CN115843682B - Method for inducing and regenerating tulip hypocotyl callus - Google Patents
Method for inducing and regenerating tulip hypocotyl callus Download PDFInfo
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- CN115843682B CN115843682B CN202211339300.3A CN202211339300A CN115843682B CN 115843682 B CN115843682 B CN 115843682B CN 202211339300 A CN202211339300 A CN 202211339300A CN 115843682 B CN115843682 B CN 115843682B
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- 241000722921 Tulipa gesneriana Species 0.000 title claims abstract description 67
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000001939 inductive effect Effects 0.000 title claims abstract description 14
- 230000001172 regenerating effect Effects 0.000 title claims abstract description 14
- 230000006698 induction Effects 0.000 claims abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 35
- 230000035784 germination Effects 0.000 claims abstract description 12
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- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 238000004140 cleaning Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000008223 sterile water Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 claims description 4
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims description 4
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims description 4
- 229960001669 kinetin Drugs 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
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- 238000012258 culturing Methods 0.000 claims 1
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- 241001048456 Tulipa sylvestris Species 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
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- 241000234280 Liliaceae Species 0.000 description 1
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Abstract
The invention relates to a method for inducing and regenerating tulip hypocotyl callus, which comprises the following steps: 1. cleaning and sterilizing tulip seeds, and inoculating the tulip seeds onto a germination culture medium, wherein the germination culture medium is a 1/2MS culture medium; 2. after the tulip seeds germinate, selecting a part of hypocotyls emitted by the tulip seeds as a callus induction explant, inoculating the callus induction explant to a callus induction culture medium, and performing dark culture, and 3, cutting the hypocotyls into small sections of 0.2-0.3 cm, and inoculating the hypocotyls to the original induction culture medium to continue dark culture; 4. after 15 days of induction culture, selecting calli with vigorous growth for subculture to obtain calli induced by tulip hypocotyl; 5. transferring the callus induced by the tulip hypocotyl into an adventitious bud induction culture medium to induce and generate adventitious buds. The explant adopted by the invention is an hypocotyl, is derived from an embryogenic organ after embryo germination, has higher activity, strong meristematic capacity and great dedifferentiation potential.
Description
Technical Field
The invention relates to the technical field of rapid plant propagation, in particular to a method for inducing and regenerating tulip hypocotyl callus.
Background
Tulip (Tulipa gesneriana l.) is a bulb flower of tulip genus of family liliaceae. The tulip flower type flower is unique and bright in color, is popular with people in the world, is widely applied to fresh cut flowers, potted plants and landscaping, and has high ornamental value and economic value. The breeding mode of tulip includes seeding breeding and ball-separating breeding. Wherein, the sowing and propagation period is long, most cultivated species need 3-5 years from seeds to forming flowering balls, and the time period is long, which restricts the breeding of tulips and expands the reproduction efficiency. The bulb is a main nutrition propagation organ of tulip, and the seed ball propagation coefficient of one commodity ball is generally 1-6 and is greatly lower than the propagation efficiency of other biennial herbaceous plants, so that most cultivated species have the problems of low seed ball propagation coefficient, seed ball degeneration and the like. Meanwhile, due to the influences of environmental climate, virus accumulation, cultivation technology and the like, the tulip seed balls are difficult to flower, and a large amount of commodity balls are required to be imported from abroad each year. How to improve the propagation efficiency of tulips is a technical bottleneck in the breeding and cultivation fields thereof. In recent years, tissue culture by taking flower stems, axillary buds, stem segments, bulbs and the like as explants is studied, and an efficient tissue culture rapid propagation technical system is attempted to be established so as to overcome the problem of low tulip propagation efficiency. It is reported that the current tulip tissue culture rapid propagation technology mainly utilizes stems and flower stems to directly induce and regenerate bulblets, or utilizes seeds and scales to firstly dedifferentiate and obtain callus, and then induces the occurrence and expansion of bulblets through regeneration bud differentiation. The technology has great difference in callus induction and regeneration efficiency due to different varieties, different sampling positions and different sampling times of explants.
The inventor establishes a tissue culture rapid propagation technology system (ZL 2020 1 0204397.1) of a wild tulip taking embryo as an explant in the early stage, but the adaptability of the technology system to tulip cultivars is low, which indicates that different tissue culture rapid propagation technology modes possibly exist between the wild species and cultivars due to large genetic background difference. In the wild tulip, although the callus can be directly induced by utilizing the embryo, one embryo can only induce one callus during primary culture, the problems of limited number of explants and low utilization rate exist, and the application space of the method in the tissue culture and rapid propagation of tulip is limited to a certain extent. Therefore, there is a need to establish a tissue culture rapid propagation technique that is relatively abundant in explant source and suitable for cultivars. The research and development of the technology has important significance for improving the propagation efficiency of tulip cultivars, shortening the breeding period and reducing the production cost. In addition, the tissue culture rapid propagation technology system with wide variety applicability is established, so that the problem of seed ball degradation can be solved, the dependence on foreign seed balls is reduced, the tulip breeding efficiency in China can be accelerated, the competitiveness of the tulip industry in China is fundamentally improved, and technical reserve is provided for realizing the localization of the seed balls.
