CN114431150A - Jute tissue culture method taking cotyledonary node as explant - Google Patents
Jute tissue culture method taking cotyledonary node as explant Download PDFInfo
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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
-
- 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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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of plant tissue culture, and particularly relates to a jute tissue culture method using jute cotyledonary nodes as explants. The method comprises the steps of firstly, disinfecting jute seeds, inoculating the jute seeds into an MS culture medium, cutting off cotyledonary nodes of aseptic seedlings after 10 days of culture, transferring the cut cotyledonary nodes to a germination culture medium to induce germination, cutting off obtained adventitious buds, transferring the cut adventitious buds to a rooting culture medium to induce rooting, and then hardening and transplanting the seedlings to obtain regenerated plants. The method for regenerating the plant of the leaf node of the jute provides technical support for genetic transformation and creation of new varieties of jute.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for culturing juteC. capsularisL.) a jute tissue culture method using cotyledonary node as explant, which can be applied to genetic transformation of jute.
Background
Jute (Corchorusspp.), the bast fiber crop of Corchorus of Malvaceae, also known as green hemp, Siberian hemp, and important raw material of hemp spinning industry, the yield and planting area of which are second to cotton in the world. Among the numerous species of the genus Corchorus, the species of the long fruit having cultivation value ((C. olitorius) And round fruit seed: (C. capsularis). The jute fiber has the advantages of soft texture, high fiber strength, good water absorption, quick water dispersion, low price, reproducibility, degradability and the like, thereby being used for manufacturing products such as gunny bags, ropes, mattresses, curtains, adsorbing materials and the like. Jute pleased warmThe humid climate is currently mainly cultivated in China, Bengal, Thailand, India, Burma, etc. However, due to biotic and abiotic stresses, jute fiber yield suffers tremendous losses each year. Traditional breeding mainly depends on phenotypic selection of plants, and the breeding period is long and is easily influenced by environmental conditions, gene interaction and the like. The excellent transgenic strain of jute can be obtained quickly by applying genetic transformation, but the genetic transformation of jute is not realized due to the restriction of regeneration system technology.
At present, the research on a hemp regeneration system is mainly to use hypocotyls, cotyledons, endosperm and the like as explants, but compared with cotyledonary nodes, the hypocotyls have poor redifferentiation capability and regenerated buds are difficult to induce; the wounds of the cotyledon are easy to brown and curl; endosperm explant sampling operation is complex, and in short, a stable regeneration system is difficult to obtain. However, research on jute regeneration systems is less, and a mature and efficient jute regeneration system cannot be explored. Although a system relating to this method has been reported in the literature in recent years, experimental reproducibility is poor and the adventitious bud regeneration rate is not high. Internationally, only Bharadwaj Pushyami and the like report 1 time in a regeneration system of jute, wherein jute peduncle cotyledons are taken as explants, MS culture medium is taken as a basic culture medium, 0.5 mg/L1-naphthylacetic acid (NAA), 0.5 mg/L6-Benzylaminopurine (BAP) and 36 g/L sucrose are added to obtain adventitious buds, but the effect is poor when the experiment is repeated. The research takes jute cotyledonary nodes as explants, finds out the most appropriate concentration of the plant growth regulator, establishes a high-efficiency in vitro regeneration system for jute and lays a foundation for genetic transformation.
Disclosure of Invention
The invention aims to provide a jute tissue culture method taking cotyledonary nodes as explants, which can obtain a large amount of regenerated seedlings in a short time by generating adventitious bud clusters, thereby realizing the in vitro regeneration of jute.
