Corymbosae of the present invention, comprises the following steps:
(1) selection of explant and sterilization: get the non-dehiscent fruit of maturation of Herba Hedyotidis Corymbosae as explant, use 2% (v/v) liquid detergent aqueous solution soaking 5min, wire tap water 15-30min successively, with the addition of 2-3 and drip 100 milliliter of 0.1% (v/v) mercuric chloride sterilization 8-10min of Tween-20, aseptic water washing 3-5 time, finally remove surface moisture with sterilized filter paper, obtain explant, wherein sterile water is through autoclaved distilled water
(2) seed germination obtains in vitro cuttings: the explant strip off on superclean bench step (1) obtained, seed is inoculated in MS medium, be 23-27 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 30 days under the condition of 8-10 hour/day, obtains in vitro cuttings, wherein add benzyladenine 6-BA, 30g/L sucrose of 0.2mg/L and the agar of 3.4g/L in MS medium after seed sprouting, the pH value of medium is 5.8
(3) test-tube plantlet Multiple Buds Fast-propagation is cultivated: the in vitro cuttings obtained in step (2) is placed in MS propagating culture medium, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate under the condition of 8-10 hour/day to obtain test-tube plantlet Multiple Buds in 30 days, wherein add kinetin KT, 30g/L sucrose of heteroauxin IAA, 0.5mg/L and the agar of 3.4g/L of benzyladenine 6-BA, 0.2mg/L of 1.5mg/L in MS propagating culture medium, the pH value of medium is 5.8, adventitious buds proliferation coefficient is 25.8
(4) Multiple Buds culture of rootage: the stem section Multiple Buds obtained in step (3) being cut into band terminal bud or leaf bud, be placed in 1/2MS root media, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate the whole plant obtaining for 30 days being with root under the condition of 12-14 hour/day, wherein add root-inducing powder ABT, 30g/L sucrose of heteroauxin IAA, 0.2mg/L and the agar of 3.4g/L of 0.5mg/L in 1/2MS root media, the pH value of medium is 5.8, rooting rate is 96.0%
(5) hardening and transplanting: after step (4) obtains the whole plant being with root, bottle cap is opened in the indoor being 25 DEG C in room temperature, a small amount of running water is added in bottle, hardening 2-4 days, after surface horny is formed, seedling is taken out, clean root medium, be transplanted to immediately in seedbed, transplant land for growing field crops grow one month in seedbed after, transplanting survival rate is 94.0%.。
Embodiment 4
Another example of the quick breeding method for tissue culture of Herba Hedyotidis Corymbosae of the present invention, comprises the following steps:
(1) selection of explant and sterilization: get the non-dehiscent fruit of maturation of Herba Hedyotidis Corymbosae as explant, use 2% (v/v) liquid detergent aqueous solution soaking 5min, wire tap water 15-30min successively, with the addition of 2-3 and drip 100 milliliter of 0.1% (v/v) mercuric chloride sterilization 8-10min of Tween-20, aseptic water washing 3-5 time, finally remove surface moisture with sterilized filter paper, obtain explant, wherein sterile water is through autoclaved distilled water
(2) seed germination obtains in vitro cuttings: the explant strip off on superclean bench step (1) obtained, seed is inoculated in MS medium, be 23-27 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 30 days under the condition of 8-10 hour/day, obtains in vitro cuttings, wherein add benzyladenine 6-BA, 30g/L sucrose of 0.2mg/L and the agar of 3.4g/L in MS medium after seed sprouting, the pH value of medium is 5.8
(3) test-tube plantlet Multiple Buds Fast-propagation is cultivated: the in vitro cuttings obtained in step (2) is placed in MS propagating culture medium, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate under the condition of 8-10 hour/day to obtain test-tube plantlet Multiple Buds in 30 days, wherein add kinetin KT, 30g/L sucrose of heteroauxin IAA, 0.5mg/L and the agar of 3.4g/L of benzyladenine 6-BA, 0.3mg/L of 2.5mg/L in MS propagating culture medium, the pH value of medium is 5.8, adventitious buds proliferation coefficient is 28.2
(4) Multiple Buds culture of rootage: the stem section Multiple Buds obtained in step (3) being cut into band terminal bud or leaf bud, be placed in 1/2MS root media, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate the whole plant obtaining for 30 days being with root under the condition of 12-14 hour/day, wherein add root-inducing powder ABT, 30g/L sucrose of heteroauxin IAA, 0.2mg/L and the agar of 3.4g/L of 0.4mg/L in 1/2MS root media, the pH value of medium is 5.8, rooting rate is 97.5%
(5) hardening and transplanting: after step (4) obtains the whole plant being with root, bottle cap is opened in the indoor being 25 DEG C in room temperature, a small amount of running water is added in bottle, hardening 2-4 days, after surface horny is formed, seedling is taken out, clean root medium, be transplanted to immediately in seedbed, transplant land for growing field crops grow one month in seedbed after, transplanting survival rate is 96.5%.
