CN108770695B - Method for in-vitro tissue culture and rapid seedling establishment of red-heart pitaya - Google Patents
Method for in-vitro tissue culture and rapid seedling establishment of red-heart pitaya Download PDFInfo
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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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention belongs to the technical field of plant propagation, and particularly relates to a method for in-vitro tissue culture and rapid seedling establishment of red-heart pitaya. The improved thorn seats are immersed in the culture medium to remarkably accelerate the sprouting and the quantity of the small buds, and in the process of the rooting culture of the small buds, the adventitious root is primarily transplanted, so that the production period of the red-heart pitaya seedlings is remarkably shortened, the multiplication coefficient is 3-5 times, the rooting rate is 100%, the survival rate after transplanting is over 96%, and the seedling production period is about 90 days.
Description
Technical Field
The invention belongs to the technical field of plant propagation, and particularly relates to a method for in-vitro tissue culture and rapid seedling establishment of red-heart pitaya.
Background
The red-core dragon fruit is also called red dragon fruit and is dragon fruit: (Hylocereus undatus) The pulp of the novel fruit with good health care effect contains a large amount of natural anthocyanin and shows red. The red heart pitaya has rich nutrition, contains rich mineral substances such as calcium, phosphorus, iron and the like and vitamin groups, is also rich in plant albumin, anthocyanin and high-amount water-soluble dietary fiber, has the sweetness far higher than that of the common pitaya, changes the common understanding of the prior dragon fruit of being good-looking and not delicious, is popular among people, and has the advantages that the red heart pitaya has rich nutrition, and the red heart pitaya contains the plant albumin, the anthocyanin and the high-amount water-soluble dietary fiberThe tropical and subtropical areas of China are widely introduced and cultivated, the seedling demand is large, and the seedling supply is not in demand.
The dragon fruit is mainly propagated by the modes of seeds, cuttage, grafting and the like, and the propagation speed of the methods is low and is limited by the supply of propagation materials, so that the market demand is difficult to meet. In recent years, researchers have also studied and made certain progress in tissue culture of pitaya. For example, patent application No. 201410135613.6 entitled "method for increasing seedling rate of tissue culture seedling of dragon fruit" discloses a method for increasing seedling rate of tissue culture seedling of dragon fruit, wherein 35-40 days are required for sprouting from the thorn of explant, and the seedling period is about 100 days. The invention patent with the patent application number of 201510297283.5 discloses a tissue culture and rapid propagation method of pitaya, wherein the time for inducing buds through explants is 28-32d, and the seedling period is more than 100 d. Different genotypes of the same species have different requirements on the tissue culture and rapid propagation technical system. The methods are all tissue culture methods of common pitaya, and the methods have poor effect in tissue culture application of red-heart pitaya.
At present, a stable and efficient in-vitro rapid propagation system for red-heart pitaya is not provided. Meanwhile, the method can not further shorten the culture time of each link in the tissue culture of the red-heart pitaya and ensure the quality of the red-heart pitaya, and can directly influence the goal of whether the tissue culture production of the red-heart pitaya can realize industrial production.
Disclosure of Invention
The invention aims to: aiming at the problems, the method for tissue culture and rapid seedling establishment of the red-heart pitaya in vitro provides technical support for industrialized, large-scale and intensive production of the red-heart pitaya seedling.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for in vitro tissue culture and rapid seedling formation of red-heart pitaya is characterized by comprising the following steps: taking a stem section with a thorn seat of the red-heart pitaya as an explant, removing the small thorn after disinfection treatment, and respectively carrying out induction culture, proliferation culture, rooting culture and transplanting culture.
The disinfection method comprises the following steps: soaking the explant with liquid detergent, brushing the part of the spinous seat with a brush, and washing with clear water for 30 min; then soaking the explant in 75% alcohol for 10s, taking out, washing with sterile water for 2 times, putting the explant into 0.1% mercuric chloride solution for detoxification for 5-8min, taking out, and repeatedly washing with sterile water for 6-8 times.
The method for inducing culture comprises the following steps: vertically inoculating the sterilized explant with the skin prick removed on an induction culture medium, and performing illumination culture for 10-15d under the conditions of illumination intensity of 1600-.
Wherein the explant is vertically inserted into the induction medium and the spinous process part is immersed in the induction medium. The induction culture medium is MS + 6-BA 2.0-3.0 mg/L + NAA 1.0-1.5 mg/L + sucrose 20-30 g/L + agar 4.0-6.0 g/L, and the pH value is 5.8-6.2.
