CN114467756B - Kenaf tissue culture method using cotyledon petiole as explant - Google Patents
Kenaf tissue culture method using cotyledon petiole as explant Download PDFInfo
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- CN114467756B CN114467756B CN202210216212.8A CN202210216212A CN114467756B CN 114467756 B CN114467756 B CN 114467756B CN 202210216212 A CN202210216212 A CN 202210216212A CN 114467756 B CN114467756 B CN 114467756B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to the technical field of plant tissue culture, and particularly relates to a kenaf tissue culture method using kenaf cotyledon stalks as explants. The method comprises the steps of placing kenaf seeds on an MS culture medium after disinfection for growth, cutting off cotyledon stalks after 12 days, placing the cut kenaf stalks in a germination induction culture medium, cutting off the kenaf seeds after adventitious buds grow, placing the kenaf seeds in a rooting culture medium for culturing until roots grow, and then hardening and transplanting the seedlings to obtain regenerated seedlings. The method provided by the invention has no genotype limitation among varieties, short culture period and normal growth of regenerated plants, and is suitable for tissue culture of various kenaf varieties.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a kenaf tissue culture method using cotyledon petioles as explants.
Background
Kenaf (A)Hibiscus) The fiber crop is an annual fiber crop of hibiscus of malvaceae, is the third natural fiber crop in the world after cotton and jute, has the characteristics of drought resistance, salt and alkali resistance, wide adaptability, large biomass and the like, has good application value in the aspects of papermaking, spinning, building materials, composite materials, water and soil conservation, adsorbents, feeds and the like, and is planted in subtropical and tropical regions. Because the kenaf bast fiber has the characteristics of bacteriostasis, ventilation, good hygroscopicity, degradability and the like, the kenaf bast fiber is regarded as a potential dominant crop in the 21 st century. Although the conventional genetic breeding method can improve the quality of the kenaf and improve important agronomic traits such as disease resistance, the period is long, and a large amount of manpower and material resources are also required to be invested. The introduction of exogenous genes for controlling effective characters into the kenaf by means of a plant transgenic technology or the directional targeted modification of a genome can be one of the efficient ways of kenaf molecular breeding. The plant transgenic technology cannot leave a high-efficiency stable genetic transformation system, and the key factor for restricting the genetic transformation of the kenaf is the lack of a high-efficiency in vitro regeneration system.
At present, more main crops establish a stable and efficient in-vitro regeneration system, and kenaf has no related report. Most of the plants reported that the explants of the regeneration system are cotyledons, hypocotyls and stem tips, and the cotyledons are less prone to blooming during the regeneration process than the cotyledons; the cotyledon petiole is a cell aggregation area with stronger division energy compared with the hypocotyl, and the cells have more potential to be converted into embryonic cells; cotyledon stalks are more accessible to homozygous plants than shoot tips in the genetic transformation system than to chimeras. Therefore, compared with cotyledon, hypocotyl and stem tip, the cotyledon stem has the advantages of higher regeneration rate, shorter period, capability of generating more adventitious buds and the like, and can lay a foundation for establishing a regeneration system.
Disclosure of Invention
The invention aims to provide a tissue culture method of kenaf by taking cotyledon petioles as explants, which can obtain a large amount of regenerated seedlings in a short time by generating adventitious bud clusters so as to realize in-vitro regeneration of kenaf.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kenaf tissue culture method using cotyledon petioles as explants comprises the following steps:
(1) Preparation of aseptic seedlings: carrying out disinfection and sterilization treatment on kenaf seeds, inoculating the kenaf seeds on an MS culture medium, and culturing until the kenaf seeds germinate to obtain kenaf sterile seedlings;
(2) Callus and adventitious bud induction: taking the cotyledon petiole of the aseptic seedling obtained by culturing in the step (1) as an explant, inoculating the cotyledon petiole into an induced sprouting culture medium for culturing, and periodically replacing the culture medium during the culturing to obtain an adventitious bud cluster;
(3) Bud elongation: stripping the adventitious bud clusters obtained in the step (2) from the callus, inoculating the adventitious bud clusters to a bud elongation culture medium for culture, periodically replacing the culture medium in 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: taking the single elongated adventitious bud seedling obtained in the step (3), inoculating the single elongated adventitious bud seedling to a rooting induction culture medium, and culturing the bud seedling until the bud seedling roots to obtain a regenerated seedling;
(5) Hardening and transplanting seedlings: and (4) hardening the regenerated seedlings obtained in the step (4), taking out the regenerated seedlings after hardening, washing off a root culture medium, transplanting the seedlings into nutrient soil, and placing the seedlings in a greenhouse for culture.
