CN111771729A - Tissue culture and rapid propagation method for pitaya - Google Patents
Tissue culture and rapid propagation method for pitaya Download PDFInfo
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- CN111771729A CN111771729A CN202010876848.6A CN202010876848A CN111771729A CN 111771729 A CN111771729 A CN 111771729A CN 202010876848 A CN202010876848 A CN 202010876848A CN 111771729 A CN111771729 A CN 111771729A
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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
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention provides a tissue culture and rapid propagation method of pitaya, which comprises the following steps: the method has the advantages of simple and easy operation, effective improvement of the rooting rate, the number of roots, the root length and the seedling height of the pitaya, strong and thick seedling, less branches and good multiplication effect.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a tissue culture and rapid propagation method for pitaya.
Background
Pitaya (Hylocereus undatus Britt.) also known as red dragon fruit, sweet fruit, sesame fruit, sweet fruit, green dragon fruit, etc., is a plant of trigonosteaceae (Cactaceae) genus trigonella, native to the tropical desert areas of Central America such as Brazil and Mexico, and is a relatively popular fruit in local areas. Due to long-term growth in tropical desert areas, leaves of the dragon fruits are degenerated, the photosynthesis function is borne by stems, and a large number of thin-walled cells full of viscous liquid are arranged inside the stems, so that the dragon fruits are beneficial to absorbing as much water as possible in rainy seasons. Although the white-pulp dragon fruit is rich in nutrition, the pulp is light and tasteless, and the taste is not good. The red-core pitaya is used as a rare variety of the pitaya, the fruit shape is perfectly round, the peel is rose, the pulp is dark purple red, the meat quality is tender, the taste is cool, and the red-core pitaya contains rich anthocyanin, multiple vitamins, water-soluble dietary fiber, glucose, mineral substances and the like.
At present, the dragon fruit is mainly cultivated in a propagation mode of seedling, cuttage, grafting and the like in production, and the red-core dragon fruit is propagated by cuttage. The propagation coefficient of cutting seedling is low and is limited by propagation materials, and the tissue propagation method in the prior art has the conditions of high pollution rate, difficult rooting, slow growth, low survival rate of tissue culture seedlings and the like when propagating plants. Can not meet the requirement of red-heart dragon fruit industry development on seedlings.
Disclosure of Invention
In view of the above, the present invention provides a tissue culture and rapid propagation method for pitaya, which solves the above problems.
The technical scheme of the invention is realized as follows: a tissue culture and rapid propagation method of pitaya comprises the following steps:
s1, explant selection: selecting tender branches and stem tips of red-heart pitaya which grow robustly and well and have no diseases and pests;
s2, disinfection of explants: sterilizing the shoot tip of the twig by using 60-80% ethanol and paclitaxel solution for 20-40 s, and then sterilizing for 15-20 min by using 0.1-0.3% liter pump;
s3, induction culture: shearing the disinfected dragon fruit stem segments into small stem segments of 1-3 cm, wherein each stem segment is provided with 1-2 thorn seats, the stem segments are horizontally placed and inoculated into an induction culture medium, the thorn seats face upwards and are placed into a culture chamber for culture, the temperature is controlled to be 20-26 ℃, the illumination intensity is 2200-2500 Lx, the illumination time is 12-18 h/d, the culture period is 30-50 days, and the induction culture medium comprises 2-9 parts of MS basic culture medium, 0.1-1.3 parts of 6-BA, 0.3-2.6 parts of NAA, 0.01-0.38 part of 4-terpinenol and 0.04-0.09 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induced culture, inoculating the young buds into a multiplication culture medium, culturing at the temperature of 22-30 ℃, the pH value of 6-8 and the humidity of 60-80%, culturing for 3-8 days in a dark place, controlling the illumination intensity to be 1500-2000 Lx, continuously culturing for 20-30 days, increasing the illumination intensity to be 3000-6000 Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium comprises the following components: 3-11 parts of WPM basic culture medium, 1-3 parts of diatom ooze, 1.5-2.5 parts of chitin and 0.03-0.12 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings.
