CN114009341A - Tissue culture and rapid propagation method for excellent single-plant jack fruit - Google Patents
Tissue culture and rapid propagation method for excellent single-plant jack fruit Download PDFInfo
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- CN114009341A CN114009341A CN202111473950.2A CN202111473950A CN114009341A CN 114009341 A CN114009341 A CN 114009341A CN 202111473950 A CN202111473950 A CN 202111473950A CN 114009341 A CN114009341 A CN 114009341A
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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
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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
- A01H4/002—Culture media for tissue culture
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Abstract
The invention provides a method for rapidly propagating excellent single-plant tissue culture of jack fruit, and belongs to the technical field of jack fruit seedling propagation. The propagation method of the invention comprises the following steps: (1) after the explant is disinfected, inoculating the explant into a callus induction culture medium to obtain callus; (2) inoculating the callus into a bud induction culture medium to obtain cluster buds; (3) inoculating the cluster buds into a bud multiplication culture medium and stimulating for 1-3 days; (4) propagating and culturing the cluster buds stimulated in the step (3) for 20-25 days; (5) inoculating the cluster buds cultured in the step (4) in a rooting culture medium to obtain a complete plant; (6) and (4) hardening off the whole plant and transplanting. The excellent single-plant tissue culture rapid propagation method for the jackfruit provided by the invention can be used for propagating a large number of excellent jackfruit seedlings and buds in a short time.
Description
Technical Field
The invention relates to the technical field of jackfruit seedling propagation, in particular to a method for rapidly propagating jackfruit by excellent single-plant tissue culture.
Background
The jackfruit is the heaviest fruit in the world, and has fresh and tender meat quality, faint scent and sweet taste and super delicious taste. The planting history of more than one thousand years in China. The jackfruit is generally propagated by seeds, but the propagation time of the seeds is long, and the offspring variation is extremely large, so that researches show that only more than one dozen of jackfruit single plants with excellent quality can be obtained by using the seeds to propagate 2 ten thousand jackfruit. In order to maintain the excellent properties of the jackfruit, people begin to try grafting technology, and at present, jackfruit grafting methods mainly comprise grafting, cutting grafting and abdominal grafting. However, the survival rate of the traditional methods such as grafting, cutting and web grafting is low, and the survival rate of grafting and cutting grafting is generally below 20%. In the actual variety improvement process of the jackfruit, the excellent jackfruit variety is basically a single plant, the number of grafted buds is very small, the requirement of large-area planting production cannot be met, and the jackfruit variety is not suitable for commercial use.
Disclosure of Invention
Aiming at the problems, the invention uses the tissue culture technology to carry out tissue culture by taking the bud point of the excellent single plant of the jack fruit as an explant, and can propagate a large amount of cluster buds and the excellent single plant in a short time.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for culturing and rapidly propagating excellent single-plant tissue of jackfruit, which comprises the following steps:
(1) after the explant is disinfected, inoculating the explant into a callus induction culture medium, and carrying out dark culture at the temperature of 28-35 ℃ for 5-10 days to obtain a callus;
(2) inoculating the callus into a bud induction culture medium, and culturing for 18-22 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain cluster buds;
(3) inoculating the cluster buds into a bud multiplication culture medium, and stimulating for 1-3 days under the conditions of temperature of 10-15 ℃, illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day;
(4) placing the cluster buds stimulated in the step (3) under the conditions of illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day at 28-35 ℃ for propagation culture for 20-25 days;
(5) inoculating the cluster buds subjected to propagation culture in the step (4) into a rooting culture medium, and culturing for 22-28 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain a complete plant;
(6) and (4) hardening off the whole plant and transplanting.
Preferably, the explant is the bud of jackfruit.
Preferably, the callus induction culture medium is formed by adding 1.5-2.5 mg/l IBA, 0.1-0.5 mg/l GA3 and 10-20 mg/l sucrose to 1/2MS culture medium.