In order to achieve the purpose, the invention adopts the following technical scheme:
a jute tissue culture method taking cotyledonary nodes as explants comprises the following steps:
1) preparation of sterile seedlings: selecting mature and plump jute seeds, sterilizing, inoculating the jute seeds into an MS culture medium, and culturing until the jute seeds germinate to obtain aseptic seedlings;
2) callus and adventitious bud induction: taking the sterile seedling obtained in the step 1), taking off complete cotyledonary nodes by using sterile scissors as explants, inoculating the explants to a germination culture medium for culture, and periodically replacing the culture medium during the period to obtain adventitious bud clusters;
3) bud elongation: stripping the adventitious bud clusters obtained in the step 2) from the callus, transferring the adventitious bud clusters to a bud elongation culture medium, continuously culturing, periodically replacing the culture medium during the period, and separating the adventitious bud clusters when the adventitious buds grow to a certain height to obtain a single elongated adventitious bud seedling;
4) rooting induction: transferring the single elongated adventitious bud seedling obtained in the step 3) to a rooting culture medium, and culturing until the bud seedling takes root to obtain a regenerated seedling;
5) hardening and transplanting seedlings: carrying out water culture hardening treatment on the regenerated seedlings obtained in the step 4), taking out the regenerated seedlings after hardening, washing off a root culture medium, transplanting the regenerated seedlings into nutrition, and culturing the regenerated seedlings in a greenhouse.
Further, all the operations of the jute tissue culture method taking cotyledonary nodes as explants are carried out in a sterile environment.
Further, the jute was named "Meifeng No. 4".
Further, the method for obtaining the sterile seedlings in the step 1) comprises the following steps: soaking jute seeds in 75% ethanol for 30s, soaking in 50% sodium hypochlorite for 20min, cleaning with sterile water for 3-4 after soaking, then sucking water on the seeds with sterile absorbent paper, inoculating the seeds into an MS culture medium, and culturing at the culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternating treatment and 2000Lux until the seeds germinate, so as to obtain sterile seedlings; wherein, the formula of the MS culture medium is as follows: MS powder 4.46g/L, sucrose 30g/L, agar powder 8g/L, pH = 5.8.
Further, the method for obtaining the adventitious bud cluster in the step 2) comprises the following steps: taking the sterile seedlings germinated for 8-10 days in the step 1), taking off complete cotyledonary nodes by using sterile scissors as explants, inoculating the explants to a germination culture medium, and culturing for 20-30 days under the conditions of culture temperature of 25 ℃, 16h illumination and 8h darkness alternation treatment and 2000Lux, wherein the culture medium is replaced every 15 days, so as to obtain adventitious bud clusters; wherein the germination culture medium takes an MS culture medium as a basic culture medium, and 2 mg/L6-benzylaminopurine and 0.5mg/L indoleacetic acid are added.
Further, the method for obtaining single elongated adventitious bud seedling in the step 3) comprises the following steps: stripping the adventitious bud clusters obtained in the step 2) from the callus, transferring the adventitious bud clusters to a bud elongation culture medium, culturing at the culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternate treatment and 2000Lux, replacing the culture medium every 15 days in the period, and separating the adventitious bud clusters when the adventitious buds grow to 3-5cm high, namely obtaining single elongated adventitious bud seedlings; wherein the bud elongation culture medium takes an MS culture medium as a basic culture medium and is added with 2 mg/L6-benzylaminopurine and 0.5mg/L indoleacetic acid.
Further, the method for obtaining the regenerated plantlet in the step 4) comprises the following steps: transferring the single elongated adventitious bud seedling to a rooting culture medium, and culturing at the culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternate treatment and 2000Lux until 2-3 roots grow, so as to obtain a regenerated seedling; wherein the rooting culture medium takes an MS culture medium as a basic culture medium.
Further, the method for hardening off and transplanting the seedlings in the step 5) specifically comprises the following steps: opening a bottle cap of a culture bottle for culturing the regenerated seedlings in the step 4), adding 25-30mL of tap water into the bottle, placing the bottle at room temperature under natural illumination for 12 hours, taking out the regenerated seedlings in the culture bottle, washing a culture medium attached to the base parts of the regenerated seedlings by using the tap water, placing the regenerated seedlings with the washed culture medium in a test tube with the roots facing downwards and upwards, injecting the tap water into the bottom of the test tube until the liquid level of the tap water submerges the base parts of the roots and the stems of the regenerated seedlings, placing the test tube under the room temperature and natural illumination for 24 hours, deepening the leaf color of the regenerated seedlings in the test tube, and finishing the water culture and seedling hardening; after hardening, transplanting the hardened jute tissue culture seedlings into nutrient soil, placing the jute tissue culture seedlings into a greenhouse, alternately treating the jute tissue culture seedlings by 16h of light and 8h of dark at the temperature of 25 ℃, continuously culturing the jute tissue culture seedlings under the condition of 2000Lux, managing the jute tissue culture seedlings according to conventional fertilizer and water until the jute tissue culture seedlings grow into finished seedlings, and outplanting the seedlings; wherein the nutrient soil is mixed with tap water according to the mass ratio of 5:1 before use so as to be completely wetted.