Further, the volume ratio of the ethanol to the paclitaxel solution in the step S2 is 1: 2-5.
Further, the concentration of the paclitaxel solution is 12-18 mg/L.
Further, in the S3 induction culture, the formula of an induction culture medium comprises 6 parts of MS minimal medium, 0.8 part of 6-BA, 1.5 parts of NAA, 0.22 part of 4-terpinenol and 0.06 part of rapeseed polypeptide.
Furthermore, in the proliferation culture of S4, the formula of the proliferation culture medium comprises 8 parts of WPM minimal medium, 2 parts of diatom ooze, 2 parts of chitin and 0.07 part of rapeseed polypeptide.
Further, the culture environment for S5 rooting culture is 22-30 ℃, the illumination intensity is 2000-3000 Lx, the illumination is 12-15 h/d, and the culture lasts 16-20 days.
Compared with the prior art, the invention has the beneficial effects that:
the tissue culture and rapid propagation method of the dragon fruit, disclosed by the invention, has the advantages that high-quality seed sources are selected, the operation is easy, the rooting rate, the root number, the root length and the seedling height of the dragon fruit are effectively improved through induction, proliferation and rooting culture, the seedlings of the rooted seedlings are strong in root, few in branches and good in proliferation effect; the method comprises the following steps of sterilizing pitaya explants, adding a taxol solution and ethanol for combination, sterilizing by matching with an ascending pump, reducing the pollution of the explants, and effectively removing the pollution of the thorn positions of the explants under the condition of not damaging the incision positions and tender parts of the explants; the raw materials in the induction culture medium and the proliferation culture medium are reasonably selected and scientifically proportioned, and the dragon fruit is induced and cultured under a specific proportioning to achieve the optimal proliferation and rooting effects.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1
A tissue culture and rapid propagation method of pitaya comprises the following steps:
s1, explant selection: selecting tender branches and stem tips of red-heart pitaya which grow robustly and well and have no diseases and pests;
s2, disinfection of explants: sterilizing shoot tip of tender branch with 55% ethanol and 12mg/L paclitaxel solution at a volume ratio of 1:2 for 20s, and sterilizing with 0.1% pump for 15 min;
s3, induction culture: cutting the stem segments of the disinfected pitaya into small stem segments of 1-3 cm, wherein each stem segment is provided with 1-2 thorn seats, the stem segments are horizontally placed and inoculated into an induction culture medium, the thorn seats face upwards and are placed into a culture chamber for culture, the temperature is controlled to be 20 ℃, the illumination intensity is 2200Lx, the illumination time is 12h/d, the culture period is 30 days, and the induction culture medium comprises 2 parts of MS basic culture medium, 0.1 part of 6-BA, 0.3 part of NAA, 0.01 part of 4-terpinenol and 0.04 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induction culture, inoculating the young buds into a multiplication culture medium, culturing for 3 days in a dark place at the culture temperature of 22 ℃, the pH value of 6 and the humidity of 60%, controlling the illumination intensity to be 1500Lx, continuously culturing for 20 days, increasing the illumination intensity to be 3000Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium is as follows: 3 parts of WPM basic culture medium, 1 part of diatom ooze, 1.5 parts of chitin and 0.03 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings; the temperature of the rooting culture is 22 ℃, the illumination intensity is 2000Lx, the illumination is 12h/d, and the culture is carried out for 16 days.