Preferably, the bud induction culture medium is added with 1.0-4.0 mg/l of 6-BA and 0.5-1.0 mg/l of IBA on the basis of MS culture.
Preferably, the bud multiplication culture medium is formed by adding 0.5-1.5 mg/l6-BA, 0.1-0.3 mg/l 2,4-D and 15-25 mg/l sucrose on the basis of an MS culture medium.
Preferably, the rooting medium is 1/2MS medium added with 0.1-0.5 mg/l NAA, 0.2-0.4 mg/l GA3 and 10-20 mg/l sucrose.
The excellent single-plant tissue culture rapid propagation method for the jackfruit provided by the invention can be used for propagating a large number of excellent jackfruit seedlings and buds in a short time. The callus inductivity of the jackfruit bud points during the propagation of jackfruit tissue culture seedlings is more than 98 percent, the adventitious bud differentiation rate is 100 percent, the average multiplication coefficient is 3.53, the average seedling height is 4.49cm, the average number of roots is 6.76, the average root length is 6.55cm, the longest root length is 12.5cm, and the field transplantation survival rate can reach 98.00 percent.
Detailed Description
The invention provides a method for culturing and rapidly propagating excellent single-plant tissue of jackfruit, which comprises the following steps:
(1) after the explant is disinfected, inoculating the explant into a callus induction culture medium, and carrying out dark culture at the temperature of 28-35 ℃ for 5-10 days to obtain a callus;
(2) inoculating the callus into a bud induction culture medium, and culturing for 18-22 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain cluster buds;
(3) inoculating the cluster buds into a bud multiplication culture medium, and stimulating for 1-3 days under the conditions of temperature of 10-15 ℃, illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day;
(4) placing the cluster buds stimulated in the step (3) under the conditions of illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day at 28-35 ℃ for propagation culture for 20-25 days;
(5) inoculating the cluster buds subjected to propagation culture in the step (4) into a rooting culture medium, and culturing for 22-28 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain a complete plant;
(6) and (4) hardening off the whole plant and transplanting.
The method comprises the steps of disinfecting an explant, inoculating the sterilized explant to a callus induction culture medium, and carrying out dark culture at the temperature of 28-35 ℃ for 5-10 days to obtain the callus.
In the present invention, the explant is preferably the shoot of a elite individual of jack fruit.
In the invention, the disinfection is preferably carried out by soaking in 0.1-0.2% mercuric chloride solution for 5-15 min, more preferably by soaking in 0.15% mercuric chloride solution for 10min, and then washing with tap water.
In the invention, the temperature for callus induction culture is 28-35 ℃, preferably 30-34 ℃, and more preferably 33 ℃.
In the present invention, the callus induction culture time is 5 to 10 days, preferably 6 to 9 days, and more preferably 8 days.
In the invention, the callus induction culture medium is preferably prepared by adding 1.5-2.5 mg/l IBA, 0.1-0.5 mg/l GA3 and 10-20 mg/l sucrose to 1/2MS culture medium.
In the invention, the concentration of IBA in the callus induction culture medium is more preferably 1.8-2.3 mg/l, and still more preferably 2.0 mg/l.
In the present invention, the concentration of GA3 in the callus induction medium is more preferably 0.2 to 0.4mg/l, and still more preferably 0.3 mg/l.
In the invention, the concentration of sucrose in the callus induction culture medium is more preferably 12-18 mg/l, and still more preferably 15 mg/l.
After obtaining the callus, inoculating the callus into a bud induction culture medium, and culturing for 18-22 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain cluster buds.
In the invention, the temperature for inducing and culturing the cluster buds is 22-26 ℃, preferably 23-25 ℃, and more preferably 24 ℃.
In the invention, the illumination intensity of the cluster bud induction culture is 1200-1500 lux, and 1300lux is preferred.
In the invention, the illumination time for inducing and culturing the cluster buds is 8-12 h/day, preferably 9-11 h/day, and more preferably 10 h/day.
In the invention, the time for inducing and culturing the cluster buds is 18-22 days, and preferably 20 days.