The application of the jute tissue culture method taking cotyledonary nodes as explants in large-scale jute culture is provided.
The invention has the following remarkable advantages:
the invention has the positive effects that: the invention takes cotyledon nodes of jute as an explant, is a key technology for realizing the efficient regeneration of jute, can generate clustered adventitious buds by being assisted with a proper sprouting culture medium, a bud elongation culture medium and a rooting culture medium, and has the characteristics of good repeatability, high sprouting rate and short regeneration period.
Drawings
FIG. 1: germinating the aseptic seedlings for 8-10 days.
FIG. 2: whole cotyledonary nodes cut from the sterile seedlings.
FIG. 3: adventitious bud clusters.
FIG. 4: adventitious bud seedling of 20 days of elongation.
FIG. 5: and (5) regenerating seedlings.
FIG. 6: schematic diagram of water culture hardening seedling.
FIG. 7: and (4) transplanting jute tissue culture seedlings into nutrient soil.
Detailed Description
The invention is further illustrated by the following examples, which are intended only for a better understanding of the invention and do not limit the scope of the invention.
Example 1
(1) Preparation of sterile seedlings: in a sterile environment, selecting mature, full and clean jute seeds of 'Meifeng No. 4' and soaking the seeds in 75 percent by volume of ethanol for 30s, then soaking the seeds in 50 percent by mass of sodium hypochlorite disinfectant for 20min, washing the seeds with sterile water for 3 to 4 times after soaking, washing the seeds with sterile water for 3 minutes each time, absorbing the water on the surfaces of the seeds by sterile water absorbing paper, inoculating the seeds into an MS culture medium, and culturing the seeds under the conditions of culture temperature of 25 ℃, 16-hour illumination and 8-hour darkness alternation, and 2000Lux until the seeds germinate to obtain sterile seedlings; wherein, the preparation method of the MS culture medium comprises the following steps: MS powder 4.46g/L, sucrose 30g/L, agar powder 8g/L, medium pH value to 5.8.
(2) Callus and adventitious bud induction: selecting a sterile seedling (shown in figure 1) which germinates for 8-10 days in the step (1) in a sterile environment, taking off a complete cotyledon node (shown in figure 2) by using sterile scissors as an explant, transferring the explant to a germination culture medium, culturing for 20-30 days under the conditions of culture temperature of 25 ℃, 16h illumination and 8h darkness alternating treatment and 2000Lux, replacing a fresh germination culture medium every 15 days to obtain an adventitious bud cluster (shown in figure 3), and counting the induction rate of the adventitious bud to be 41.7% after culturing for 30 days; wherein the germination medium takes an MS medium as a basic medium and is added with 2 mg/L6-benzylaminopurine (6-BA) and 0.5mg/L indoleacetic acid (IAA); the preparation method of the MS culture medium comprises the following steps: MS powder 4.46g/L, sucrose 30g/L, agar powder 8g/L, medium pH value to 5.8.