By adopting the induction culture medium and the mode of vertically inoculating the explant on the induction culture medium for induction, and compared with the prior art, the number of days for sprouting can be obviously shortened, the inventor carries out a comparative test on the disclosed induction culture medium by adopting the mode of positioning the traditional acanthosis position on the culture medium, and the results are as follows:
the proliferation culture method comprises the following steps: cutting the bud to induce germination from explant, inoculating to proliferation culture medium, and culturing under illumination intensity of 1600-1800Lux for 12 hr/day and temperature of 25 + -2 deg.C for 25-30d to sprout 3-5 clump buds with height of 4-5cm from bud base. Wherein the enrichment medium is MS + 6-BA 1.0-2.0 mg/L + NAA 0.5-1.0 mg/L + sucrose 20-30 g/L + agar 4.0-7.0 g/L, and the pH value is 5.8-6.2.
Compared with the components of the disclosed proliferation culture medium, the proliferation culture medium has the following differences:
the proliferation culture medium adopted by the invention combines the low-concentration cytokinin 6-BA and the auxin NAA, generates obvious synergistic interaction, has the proliferation coefficient reaching 3-5 times, has vigorous growth vigor and height reaching 4-5cm, can be used for rooting induction culture without a strong seedling culture link, shortens the production period of the tissue culture seedling of the red-heart pitaya, and also saves the production cost.
The rooting culture method comprises the following steps: cutting the cluster buds obtained by propagation culture into single buds, transferring the single buds to a rooting culture medium, performing illumination culture for 10-15 days under the conditions of illumination intensity of 1600 plus 1800Lux, illumination for 12 h/day and temperature of 25 +/-2 ℃, and transplanting the single buds to a seedling culture plug tray for transplanting culture when 2-3 adventitious roots sprout at the base of the single buds.
Wherein the rooting medium is 1/2MS + IBA 0.2-1.0 mg/L + IAA 0.1-0.5 mg/L + sucrose 20-30 g/L + agar 4.0-7.0 g/L, and the pH value is 5.8-6.2.
Compared with the components of the rooting induction culture medium disclosed by the invention:
the 1/2MS with low salt content and 2 auxin exogenous hormones with low concentration are used in proportion, so that a relatively obvious synergistic effect is exerted, the inoculated buds can be promoted to sprout 2-3 adventitious roots at the base part within 10-15 days, the rooting rate reaches 100%, the buds grow strongly, and the method can be used for transplanting seedling culture, and the production period is shortened by more than 15 days compared with the prior art.
The transplanting culture method comprises the following steps: transplanting the seedlings into a container filled with red soil: humus soil: 1 seedling in each hole of a seedling culture hole tray with vermiculite =1:1:1 matrix, watering enough water, and placing the seedling culture hole tray in a double-layer arched shed in a greenhouse to culture for 30d to obtain the red-heart pitaya seedlings with the height of 8-12cm, the number of the roots of 6-8, the length of the roots of 10-12cm and the stout root systems.
The double-layer arched shed in the greenhouse comprises a small arched shed which is built at first and is 50-60cm high, a layer of white plastic film covers the small arched shed, and then an arched shed which is built at the outer layer of the small arched shed and is 100cm high is built, and a layer of white plastic film covers the small arched shed.
The invention establishes a stable and efficient in-vitro tissue culture and rapid seedling establishment technical system of the red-heart pitaya by taking the stem section with the thorn seat of the red-heart pitaya as an explant, removing the small thorn after disinfection treatment, and respectively carrying out the steps of small bud induction culture, proliferation culture, rooting culture, transplanting culture and the like. Wherein, the spiny part of the explant is immersed in the culture medium in the step (2), so that the induction time of the small bud can be shortened by about 15d compared with the prior art; the proliferation culture in the step (3) realizes the proliferation coefficient of 3-5 times; when the rooting culture in the step (4) is carried out, transplanting culture is carried out until 2-3 adventitious roots are initially germinated at the base part of the small bud, the culture time is shortened by about 15d compared with the prior art, and the rooting rate reaches 100%; the survival rate of the transplanting culture in the step (5) is more than 96 percent. By comprehensively applying the technology, the nursery stock meeting the production requirement can be obtained within 90 days, and more than 100 thousands of seedlings can be produced per year.