Claims (1)
1. a method for quickly breeding for Herba Hedyotidis Corymbosae plantlet in vitro, is characterized in that: the method comprises the following steps:
(1) selection of explant and sterilization: get the non-dehiscent fruit of maturation of Herba Hedyotidis Corymbosae as explant, use 2% (v/v) liquid detergent aqueous solution soaking 5min, wire tap water 15-30min successively, with the addition of 2-3 and drip 100 milliliter of 0.1% (v/v) mercuric chloride sterilization 8-10min of Tween-20, aseptic water washing 3-5 time, finally remove surface moisture with sterilized filter paper, obtain explant, wherein sterile water is through autoclaved distilled water
(2) seed germination obtains in vitro cuttings: the explant strip off on superclean bench step (1) obtained, seed is inoculated in MS medium, be 23-27 DEG C in cultivation temperature, intensity of illumination 1500lux, light application time is cultivate 30 days under the condition of 8-10 hour/day, obtains in vitro cuttings, wherein add benzyladenine 6-BA, 30g/L sucrose of 0.2mg/L and the agar of 3.4g/L in MS medium after seed sprouting, the pH value of medium is 5.8
(3) test-tube plantlet Multiple Buds Fast-propagation is cultivated: the in vitro cuttings obtained in step (2) is placed in MS propagating culture medium, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate under the condition of 8-10 hour/day to obtain test-tube plantlet Multiple Buds in 30 days, wherein add kinetin KT, 30g/L sucrose of heteroauxin IAA, 0.1-1.0mg/L and the agar of 3.4g/L of benzyladenine 6-BA, 0.1-0.3mg/L of 0.5-3.0mg/L in MS propagating culture medium, the pH value of medium is 5.8
(4) Multiple Buds culture of rootage: the stem section Multiple Buds obtained in step (3) being cut into band terminal bud or leaf bud, be placed in 1/2MS root media, at cultivation temperature 23-27 DEG C, intensity of illumination 1500lux, light application time is cultivate the whole plant obtaining for 30 days being with root under the condition of 12-14 hour/day, wherein add root-inducing powder ABT, 30g/L sucrose of heteroauxin IAA, 0.1-0.3mg/L and the agar of 3.4g/L of 0.1-1.0mg/L in 1/2MS root media, the pH value of medium is 5.8
(5) hardening and transplanting: after step (4) obtains the whole plant being with root, bottle cap is opened in the indoor being 25 DEG C in room temperature, a small amount of running water is added in bottle, hardening 2-4 days, after surface horny is formed, seedling is taken out, clean root medium, be transplanted to immediately in seedbed, grow one month in seedbed after, transplant land for growing field crops.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111279915A (en) * | 2018-12-07 | 2020-06-16 | 广西作物遗传改良生物技术重点开放实验室 | Method for inducing amphoteric flowers of momordica grosvenori |
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Non-Patent Citations (2)
| Title |
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| 李国平等: "白花蛇舌草叶片离体培养及试管无性系的建立", 《广西植物》 * |
| 黄碧兰等: "乌檀的组织培养", 《植物生理学通讯》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111279915A (en) * | 2018-12-07 | 2020-06-16 | 广西作物遗传改良生物技术重点开放实验室 | Method for inducing amphoteric flowers of momordica grosvenori |
| CN111279915B (en) * | 2018-12-07 | 2021-09-28 | 广西作物遗传改良生物技术重点开放实验室 | Method for inducing amphoteric flowers of momordica grosvenori |
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