Further, the above-mentioned kenaf variety is selected from fu hong 952 and zan 1.
Further, all of the above operations are performed in a sterile environment.
Further, the method for obtaining the sterile seedlings in the step (1) comprises the following steps: selecting full and clean kenaf seeds, cleaning with 75% alcohol by volume fraction for 3min, cleaning with sterile water for 3min, cleaning with 84% disinfectant by volume fraction for 20min, cleaning with sterile water for 4 times (3 min each time), and sterilizing; inoculating the sterilized kenaf seeds into an MS culture medium, and culturing under the conditions of culture temperature of 25 ℃,16h illumination and 8h darkness alternation, and 2000Lux until the seeds germinate, thus obtaining aseptic 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 clusters in the step (2) comprises the following steps: taking the sterile seedlings germinated for 12-14 days in the step (1), taking off cotyledon petioles as explants by using sterile scissors, inoculating the explants to an induced germination culture medium, culturing for about 45 days under the conditions of culture temperature of 25 ℃,16h illumination and 8h darkness alternation, and 2000Lux, and replacing the culture medium every 15 days, thus obtaining adventitious bud clusters; wherein the induced germination culture medium takes an MS culture medium as a basic culture medium, and 0.1mg/L of thidiazuron and 0.1mg/L of 2, 4-dichlorophenoxyacetic 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, 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 0.1mg/L of thidiazuron and 0.1mg/L of 2, 4-dichlorophenoxyacetic acid are added.
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 a culture temperature of 25 ℃, under the conditions of alternative treatment of 16h of illumination and 8h of darkness and 2000Lux until 5-6 fibrous 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 seedling exercising and transplanting method in the step (5) specifically comprises the following steps: opening the bottle cap of the culture bottle for culturing the regenerated seedlings in the step 4), standing for 3 hours at room temperature under natural illumination, adding 25-30ml of tap water into the culture bottle, continuously standing for 12 hours at room temperature under natural illumination, and finishing seedling hardening; taking out the regenerated seedlings in the culture bottles after hardening, cleaning culture media attached to the bases of the regenerated seedlings by tap water, transplanting the regenerated seedlings into nutrient soil, placing the regenerated seedlings in a greenhouse for culture under the conditions of 25 ℃ temperature, 16h illumination and 8h darkness alternation, 2000Lux, managing according to conventional fertilizer and water until the regenerated seedlings grow into finished seedlings, and taking out of the nursery; wherein, the nutrient soil is required to be mixed with tap water according to the mass ratio of 5.
Furthermore, the tissue culture method of the kenaf by using the cotyledon petiole as the explant can be applied to large-scale cultivation of the kenaf.
The invention has the following remarkable advantages:
the invention relates to a domestic first kenaf in vitro regeneration scheme. Firstly, the invention uses aseptic seedling petioles as explants, which is one of the cores for realizing the high-efficiency in-vitro regeneration of the kenaf; secondly, the same culture medium is used for carrying out callus induction, adventitious bud differentiation and adventitious bud elongation on the kenaf petiole, the number of the obtained adventitious buds is large, and the final rooting rate can reach 90.3%; thirdly, the method is simple to operate, and has the characteristics of low investment and high yield.
Drawings
FIG. 1: mature and plump 'Fuhong 952' kenaf hemp seed.
FIG. 2: sterile shoot petioles.
FIG. 3: adventitious bud clusters.
FIG. 4 is a schematic view of: adventitious buds elongated for 30 days.
FIG. 5 is a schematic view of: and (4) rooting the regenerated seedlings.
FIG. 6: and (5) seedling hardening schematic diagram.