In conclusion, the in-vitro tissue culture and rapid seedling formation method of the red-heart pitaya is a rapid propagation technology of the red-heart pitaya, and is suitable for industrialized, large-scale and intensive production of the seedlings of the red-heart pitaya.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1 the method for in vitro tissue culture and rapid seedling establishment of the red-heart pitaya disclosed by the embodiment comprises the following steps:
(1) selecting and disinfecting explants: soaking the stem of red-core dragon fruit with thorn seat in detergent solution, brushing the thorn seat with brush, and washing with clear water for 30 min; soaking the explant in 75% alcohol for 10s, taking out, washing with sterile water for 2 times, disinfecting in 0.1% mercuric chloride solution for 5-8min, taking out, washing with sterile water for 6-8 times, and removing skin prick with disinfected forceps;
(2) and (3) induction culture: vertically inoculating the sterilized explant with skin pricks removed on an induction culture medium of MS + 6-BA 2.0mg/L + NAA 1.5 mg/L + sucrose 20g/L + agar 6.0g/L and with the pH value of 5.8-6.2, immersing the prick seat part of the explant in the culture, and culturing for 10-15 days under the conditions of illumination intensity of 1600 plus 1800Lux, illumination intensity of 12 h/day and temperature of 25 +/-2 ℃ to obtain a bud with the height of about 2-3cm at the prick seat part of the explant;
(3) and (3) proliferation culture: cutting the bud for inducing germination from the explant, inoculating the cut bud on a culture medium of MS + 6-BA 1.0mg/L + NAA 1.0mg/L + sucrose 30g/L + agar 4.0g/L and pH value of 5.8-6.2 for propagation culture, and placing the culture medium under the conditions of illumination intensity of 1600 Lux and 1800Lux, illumination intensity of 12 h/day and temperature of 25 +/-2 ℃ for illumination culture for 25-30d, and germinating 3-5 cluster buds with the height of about 4-5cm from the bud base part;
(4) rooting culture: cutting the cluster buds obtained by propagation culture into single buds, transferring the single buds to a rooting culture medium of 1/2MS + IBA 0.5mg/L + IAA 0.1mg/L + sucrose 20g/L + agar 5g/L and pH value of 5.8-6.2, placing the single buds on a rooting culture medium with illumination intensity of 1600 plus 1800Lux and illumination intensity of 12 h/day and temperature of 25 +/-2 ℃ for illumination culture for 10-15 days, and transplanting the single buds to a seedling culture tray for transplanting culture when 2-3 adventitious roots sprout at the base of the single buds;
(5) transplanting and culturing: transplanting the seedlings into a container filled with red soil: humus soil: 1 seedling is planted in each hole of a seedling culture hole tray with vermiculite =1:1:1 matrix, sufficient water is poured, and the seedling culture hole tray is placed in a double-layer arched shed in a greenhouse to be cultured for 30 days, so that the seedlings can be bagged and taken out of the nursery.
By adopting the method to carry out rapid propagation of the red-heart pitaya, the average multiplication coefficient is up to 4.6 times, the rooting rate is up to 100 percent, the average transplanting culture survival rate is 98.6 percent, and red-heart pitaya seedlings with the average seedling height of 10.6cm, the average lateral root number of 8, the average root length of 12cm and thick root systems can be obtained within 90 days.
Embodiment 2, the method for tissue culture and rapid seedling establishment in vitro of red-heart pitaya disclosed in the embodiment comprises the following steps:
(1) selecting and disinfecting explants: soaking the stem of red-core dragon fruit with thorn seat in detergent solution, brushing the thorn seat with brush, and washing with clear water for 30 min; soaking the explant in 75% alcohol for 10s, taking out, washing with sterile water for 2 times, disinfecting in 0.1% mercuric chloride solution for 5-8min, taking out, washing with sterile water for 6-8 times, and removing skin prick with disinfected forceps;
(2) and (3) induction culture: vertically inoculating the sterilized explant with skin pricks removed on an induction culture medium of MS + 6-BA 3.0mg/L + NAA 1.0mg/L + sucrose 30g/L + agar 4.0g/L and with the pH value of 5.8-6.2, immersing the prick seat part of the explant in the culture, and culturing for 10-15 days under the conditions of illumination intensity of 1600 plus 1800Lux, illumination intensity of 12 h/day and temperature of 25 +/-2 ℃ to obtain a bud with the height of about 2-3cm at the prick seat part of the explant;
(3) and (3) proliferation culture: cutting the bud for inducing germination from the explant, inoculating the cut bud on a culture medium of MS + 6-BA 2.0mg/L + NAA 0.5mg/L + sucrose 30g/L + agar 7.0g/L and pH value of 5.8-6.2 for propagation culture, and performing the propagation culture for 25-30d under the conditions of illumination intensity of 1600 Lux and illumination intensity of 1800Lux, illumination time of 12 h/day and temperature of 25 +/-2 ℃ to germinate 3-5 cluster buds with the height of about 4-5cm from the bud base part;
(4) rooting culture: cutting the cluster buds obtained by propagation culture into single buds, transferring the single buds to a rooting culture medium of 1/2MS + IBA 1.0mg/L + IAA 0.5mg/L + sucrose 30g/L + agar 6g/L and pH value of 5.8-6.2, placing the single buds on a rooting culture medium with illumination intensity of 1600 plus 1800Lux and illumination intensity of 12 h/day and temperature of 25 +/-2 ℃ for illumination culture for 10-15 days, and transplanting the single buds to a seedling culture tray for transplanting culture when 2-3 adventitious roots sprout at the base of the single buds;
(5) transplanting and culturing: transplanting the seedlings into a container filled with red soil: humus soil: 1 seedling is planted in each hole of a seedling culture hole tray with vermiculite =1:1:1 matrix, sufficient water is poured, and the seedling culture hole tray is placed in a double-layer arched shed in a greenhouse to be cultured for 30 days, so that the seedlings can be bagged and taken out of the nursery.