In the present invention, the bud induction medium is preferably supplemented with 1.0-4.0 mg/l6-BA and 0.5-1.0 mg/l IBA based on MS culture.
In the present invention, the concentration of 6-BA in the shoot induction medium is more preferably 2.0 to 3.0mg/l, and still more preferably 2.5 mg/l.
In the present invention, the concentration of IBA in the shoot induction medium is more preferably 0.6 to 0.9mg/l, and still more preferably 0.75 mg/l.
After cluster buds are obtained through induction, the cluster buds are inoculated into a bud multiplication culture medium and stimulated for 1-3 days under the conditions of temperature of 10-15 ℃, illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day.
In the invention, the temperature for stimulating and culturing the cluster buds is 10-15 ℃, and preferably 12 ℃.
In the invention, the illumination intensity during the cluster bud stimulation culture is 1200-1500 lux, and 1300lux is preferred.
In the invention, the illumination time for stimulating and culturing the cluster buds is 8-12 h/day, preferably 9-11 h/day, and more preferably 10 h/day.
In the invention, the time for stimulating and culturing the cluster buds is 1-3 days, preferably 2 days.
In the invention, the bud multiplication culture medium is preferably added with 0.5-1.5 mg/l of 6-BA, 0.1-0.3 mg/l of 2,4-D and 15-25 mg/l of cane sugar on the basis of an MS culture medium.
In the present invention, the concentration of 6-BA in the shoot growth medium is more preferably 0.8 to 1.2mg/l, and still more preferably 1.0 mg/l.
In the present invention, the concentration of 2,4-D in the shoot growth medium is more preferably 0.15 to 0.25 mg/l. Still more preferably 0.2 mg/l.
In the present invention, the concentration of sucrose in the shoot growth medium is more preferably 18 to 23mg/l, and still more preferably 20 mg/l.
After the stimulation culture is finished, directly placing the cluster buds under the conditions of the temperature of 28-35 ℃, the illumination intensity of 1200-1500 lux and the illumination time of 8-12 h/day for propagation culture for 20-25 days.
In the invention, the temperature for propagation culture of the cluster buds is 28-35 ℃, preferably 30-34 ℃, and more preferably 33 ℃.
In the invention, the illumination intensity for the propagation culture of the cluster buds is 1200-1500 lux, and 1300lux is preferred.
In the invention, the illumination time for the propagation culture of the cluster buds is 8-12 h/day, preferably 9-11 h/day, and more preferably 10 h/day.
In the present invention, the time for propagation culture of the cluster buds is 20 to 25 days, preferably 21 to 24 days, and more preferably 23 days.
After the propagation culture is finished, inoculating the cluster buds subjected to the propagation culture into a rooting culture medium, and culturing for 22-28 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain a complete plant.
In the invention, the temperature for rooting culture is 22-26 ℃, preferably 23-25 ℃, and more preferably 24 ℃.
In the invention, the illumination intensity of rooting culture is 1200-1500 lux, and 1300lux is preferred.
In the invention, the illumination time of rooting culture is 8-12 h/day, preferably 9-11 h/day, and further preferably 10 h/day.
In the invention, the rooting culture time is 22-28 days, preferably 23-26 days, and more preferably 25 days.
In the invention, the rooting medium is preferably 1/2MS medium added with 0.1-0.5 mg/l NAA, 0.2-0.4 mg/l GA3 and 10-20 mg/l sucrose.
In the invention, the concentration of NAA in the rooting medium is further preferably 0.2-0.4 mg/l, and is further preferably 0.3 mg/l.
In the invention, the concentration of GA3 in the rooting medium is preferably 0.25-0.35 mg/l, and more preferably 0.3 mg/l.
In the invention, the concentration of sucrose in the rooting medium is more preferably 12-18 mg/l, and still more preferably 15 mg/l.
After the cluster buds grow into a complete plant, the complete plant is transplanted after hardening off.