Example 2
A tissue culture and rapid propagation method of pitaya comprises the following steps:
s1, explant selection: selecting tender branches and stem tips of red-heart pitaya which grow robustly and well and have no diseases and pests;
s2, disinfection of explants: sterilizing shoot tip with 80% ethanol and 18mg/L paclitaxel solution at a volume ratio of 1:5 for 40s, and sterilizing with 0.3% pump for 20 min;
s3, induction culture: cutting the stem segments of the disinfected pitaya into small stem segments of 1-3 cm, wherein each stem segment is provided with 1-2 thorn seats, the stem segments are horizontally placed and inoculated into an induction culture medium, the thorn seats face upwards and are placed into a culture chamber for culture, the temperature is controlled to be 26 ℃, the illumination intensity is 2500Lx, the illumination time is 18h/d, the culture period is 50 days, and the induction culture medium comprises 9 parts of MS basic culture medium, 1.3 parts of 6-BA, 2.6 parts of NAA, 0.38 part of 4-terpinenol and 0.09 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induced culture, inoculating the young buds into a multiplication culture medium, culturing for 8 days in a dark place at the culture temperature of 30 ℃, the pH value of 8 and the humidity of 80%, controlling the illumination intensity to be 2000Lx, continuously culturing for 30 days, increasing the illumination intensity to be 6000Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium is as follows: 11 parts of WPM basic culture medium, 3 parts of diatom ooze, 2.5 parts of chitin and 0.12 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings; the temperature of the rooting culture is 30 ℃, the illumination intensity is 3000Lx, the illumination is 15h/d, and the culture is carried out for 20 days.
Example 3
A tissue culture and rapid propagation method of pitaya comprises the following steps:
s1, explant selection: selecting tender branches and stem tips of red-heart pitaya which grow robustly and well and have no diseases and pests;
s2, disinfection of explants: sterilizing shoot tip with 75% ethanol and 15mg/L paclitaxel solution at a volume ratio of 1:4 for 30s, and sterilizing with 0.2% pump for 18 min;
s3, induction culture: cutting the stem segments of the disinfected pitaya into small stem segments of 1-3 cm, wherein each stem segment is provided with 1-2 thorn seats, the stem segments are horizontally placed and inoculated into an induction culture medium, the thorn seats face upwards and are placed into a culture chamber for culture, the temperature is controlled to be 23 ℃, the illumination intensity is 2400Lx, the illumination time is 15h/d, the culture period is 40 days, and the induction culture medium comprises 6 parts of MS basic culture medium, 0.8 part of 6-BA, 1.5 parts of NAA, 0.22 part of 4-terpinenol and 0.06 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induced culture, inoculating the young buds into a multiplication culture medium, culturing at the temperature of 22-30 ℃, the pH value of 6-8 and the humidity of 60-80%, culturing for 3-8 days in a dark place, controlling the illumination intensity to be 1500-2000 Lx, continuously culturing for 20-30 days, increasing the illumination intensity to be 3000-6000 Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium comprises the following components: 8 parts of WPM basic culture medium, 2 parts of diatom ooze, 2 parts of chitin and 0.07 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings; the temperature of the rooting culture is 26 ℃, the illumination intensity is 2500Lx, the illumination is 13h/d, and the rooting culture is carried out for 18 days.
Example 4
A tissue culture and rapid propagation method of pitaya comprises the following steps:
s1, explant selection: selecting tender branches and stem tips of red-heart pitaya which grow robustly and well and have no diseases and pests;
s2, disinfection of explants: sterilizing shoot tip of tender branch with 75% ethanol and 12mg/L paclitaxel solution at a volume ratio of 1:2 for 20s, and sterilizing with 0.1% pump for 15 min;
s3, induction culture: cutting the stem segments of the disinfected pitaya into small stem segments of 1-3 cm, wherein each stem segment is provided with 1-2 thorn seats, the stem segments are horizontally placed and inoculated into an induction culture medium, the thorn seats face upwards and are placed into a culture chamber for culture, the temperature is controlled to be 20 ℃, the illumination intensity is 2200Lx, the illumination time is 12h/d, the culture period is 30 days, and the induction culture medium comprises 6 parts of MS basic culture medium, 0.8 part of 6-BA, 1.5 parts of NAA, 0.22 part of 4-terpinenol and 0.06 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induction culture, inoculating the young buds into a multiplication culture medium, culturing for 3 days in a dark place at the culture temperature of 22 ℃, the pH value of 6 and the humidity of 60%, controlling the illumination intensity to be 1500Lx, continuously culturing for 20 days, increasing the illumination intensity to be 3000Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium is as follows: 8 parts of WPM basic culture medium, 2 parts of diatom ooze, 2 parts of chitin and 0.07 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings; the temperature of the rooting culture is 22 ℃, the illumination intensity is 2000Lx, the illumination is 12h/d, and the culture is carried out for 16 days.