By adopting the method to carry out rapid propagation of the red-heart pitaya, the average multiplication coefficient is 5 times, the rooting rate is 100 percent, the average transplanting culture survival rate is 96.7 percent, and red-heart pitaya seedlings with the average seedling height of 11.8cm, the average lateral root number of 6.9 roots, the average root length of 11.6cm and thick root systems can be obtained within 90 days.
Claims (3)
1. A method for in vitro tissue culture and rapid seedling formation of red-heart pitaya specifically comprises the following steps:
(1) selecting and disinfecting explants: soaking the stem of red-core dragon fruit with thorn seat in detergent solution, brushing the thorn seat with brush, and washing with clear water for 30 min; soaking the explant in 75% alcohol for 10s, taking out, washing with sterile water for 2 times, sterilizing in 0.1% mercuric chloride solution for 5-8min, taking out, washing with sterile water for 6-8 times, and removing skin prick with sterilized forceps;
(2) and (3) induction culture: vertically inoculating the explant which is disinfected and has skin pricks removed on an induction culture medium, immersing the prick seat part of the explant in the culture medium, and culturing for 10-15 days under the conditions of illumination intensity of 1600-1800Lux, illumination for 12 h/day and temperature of 25 +/-2 ℃ to obtain the bud with the height of about 2-3cm at the prick seat part of the explant;
(3) and (3) proliferation culture: cutting the bud induced to sprout from the explant, inoculating the cut bud on a proliferation culture medium for proliferation culture, and performing the proliferation culture under the conditions of illumination intensity of 1600-;
(4) rooting culture: cutting the cluster buds obtained by propagation culture into single buds, transferring the single buds to a rooting culture medium, performing illumination culture for 10-15 days under the conditions of illumination intensity of 1600 plus 1800Lux, illumination for 12 h/day and temperature of 25 +/-2 ℃, and transplanting the single buds to a seedling culture plug tray for transplanting culture when 2-3 adventitious roots sprout at the base of the single buds;
(5) transplanting and culturing: transplanting the seedlings into a container filled with red soil: humus soil: 1 seedling is planted in each hole of a seedling culture hole tray with vermiculite =1:1:1 matrix, sufficient water is poured, and the seedling culture hole tray is placed in a double-layer arched shed in a greenhouse to be cultured for 30 days, so that the seedlings can be bagged and taken out of the nursery.
2. The method for in vitro tissue culture and rapid seedling establishment of the red-core pitaya as claimed in claim 1, wherein the induction culture medium adopts MS + 6-BA 2.0-3.0 mg/L + NAA 1.0-1.5 mg/L + sucrose 20-30 g/L + agar 4.0-6.0 g/L, and the pH value is 5.8-6.2.
3. The method for in vitro tissue culture and rapid seedling establishment of the red-core pitaya as claimed in claim 1, wherein the proliferation culture medium adopts MS + 6-BA 1.0-2.0 mg/L + NAA 0.5-1.0 mg/L + sucrose 20-30 g/L + agar 4.0-7.0 g/L, and the pH value is 5.8-6.2.
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| CN111771729A (en) * | 2020-08-27 | 2020-10-16 | 海南瑞民农业科技有限公司 | Tissue culture and rapid propagation method for pitaya |
| CN113229146A (en) * | 2021-06-02 | 2021-08-10 | 中国热带农业科学院橡胶研究所 | Tissue culture, transplantation and domestication method for red-meat pitaya |
| CN113170734B (en) * | 2021-06-02 | 2022-11-11 | 中国热带农业科学院橡胶研究所 | Tissue culture, transplantation and domestication method for bird's nest fruit |
| CN116472959A (en) * | 2023-04-11 | 2023-07-25 | 广西硕果农业开发有限公司 | Culture method for bird's nest fruit seedlings |
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