In the invention, the seedling exercising is preferably carried out according to the following steps: adding 2-4 ml of tap water, preferably 3ml of tap water into a culture bottle filled with a rooting culture medium of a complete plant, opening a bottle mouth, and hardening seedlings for 3-7 days, preferably 5 days;
in the invention, the tissue culture seedlings after hardening off are carefully taken out from the culture bottle, the root culture medium is cleaned, and the tissue culture seedlings are transplanted to a sand bed for one month and then are transplanted to a field.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The jackfruit bud points used in this example were from the apical and axillary buds of a elite single plant of jackfruit. Placing the collected jackfruit bud points in a sterile cup, sterilizing on an ultra-clean workbench, pouring 0.15% mercuric chloride solution into the sterile cup, soaking for 10min, pouring out the mercuric chloride solution, and washing with tap water for 3 times. Inoculating the sterilized bud points into a callus induction culture medium, culturing in the dark at 33 ℃ for 8 days, and carrying out dedifferentiation on the bud points to form callus. Of the 200 shoots inoculated, callus formation was induced except for 3 that were contaminated. Therefore, the callus induction success rate of the jackfruit bud point in the embodiment is 98.5%.
And respectively inoculating 197 callus tissues successfully induced into a bud induction culture medium, culturing for 20 days under the conditions of 24 ℃, 1300lux of illumination intensity and 10 h/day of illumination time, differentiating the callus tissues to form adventitious buds and cluster buds, and counting the differentiation rate of the adventitious buds to be 100%.
Inoculating the differentiated adventitious buds and the cluster buds into bud multiplication culture, dividing the cluster buds with a plurality of adventitious buds, then respectively inoculating the cluster buds into the bud multiplication culture, ensuring that only one adventitious bud seedling exists in one culture bottle, and finally obtaining 396 adventitious buds of the culture bottle. Then, the culture was stimulated for 2 days at a temperature of 12 ℃, a light intensity of 1300lux and a light time of 10 h/day. The purpose of low-temperature stimulation is to improve the proliferation rate of buds and culture strong seedlings. After the stimulation culture is finished, the culture bottle is placed at the temperature of 33 ℃, the illumination intensity is 1300lux, the illumination time is 10 h/day, the propagation culture is carried out for 23 days, and the number of the proliferated adventitious buds is counted to be 1528 strains. Therefore, the growth coefficient was 3.86, and the average seedling height was 4.53 cm.
And (3) inoculating the cluster buds after propagation culture into a rooting culture medium, and culturing for 25 days under the conditions of 24 ℃, 1300lux of illumination intensity and 10 h/day of illumination time to obtain a complete plant. And counting the rooting condition, and obtaining the result that the cluster buds in 396 culture bottles have root systems, namely the rooting rate is 100%. And counting the number of roots and the length of the roots to obtain the average number of the roots per bottle of 6.23, the average length of the roots of 6.58cm and the length of the longest root of 12.3 cm.
And placing the obtained complete plants in an open place, adding 3ml of tap water into each culture bottle for hardening seedlings, dividing all the plants into single plants after hardening the seedlings for 5 days, and culturing the single plants in a transplanting sand bed for one month, wherein the transplanting survival rate in the sand bed is 94.96%. Transplanting the seedlings into a field after one month, and counting the transplanting survival rate of the field to be 98.00 percent after 2 months of field cultivation.
In this example, the callus induction medium was 1/2MS +2.0mg/l IBA +0.3mg/l GA3+15mg/l sucrose; the bud induction culture medium is MS +2.5mg/l 6-BA +0.75mg/l IBA; the bud multiplication culture medium is MS +1.0mg/l 6-BA +0.2mg/l 2,4-D +20mg/l sucrose; the rooting medium is 1/2MS +0.3mg/l NAA +0.3mg/l GA3+15mg/l sucrose.