(3) Bud elongation: in a sterile environment, taking the adventitious bud clusters obtained in the step (2), cutting off the callus, transferring the adventitious bud clusters to a bud elongation culture medium for culture, and replacing a fresh bud elongation culture medium every 15 days, wherein the culture conditions are as follows: culturing at 25 deg.C under the conditions of 16h illumination and 8h darkness alternately and 2000Lux for about 20 days until adventitious bud grows to 3-5cm (FIG. 4), and separating adventitious bud cluster to obtain single elongated adventitious bud seedling; wherein the bud elongation culture medium takes an MS culture medium as a basic culture medium, and 2 mg/L6-benzylaminopurine (6-BA) and 0.5mg/L indoleacetic acid (IAA) are added; the preparation method of the MS culture medium comprises the following steps: MS powder 4.46g/L, sucrose 30g/L, agar powder 8g/L, medium pH value to 5.8.
(4) Rooting induction: inoculating the single elongated adventitious bud seedling obtained in the step (3) into a rooting culture medium in an aseptic environment, and culturing at a culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternate treatment and 2000Lux until 2-3 roots grow out to obtain a regenerated seedling (figure 5); wherein the rooting culture medium takes an MS culture medium as a basic culture medium; the preparation method of the MS culture medium comprises the following steps: MS powder 4.46g/L, sucrose 30g/L, agar powder 8g/L, medium pH value to 5.8.
(5) Hardening and transplanting seedlings: opening a bottle cap of a culture bottle for culturing the regenerated seedlings which grow 2-3 roots and have strong growth vigor in the step (4), adding 25-30ml of tap water into the bottle, placing the bottle at room temperature under natural illumination for 12 hours, taking the regenerated seedlings out of the culture bottle, cleaning a culture medium attached to the base parts of the regenerated seedlings by using the tap water, placing the roots, downward leaves and upward leaves of the regenerated seedlings, which are washed with the culture medium, into a new test tube, injecting tap water into the bottom of the test tube until the liquid level of the tap water submerges the root and stem base parts of the regenerated seedlings (figure 6), placing the test tube at room temperature under natural illumination for 24 hours, deepening the leaf color of the regenerated seedlings in the test tube, and finishing water culture and seedling hardening; after hardening, transplanting the hardened jute tissue culture seedlings into nutrient soil (figure 7), placing the jute tissue culture seedlings into a greenhouse, alternately treating the jute tissue culture seedlings by 16h of light and 8h of dark at the temperature of 25 ℃, continuously culturing the jute tissue culture seedlings under the condition of 2000Lux, managing the jute tissue culture seedlings according to conventional fertilizer and water until the jute tissue culture seedlings grow into finished seedlings, and outplanting the seedlings; wherein the nutrient soil is mixed with tap water according to the mass ratio of 5:1 before use so as to be completely wetted.
Claims (9)
1. A jute tissue culture method taking cotyledonary nodes as explants is characterized in that: the method comprises the following steps:
1) preparation of sterile seedlings: selecting mature and plump jute seeds, sterilizing, inoculating the jute seeds into an MS culture medium, and culturing until the jute seeds germinate to obtain aseptic seedlings;
2) callus and adventitious bud induction: taking the sterile seedling obtained in the step 1), taking off complete cotyledonary nodes by using sterile scissors as explants, inoculating the explants to a germination culture medium for culture, and periodically replacing the culture medium during the period to obtain adventitious bud clusters;
3) bud elongation: stripping the adventitious bud clusters obtained in the step 2) from the callus, transferring the adventitious bud clusters to a bud elongation culture medium, continuously culturing, periodically replacing the culture medium during the period, and separating the adventitious bud clusters when the adventitious buds grow to a certain height to obtain single elongated adventitious bud seedlings;
4) rooting induction: transferring the single elongated adventitious bud seedling obtained in the step 3) to a rooting culture medium, and culturing until the bud seedling takes root to obtain a regenerated seedling;
5) hardening and transplanting seedlings: carrying out water culture hardening treatment on the regenerated seedlings obtained in the step 4), taking out the regenerated seedlings after hardening, washing off a root culture medium, transplanting the regenerated seedlings into nutrition, and culturing the regenerated seedlings in a greenhouse.
2. The method for culturing the jute tissue taking cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: the jute is named as Meifeng No. 4.
3. The method for culturing the jute tissue taking cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: all manipulations were performed in a sterile environment.