Example 5
A tissue culture and rapid propagation method of pitaya comprises the following steps:
s1, explant selection: selecting tender branches and stem tips of red-heart pitaya which grow robustly and well and have no diseases and pests;
s2, disinfection of explants: sterilizing shoot tip with 75% ethanol and 18mg/L paclitaxel solution at a volume ratio of 1:5 for 40s, and sterilizing with 0.1% pump for 20 min;
s3, induction culture: shearing the disinfected dragon fruit stem into small stem segments of 1-3 cm, wherein each stem segment is provided with 1-2 thorn seats, the stem segments are horizontally placed and inoculated into an induction culture medium, the thorn seats face upwards and are placed into a culture chamber for culture, the temperature is controlled to be 26 ℃, the illumination intensity is 2500Lx, the illumination time is 18h/d, the culture period is 50 days, and the induction culture medium comprises 2-9 parts of MS basic culture medium, 0.1-1.3 parts of 6-BA, 0.3-2.6 parts of NAA, 0.01-0.38 part of 4-terpinenol and 0.04-0.09 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induced culture, inoculating the young buds into a multiplication culture medium, culturing for 8 days in a dark place at the culture temperature of 30 ℃, the pH value of 8 and the humidity of 80%, controlling the illumination intensity to be 2000Lx, continuously culturing for 30 days, increasing the illumination intensity to be 6000Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium is as follows: 3-11 parts of WPM basic culture medium, 1-3 parts of diatom ooze, 1.5-2.5 parts of chitin and 0.03-0.12 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings; the temperature of the rooting culture is 30 ℃, the illumination intensity is 3000Lx, the illumination is 15h/d, and the culture is carried out for 20 days.
Example 6
The difference between the present embodiment and embodiment 3 is that the volume ratio of ethanol to paclitaxel solution in the step S2 is 1: 2-5.
Comparative example 1
This comparative example differs from example 3 in that in the disinfection of the explant, the shoot tips were disinfected first with 75% ethanol for 30s and then with 0.1% liter pump for 18min, without using paclitaxel solution.
Comparative example 2
The comparative example differs from example 3 in that the formulation of the induction medium in the induction culture is: 10 parts of MS basic culture medium, 1.4 parts of 6-BA, 0.2 part of NAA, 0.03 part of 4-terpinenol and 0.03 part of rapeseed polypeptide.
Comparative example 3
The difference between the comparative example and the example 3 is that in the induction culture, the formula of the induction culture medium does not contain 4-terpinenol and rapeseed polypeptide.
Comparative example 4
This comparative example differs from example 3 in that, in the proliferation culture, the proliferation medium formulation: 2 parts of WPM basic culture medium, 5 parts of diatom ooze, 1.0 part of chitin and 0.02 part of rapeseed polypeptide.
Comparative example 5
The difference between the comparative example and the example 3 is that the proliferation culture medium formula does not contain diatom ooze, chitin and rapeseed polypeptide in the proliferation culture.
Comparative example 6
This comparative example differs from example 3 in that light-shielding culture was not performed in the proliferation culture.