Disclosure of Invention
According to the problems existing in the prior art, the invention discloses a method for inducing and regenerating tulip hypocotyl callus, which takes the hypocotyl after germination of tulip seeds as an explant, and the explant can obtain a large amount of callus in a short time on a callus induction culture medium, and obtain regenerated buds after differentiation culture, thus providing important technical support for proliferation, propagation and genetic transformation of tulip.
In order to solve the technical problems, the invention adopts the following technical scheme:
the inducing and regenerating process of tulip hypocotyl callus includes the following steps:
step 1, cleaning and sterilizing tulip seeds, and inoculating the tulip seeds to a germination culture medium, wherein the germination culture medium is a 1/2MS culture medium;
step 2, after germination of tulip seeds, selecting a part of hypocotyls emitted by the tulip seeds as a callus induction explant, cutting the callus induction explant into small sections of 0.5cm, and inoculating the small sections to a callus induction culture medium for dark culture, wherein the callus induction culture medium is an MS culture medium and contains 1.0-3.0 mg/L of dichlorophenoxyacetic acid, 0.5-2.0mg/L of 6-benzylaminopurine and 0.1-0.3 mg/L of kinetin;
step 3, after dark culture for 8-12 days, cutting the hypocotyl into shorter small sections of 0.2-0.3 cm, and inoculating the small sections on an original induction culture medium to continue dark culture;
step 4, selecting callus with light yellow color, compact structure and vigorous growth after 15 days of induction culture for subculture, and carrying out subculture once every 30 days to obtain tulip hypocotyl-induced callus;
and 5, transferring the callus induced by the tulip hypocotyl into an adventitious bud induction culture medium to induce and generate adventitious buds.
Further, in the step 1, the specific method for cleaning and sterilizing the tulip seeds comprises the following steps: soaking tulip seed in solution with 0.1% Tween-20 and 2-3 drops of detergent for 20min, washing with water for 2h, soaking with sterile water for 8-10 h, sterilizing with 75% ethanol in an ultra clean bench for 1min, washing with sterile water for three times, sterilizing with 2% sodium hypochlorite solution for 20min, and washing with sterile water for 5-6 times.
Further, in the step 1, the culture environment is dark culture at 4 ℃ for 40-60 days.
Further, in the step 2, when the tulip hypocotyl grows to 6-8 cm, the white part of the hypocotyl is selected as the callus-induced explant.
Further, the secondary culture medium is an MS culture medium and comprises 1.0mg/L of dichlorophenoxyacetic acid, 0.5mg/L of 6-benzylaminopurine and 0.1mg/L of kinetin.
Further, the germination culture medium, the callus induction culture medium and the adventitious bud induction culture medium comprise 30g/L of sucrose and 8g/L of agar, and the pH value is 5.7-5.8.
Further, the culture condition in the step 5 is that the illumination time is 14-16 h/d, the illumination intensity is 1600-2000Lx, and the temperature is 23+/-2 ℃.
The beneficial effects of the invention are as follows:
1. the explant has sufficient source and strong dedifferentiation capacity. The explant adopted by the invention is an hypocotyl, is derived from an embryogenic organ after embryo germination, has higher activity, strong meristematic capacity and great dedifferentiation potential. Compared with other explants, the method has the advantages that the callus is easier to induce, the callus is not influenced by environmental seasons, the acquisition and the preservation are simple, and the source of the explants is sufficient.
2. The callus induction period is short and the efficiency is high. The callus induction culture medium adopted by the invention can induce callus after the explant is inoculated for 15 days, has high induction efficiency, and greatly shortens the induction callus period compared with other explants and induction methods.
3. The invention is suitable for a plurality of tulip cultivars. Due to the difference of genetic background, the tulip tissue culture rapid propagation technology often has the phenomenon of variety-to-variety, and the same tissue culture technology system is difficult to be suitable for a plurality of varieties or strains. The method is suitable for tissue culture and rapid propagation of a plurality of tulip cultivars, expands variety applicability and has wider application prospect.
The invention will now be described in detail with reference to the drawings and examples.
Drawings
FIG. 1 is a technical flow of the invention, taking tissue culture rapid propagation and callus induction of tulip varieties 'Fan Landi', 'daojones' and 'pure gold' as examples, and comparing the induction efficiency of callus at different parts of hypocotyls after germination of embryo.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
As shown in figure 1, the induction and regeneration method of tulip hypocotyl callus comprises the following specific implementation steps:
(1) The tulip variety 'Fan Landi' ('Verandi') (variety 1) is selected as a material, and the variety has excellent ornamental and resistance properties and is a cut flower and garden application variety popular in the market. And selecting 'Fan Landi' seeds with consistent shape and size, full growth and good embryo.