Example 2
The jackfruit bud points used in this example were from the apical and axillary buds of a elite single plant of jackfruit. Placing the collected jackfruit bud points in a sterile cup, sterilizing on an ultra-clean workbench, pouring 0.1% mercuric chloride solution into the sterile cup, soaking for 15min, pouring out the mercuric chloride solution, and washing with tap water for 3 times. Inoculating the sterilized bud points into a callus induction culture medium, culturing in the dark at 28 ℃ for 10 days, and carrying out dedifferentiation on the bud points to form callus. In the present example, the callus induction success rate of the jackfruit bud point is 98.0%.
Respectively inoculating the successfully induced callus into a bud induction culture medium, culturing for 18 days at the temperature of 26 ℃, the illumination intensity of 1500lux and the illumination time of 8 h/day, and enabling the callus to differentiate to form adventitious buds and cluster buds, wherein the differentiation rate of the adventitious buds is counted to be 100%.
Inoculating the differentiated adventitious buds and the cluster buds into bud multiplication culture, dividing the cluster buds with a plurality of adventitious buds, then respectively inoculating the cluster buds into the bud multiplication culture, ensuring that only one adventitious bud seedling exists in one culture bottle, and finally obtaining culture bottles with 432 adventitious buds. Then, the culture was stimulated for 3 days at a temperature of 10 ℃, a light intensity of 1500lux and a light time of 8 h/day. After the stimulation culture is finished, the culture bottle is placed at 35 ℃, the illumination intensity is 1500lux, the illumination time is 8 h/day, the propagation culture is carried out for 25 days, and the number of the proliferated adventitious buds is 1435. Therefore, the growth coefficient was 3.32, and the average seedling height was 4.36 cm.
And (3) inoculating the cluster buds after propagation culture into a rooting culture medium, and culturing for 28 days at the temperature of 26 ℃, the illumination intensity of 1500lux and the illumination time of 8 h/day to obtain a complete plant. The statistical rooting rate is 100%. And counting the number of roots and the length of the roots to obtain the average number of the roots per bottle of 7.22, the average length of the roots of 6.35cm and the length of the longest root of 12.1 cm.
And placing the obtained complete plants in an open place, adding 2ml of tap water into each culture bottle for hardening seedlings, dividing all the plants into single plants after hardening seedlings for 7 days, and culturing the single plants in a transplanting sand bed for one month, wherein the transplanting survival rate in the sand bed is 95.40%. Transplanting the seedlings into a field after one month, and counting the transplanting survival rate of the field to be 97.57 percent after 2 months of field cultivation.
In this example, the callus induction medium was 1/2MS +1.5mg/l IBA +0.5mg/l GA3+20mg/l sucrose; the bud induction culture medium is MS +3.0mg/l 6-BA +0.5mg/l IBA; the bud multiplication culture medium is MS +0.8mg/l 6-BA +0.15mg/l 2,4-D +15mg/l sucrose; the rooting medium is 1/2MS +0.5mg/l NAA +0.4mg/l GA3+10mg/l sucrose.
Example 3
The jackfruit bud points used in this example were from the apical and axillary buds of a elite single plant of jackfruit. Placing the collected jackfruit bud points in a sterile cup, sterilizing on an ultra-clean workbench, pouring 0.2% mercuric chloride solution into the sterile cup, soaking for 5min, pouring out the mercuric chloride solution, and washing with tap water for 3 times. Inoculating the sterilized bud points into a callus induction culture medium, culturing in the dark at 30 ℃ for 6 days, and carrying out dedifferentiation on the bud points to form callus. In the present example, the callus induction success rate of the jackfruit bud is 99.5%.
Respectively inoculating the successfully induced callus into a bud induction culture medium, culturing for 22 days under the conditions of 22 ℃ of temperature, 1500lux of illumination intensity and 8 h/day of illumination time, differentiating the callus to form adventitious buds and cluster buds, and counting the differentiation rate of the adventitious buds to be 100%.