FIG. 7 is a schematic view of: and transplanting the kenaf 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 an aseptic environment, selecting mature and plump hibiscus 952 hibiscus cannabinus seeds (figure 1), cleaning with 75% alcohol by volume fraction for 3min, cleaning with sterile water for 3min, cleaning with 50% 84 disinfectant by volume fraction for 20min, and finally cleaning with sterile water for 4 times, each time for 3min, thereby completing disinfection. Inoculating the disinfected and sterilized seeds into a germination culture medium, and culturing under the conditions of culture temperature of 25 ℃,16h illumination and 8h darkness alternation, and 2000Lux until the seeds germinate, so as to obtain aseptic seedlings; wherein, the germination culture medium is an MS culture medium, and the preparation method 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: in a sterile environment, cutting off complete cotyledon petioles of sterile seedlings germinating for 12-14 days in the step (1) by using a sterile surgical scissors (figure 2), inoculating the sterile seedlings on an adventitious bud induction culture medium, culturing the sterile seedlings under the conditions of culture temperature of 25 ℃,16h illumination and 8h darkness alternating treatment and 2000Lux for about 45 days, subculturing every 15 days to obtain an adventitious bud cluster (figure 3), and counting to obtain the adventitious bud induction rate (32.1%); wherein the adventitious bud induction culture medium takes an MS culture medium as a basic culture medium, and is added with 0.1mg/L of Thidiazuron (TDZ) and 0.1mg/L of 2, 4-dichlorophenoxyacetic acid (2, 4-D), and 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) Adventitious bud elongation: shearing the adventitious bud clusters obtained in the step (2) from the callus by using an aseptic surgical scissors in an aseptic environment, transferring the adventitious bud clusters to an adventitious bud elongation culture medium, subculturing once every 15d, culturing for about 30 days under the conditions of a culture temperature of 25 ℃,16h illumination and 8h darkness alternating treatment, 2000Lux and an adventitious bud length of 3-5cm (shown in a figure 4), and separating the adventitious bud clusters to obtain a single elongated adventitious bud seedling; wherein the adventitious bud elongation culture medium takes an MS culture medium as a basic culture medium, and is added with 0.1mg/L of Thidiazuron (TDZ) and 0.1mg/L of 2, 4-dichlorophenoxyacetic acid (2, 4-D), and 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: in an aseptic environment, transferring the single elongated adventitious bud seedling obtained in the step (3) to an adventitious bud induction rooting culture medium, and culturing at a culture temperature of 25 ℃, under conditions of alternative treatment of 16h light and 8h dark and 2000Lux until 5-6 fibrous roots grow out, so as to obtain a regenerated seedling (figure 5); the rooting induction culture medium is an MS culture medium without any hormone, and the preparation method comprises the steps of adding 4.46g/L of MS powder, 30g/L of sucrose and 8g/L of agar powder, wherein the pH value of the culture medium is 5.8.
(5) Hardening and transplanting seedlings: opening the bottle cap of the culture bottle for culturing the rooted seedling with 5 to 6 fibrous roots in the step (4), standing for 3 hours at room temperature under natural illumination, adding 25 to 30ml of tap water (shown in figure 6) into the culture bottle, continuously standing for 12 hours at room temperature under natural illumination, and finishing seedling hardening; taking out the regenerated seedlings in the culture bottles after hardening, washing the culture medium attached to the base parts of the regenerated seedlings by using tap water, transplanting the regenerated seedlings into nutrient soil (figure 7), placing the regenerated seedlings in a greenhouse for culturing under the conditions of 25 ℃ temperature, 16h illumination and 8h dark alternate treatment and 2000Lux, managing according to conventional fertilizer and water until the regenerated seedlings grow into finished seedlings, and taking out of the nursery; wherein, the nutrient soil is mixed with tap water according to the mass ratio of 5.