First, test results of tissue culture of pitaya
The proliferation effect of the pitaya tissues cultured in the examples 1 to 6 and the comparative examples 1 to 6 was measured, and the results are as follows:
the above table shows that the cultivation method can effectively improve the proliferation effect of the pitaya tissue, compared with the comparative example 1, the cultivation method of the invention has the advantages that the taxol solution and the ethanol are added for combination, the disinfection is carried out by matching with the pump, the pollution of the explant is reduced, and the pollution of the thorn seat of the explant is effectively eliminated under the condition of not damaging the incision and the tender part of the explant; compared with the comparative examples 2 and 3, the scientific proportion of the induction culture medium and the combination of all the components can effectively improve the induced germination rate and promote the subsequent proliferation culture by combining the induction culture condition in the examples 1 to 6; compared with the comparative examples 4 and 5, the component selection and the proportion of the multiplication medium are beneficial to the multiplication of the pitaya cluster buds, and compared with the comparative example 6, the stress effect of the tender buds can be effectively improved by dark culture in the examples 1 to 6.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A tissue culture and rapid propagation method for pitaya is characterized in that: the method comprises the following steps:
s1, explant selection: selecting tender dragon fruit branch and stem tips which are strong in growth, good in growth vigor and free of diseases and insect pests;
s2, disinfection of explants: sterilizing the shoot tip of the twig by using 60-80% ethanol and paclitaxel solution for 20-40 s, and then sterilizing for 15-20 min by using 0.1-0.3% liter pump;
s3, induction culture: shearing the stem segments of the disinfected pitaya into small stem segments of 1-3 cm, inoculating the small stem segments into an induction culture medium, placing the culture medium into a culture room for culture, controlling the temperature to be 20-26 ℃, the illumination intensity to be 2200-2500 Lx, the illumination time to be 12-18 h/d, and the culture period to be 30-50 days, wherein the induction culture medium comprises 2-9 parts of MS basic culture medium, 0.1-1.3 parts of 6-BA, 0.3-2.6 parts of NAA, 0.01-0.38 part of 4-terpinenol and 0.04-0.09 part of rapeseed polypeptide;
s4, proliferation culture: cutting off callus parts of young buds subjected to induced culture, inoculating the young buds into a multiplication culture medium, culturing at the temperature of 22-30 ℃, the pH value of 6-8 and the humidity of 60-80%, culturing for 3-8 days in a dark place, controlling the illumination intensity to be 1500-2000 Lx, continuously culturing for 20-30 days, increasing the illumination intensity to be 3000-6000 Lx, and continuously culturing for 3 generations, wherein the formula of the multiplication culture medium comprises the following components: 3-11 parts of WPM basic culture medium, 1-3 parts of diatom ooze, 1.5-2.5 parts of chitin and 0.03-0.12 part of rapeseed polypeptide;
s5, rooting culture: cutting off buds of the buds, performing rooting culture to obtain rooted seedlings, and transplanting the rooted seedlings.
2. The tissue culture and rapid propagation method of pitaya according to claim 1, characterized in that: and in the step S2, the volume ratio of the ethanol to the paclitaxel solution is 1: 2-5.
3. The tissue culture and rapid propagation method of pitaya according to claim 1, characterized in that: the concentration of the paclitaxel solution is 12-18 mg/L.
4. The tissue culture and rapid propagation method of pitaya according to claim 1, characterized in that: in the S3 induced culture, the formula of the induced culture medium comprises 6 parts of MS minimal medium, 0.8 part of 6-BA, 1.5 parts of NAA, 0.22 part of 4-terpinenol and 0.06 part of rapeseed polypeptide.
5. The tissue culture and rapid propagation method of pitaya according to claim 1, characterized in that: in the proliferation culture of S4, the formula of the proliferation culture medium comprises 8 parts of WPM basic culture medium, 2 parts of diatom ooze, 2 parts of chitin and 0.07 part of rapeseed polypeptide.
6. The tissue culture and rapid propagation method of pitaya according to claim 1, characterized in that: the culture environment for S5 rooting culture is 22-30 ℃, the illumination intensity is 2000-3000 Lx, the illumination is 12-15 h/d, and the culture lasts for 16-20 days.
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| CN114097610A (en) * | 2021-10-28 | 2022-03-01 | 浙江万里学院 | Pitaya suspension cell culture method |
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