Inoculating the differentiated adventitious buds and the cluster buds into bud multiplication culture, dividing the cluster buds with a plurality of adventitious buds, then respectively inoculating the cluster buds into the bud multiplication culture, ensuring that only one adventitious bud seedling exists in one culture bottle, and finally obtaining 419 adventitious buds in the culture bottle. Then stimulating and culturing for 2 days under the conditions of 15 ℃ of temperature, 1200lux of illumination intensity and 12 h/day of illumination time. After the stimulation culture is finished, the culture bottle is placed at 30 ℃, the illumination intensity is 1200lux, the illumination time is 10 h/day, the propagation culture is carried out for 20 days, and the number of the propagated adventitious buds is 1429 strains. Therefore, the growth coefficient was 3.41, and the average seedling height was 4.59 cm.
And (3) inoculating the cluster buds after propagation culture into a rooting culture medium, and culturing for 22 days under the conditions of 22 ℃ of temperature, 1200lux of illumination intensity and 10 h/day of illumination time to obtain a complete plant. The statistical rooting rate is 100%. Counting the number of roots and the length of the roots to obtain the average number of roots per bottle of 6.73, the average length of the roots of 6.71cm, wherein the length of the longest root of 12.5 cm.
And placing the obtained complete plants in an open manner, adding 2ml of tap water into each culture bottle for hardening seedlings, dividing all the plants into single plants after hardening seedlings for 3 days, and culturing the single plants in a transplanting sand bed for one month, wherein the transplanting survival rate in the sand bed is 97.20%. Transplanting the seedlings into a field after one month, and counting the transplanting survival rate of the field to be 97.65% after 2 months of field cultivation.
In this example, the callus induction medium was 1/2MS +2.5mg/l IBA +0.2mg/l GA3+10mg/l sucrose; the bud induction culture medium is MS +1.0mg/l 6-BA +1.0mg/l IBA; the bud multiplication culture medium is MS +0.5mg/l 6-BA +0.3mg/l 2,4-D +25mg/l sucrose; the rooting medium is 1/2MS +0.1mg/l NAA +0.2mg/l GA3+20mg/l sucrose.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for culturing and rapidly propagating excellent single-plant tissue of jackfruit is characterized by comprising the following steps:
(1) after the explant is disinfected, inoculating the explant into a callus induction culture medium, and carrying out dark culture at the temperature of 28-35 ℃ for 5-10 days to obtain a callus;
(2) inoculating the callus into a bud induction culture medium, and culturing for 18-22 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain cluster buds;
(3) inoculating the cluster buds into a bud multiplication culture medium, and stimulating for 1-3 days under the conditions of temperature of 10-15 ℃, illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day;
(4) placing the cluster buds stimulated in the step (3) under the conditions of illumination intensity of 1200-1500 lux and illumination time of 8-12 h/day at 28-35 ℃ for propagation culture for 20-25 days;
(5) inoculating the cluster buds subjected to propagation culture in the step (4) into a rooting culture medium, and culturing for 22-28 days under the conditions that the temperature is 22-26 ℃, the illumination intensity is 1200-1500 lux, and the illumination time is 8-12 h/day to obtain a complete plant;
(6) and (4) hardening off the whole plant and transplanting.
2. The method of claim 1, wherein the explant is the shoot of jackfruit.
3. The method according to claim 2, wherein the callus induction medium is prepared by adding 1.5-2.5 mg/l IBA, 0.1-0.5 mg/l GA3 and 10-20 mg/l sucrose to 1/2MS medium.
4. The method of claim 3, wherein the shoot induction medium is supplemented with 1.0-4.0 mg/l6-BA and 0.5-1.0 mg/l IBA based on MS culture.
5. The method according to claim 4, wherein the shoot growth medium is prepared by adding 0.5 to 1.5mg/l of 6-BA, 0.1 to 0.3mg/l of 2,4-D and 15 to 25mg/l of sucrose to the MS medium.
6. The method according to any one of claims 1 to 5, wherein the rooting medium is 1/2MS medium supplemented with 0.1-0.5 mg/l NAA, 0.2-0.4 mg/l GA3, 10-20 mg/l sucrose.
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