Claims (4)
1. A kenaf tissue culture method using cotyledon petioles as explants is characterized in that: the method comprises the following steps:
(1) Preparation of sterile seedlings: carrying out disinfection and sterilization treatment on kenaf seeds, inoculating the kenaf seeds on an MS culture medium, and culturing until the kenaf seeds germinate to obtain kenaf sterile seedlings;
(2) Callus and adventitious bud induction: taking the cotyledon petiole of the aseptic seedling obtained by culturing in the step (1) as an explant, inoculating the cotyledon petiole into an induced sprouting culture medium for culturing, and periodically replacing the culture medium during the culturing to obtain an adventitious bud cluster;
(3) Bud elongation: stripping the adventitious bud clusters obtained in the step (2) from the callus, inoculating the adventitious bud clusters to a bud elongation culture medium for culture, periodically replacing the culture medium in 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: taking the single elongated adventitious bud seedling obtained in the step (3), inoculating the single elongated adventitious bud seedling to a rooting induction culture medium, and culturing the bud seedling until the bud seedling roots to obtain a regenerated seedling;
(5) Hardening and transplanting seedlings: hardening the regenerated seedlings obtained in the step (4), taking out the regenerated seedlings after hardening, washing off root culture media, transplanting the regenerated seedlings into nutrient soil, and placing the regenerated seedlings in a greenhouse for culturing;
the method for obtaining the aseptic seedlings in the step (1) comprises the following steps: selecting full and clean kenaf seeds, firstly cleaning with 75% alcohol by volume fraction for 3min, then cleaning with sterile water for 3min, then cleaning with 84% disinfectant by volume fraction for 20min, finally cleaning with sterile water for 4 times, 3min each time, and finishing disinfection; inoculating the sterilized kenaf seeds into an MS culture medium, and culturing under the conditions of culture temperature of 25 ℃,16h illumination and 8h darkness alternating treatment and 2000Lux until the seeds germinate, thereby obtaining aseptic 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;
the method for obtaining the adventitious bud cluster in the step (2) comprises the following steps: taking the sterile seedlings germinated for 12-14 days in the step (1), taking off complete cotyledon petioles by using sterile scissors as explants, inoculating the explants to an induced sprouting culture medium, culturing for about 45 days under the conditions that the culture temperature is 25 ℃,16h illumination and 8h darkness are alternately treated, and 2000Lux is adopted, and replacing the culture medium every 15 days in the period to obtain adventitious bud clusters; wherein the induced germination culture medium takes an MS culture medium as a basic culture medium, and 0.1mg/L of thidiazuron and 0.1mg/L of 2, 4-dichlorophenoxyacetic acid are added;
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, 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 0.1mg/L thidiazuron and 0.1 mg/L2, 4-dichlorophenoxyacetic acid are added;
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 darkness alternation, and 2000Lux until 5-6 fibrous roots are grown, thus obtaining a regenerated seedling; wherein the rooting culture medium takes an MS culture medium as a basic culture medium;
the seedling hardening and transplanting method in the step (5) specifically comprises the following steps: opening the bottle cap of the culture bottle for culturing the regenerated seedlings in the step (4), standing for 3 hours at room temperature under natural illumination, adding 25-30ml of tap water into the culture bottle, continuously standing for 12 hours at room temperature under natural illumination, and finishing seedling hardening; taking out the regenerated seedlings in the culture bottles after hardening, cleaning culture media attached to the bases of the regenerated seedlings by tap water, transplanting the regenerated seedlings into nutrient soil, placing the regenerated seedlings in a greenhouse for culture under the conditions of 25 ℃ temperature, 16h illumination and 8h darkness alternation, 2000Lux, managing according to conventional fertilizer and water until the regenerated seedlings grow into finished seedlings, and taking out of the nursery; wherein, the nutrient soil is mixed with tap water according to the mass ratio of 5.
2. The kenaf tissue culture method using cotyledon petioles as explants according to claim 1, wherein: the variety of the kenaf is selected from Fuhong 952 and Zanzi No. 1.
3. The method for culturing the tissue of kenaf with high efficiency by using the cotyledon petiole as the explant according to claim 1, wherein the method comprises the following steps: all manipulations were performed in a sterile environment.
4. The use of the tissue culture method of kenaf using cotyledon petiole as explant according to claim 1 for large-scale kenaf culture.
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| US5998207A (en) * | 1997-06-13 | 1999-12-07 | Mississippi State University | Method for transformation of cotton and kenaf and organogenic regeneration |
| JP2000217457A (en) * | 1999-01-29 | 2000-08-08 | Araco Corp | Callus redifferentiation of kenaf |
| JP3916938B2 (en) * | 2001-12-05 | 2007-05-23 | 独立行政法人科学技術振興機構 | Kenaf breeding method |
| ES2390919T3 (en) * | 2006-03-31 | 2012-11-19 | Basf Plant Science Gmbh | Plants that have improved performance-related traits and a method to make them |
| CN102487818B (en) * | 2011-11-22 | 2013-10-23 | 福建农林大学 | Method for improving kenaf cotyledon adventitious bud inductivity |
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