4. The method for culturing the jute tissue taking cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: the method for obtaining the aseptic seedlings in the step 1) comprises the following steps: soaking jute seeds in 75% ethanol for 30s, soaking in 50% sodium hypochlorite for 20min, cleaning with sterile water for 3-4 after soaking, then sucking water on the seeds with sterile absorbent paper, inoculating the seeds into an MS culture medium, and culturing at the culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternating treatment and 2000Lux until the seeds germinate, so as to obtain sterile seedlings; wherein, the formula of the MS culture medium is as follows: MS powder 4.46g/L, sucrose 30g/L, agar powder 8g/L, pH = 5.8.
5. The method for culturing the jute tissue taking cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: the method for obtaining the adventitious bud cluster in the step 2) comprises the following steps: taking the sterile seedlings germinated for 8-10 days in the step 1), taking off complete cotyledonary nodes by using sterile scissors as explants, inoculating the explants to a germination culture medium, and culturing for 20-30 days under the conditions of culture temperature of 25 ℃, 16h illumination and 8h darkness alternation treatment and 2000Lux, wherein the culture medium is replaced every 15 days, so as to obtain adventitious bud clusters; wherein the germination culture medium takes an MS culture medium as a basic culture medium, and 2 mg/L6-benzylaminopurine and 0.5mg/L indoleacetic acid are added.
6. The method for culturing the jute tissue taking cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: the method for obtaining the single elongated adventitious bud seedling in the step 3) comprises the following steps: stripping the adventitious bud clusters obtained in the step 2) from the callus, transferring the adventitious bud clusters to a bud elongation culture medium, culturing at the culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternate treatment and 2000Lux, replacing the culture medium every 15 days in the period, and separating the adventitious bud clusters when the adventitious buds grow to 3-5cm high, namely obtaining single elongated adventitious bud seedlings; wherein the bud elongation culture medium takes an MS culture medium as a basic culture medium and is added with 2 mg/L6-benzylaminopurine and 0.5mg/L indoleacetic acid.
7. The method for cultivating jute tissue by using cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: the method for obtaining the regenerated seedlings in the step 4) comprises the following steps: transferring the single elongated adventitious bud seedling to a rooting culture medium, and culturing at the culture temperature of 25 ℃, under the conditions of 16h illumination and 8h dark alternate treatment and 2000Lux until 2-3 roots grow, so as to obtain a regenerated seedling; wherein the rooting culture medium takes an MS culture medium as a basic culture medium.
8. The method for culturing the jute tissue taking cotyledonary nodes as explants according to claim 1, wherein the method comprises the following steps: the seedling hardening and transplanting method in the step 5) specifically comprises the following steps: opening a bottle cap of a culture bottle for culturing the regenerated seedlings in the step 4), adding 25-30mL of tap water into the bottle, standing for 12 hours at room temperature under natural illumination, taking out the regenerated seedlings in the culture bottle, washing a culture medium attached to the base parts of the regenerated seedlings by using the tap water, placing the regenerated seedlings with the washed culture medium in a test tube with the roots facing downwards and upwards, injecting the tap water into the bottom of the test tube until the liquid level of the tap water submerges the base parts of the roots and the stems of the regenerated seedlings, standing for 24 hours at room temperature under natural illumination, deepening the leaf color of the regenerated seedlings in the test tube, and finishing water culture and seedling hardening; after hardening, transplanting the hardened jute tissue culture seedlings into nutrient soil, placing the jute tissue culture seedlings into a greenhouse, alternately treating the jute tissue culture seedlings by 16h of light and 8h of dark at the temperature of 25 ℃, culturing the jute tissue culture seedlings under 2000Lux conditions, managing the jute tissue culture seedlings according to conventional fertilizer and water until the jute tissue culture seedlings grow into finished seedlings, and outplanting the seedlings; wherein, the nutrient soil is required to be mixed with tap water according to the mass ratio of 5:1 before use so as to be completely wetted.
9. The application of the jute tissue culture method according to claim 1, wherein the jute tissue culture method takes cotyledonary nodes as explants in large-scale jute cultivation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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