(2) Soaking tulip seed in solution of Tween-20 and detergent in 0.1 wt% for 2-3 drops for 20min, flushing with running water for 2 hr, and soaking in sterile water for 8-10 hr. In an ultra-clean workbench, sterilizing with 75% ethanol for 1min, washing with sterile water for three times, then sterilizing with 2% sodium hypochlorite solution for 20min, and washing with sterile water for 5-6 times.
(3) The sterilized tulip seeds are inoculated on a 1/2MS culture medium (pH 5.7-5.8), and dark culture is carried out for 60 days (4 ℃) until the embryo germinates and the length of the hypocotyl is about 6-8 cm.
(4) In an ultra-clean workbench, placing tulip hypocotyl on clean sterile filter paper, cutting the white part and the green part of the hypocotyl into small sections of about 0.5cm respectively by using a surgical knife, inoculating on a callus induction medium (MS medium, 30g/L of sucrose, 8g/L of agar and pH of 5.7-5.8, and performing preculture at 2.4-D3.0 mg/L,6-BA 0.5mg/L and KT 0.3 mg/L). Dark culture is carried out for 10 days (23+/-2 ℃), and the expansion and elongation of the hypocotyl are 1-1.5 times of the original hypocotyl.
(5) The elongated hypocotyl is cut into small sections of about 0.2-0.3 cm, and inoculated on an original culture medium for dark culture (23+/-2 ℃). After 5 days, the generation of the callus can be observed, and after the culture is continued for 20 days, the obvious expansion of the callus can be observed, and the callus is in a milky or yellow compact particle shape.
(6) The well-grown callus is inoculated on a secondary culture medium (MS culture medium, sucrose 30g/L, agar 8g/L, pH 5.7-5.8,2.4-D1.0 mg/L,6-BA 0.5mg/L, KT 0.1 mg/L). Subculture was performed in 30 days as one cycle.
(7) The callus with good growth is selected to be inoculated on an adventitious bud induction culture medium (MS culture medium, sucrose 30g/L, agar 8g/L, pH 5.7-5.8, NAA 0.5mg/L and 6-BA 1.0 mg/L) for the induction culture of adventitious buds (culture conditions: illumination time 14-16 h/d, illumination intensity 1600-2000Lx, temperature 23+ -2 ℃). The callus induction rate and regeneration effect are shown in Table 1.
Example 2:
the inducing and regenerating process of tulip hypocotyl callus includes the following steps:
(1) The seeds of tulip variety 'daojones' (variety 2) are used as materials, and tulip seeds with consistent shape and size, full growth and good embryo are selected.
(2) - (7) the same as in example 1.
Example 3:
the inducing and regenerating process of tulip hypocotyl callus includes the following steps:
(1) The seeds of tulip variety 'pure Gold' (variety 3) are used as materials, and tulip seeds with consistent shape and size, full growth and good embryo are selected.
The callus induction medium in steps (4) and (5) was adjusted to 2.4-D1.0 mg/L,6-BA 1.0mg/L, KT 0.2mg/L, otherwise as in example 1.
As can be seen from Table 1, the white hypocotyl parts of the tulip hypocotyls from different varieties have higher callus induction rate, the induction rate is between 74% and 95%, the callus state is good, the proliferation capacity is strong, and the callus is not easy to brown; the green hypocotyl part only has lower callus induction rate in part of varieties; in the stage of cluster bud induction, the regeneration rate of the callus induced by the white hypocotyl is high, the number of the induced cluster buds is large, the regeneration efficiency is 33% -45%, and the callus induced by the green hypocotyl cannot be regenerated.
TABLE 1 induction rate of embryogenic callus and regeneration rate of multiple shoots of different varieties
The foregoing is illustrative of the best mode of carrying out the invention, and is not presented in any detail as is known to those of ordinary skill in the art. The protection scope of the invention is defined by the claims, and any equivalent transformation based on the technical teaching of the invention is also within the protection scope of the invention.
Claims (6)
1. The method for inducing and regenerating the tulip hypocotyl callus is characterized by comprising the following steps of:
step 1, cleaning and sterilizing tulip seeds, and inoculating the tulip seeds to a germination culture medium, wherein the germination culture medium is a 1/2MS culture medium;
step 2, after the tulip seeds germinate, after the hypocotyls grow to 6-8 cm, selecting white parts of the hypocotyls as callus-induced explants, cutting the callus-induced explants into small sections of 0.5cm, and inoculating the small sections to a callus-induced culture medium for dark culture, wherein the callus-induced culture medium is an MS (mass transfer) culture medium and comprises 1.0-3.0 mg/L of dichlorophenoxyacetic acid, 0.5-2.0mg/L of 6-benzylaminopurine and 0.1-0.3 mg/L of kinetin;
step 3, after dark culture for 8-12 days, cutting the hypocotyl into shorter small sections of 0.2-0.3 cm, and inoculating the small sections on an original induction culture medium to continue dark culture;
step 4, selecting callus with light yellow color, compact structure and vigorous growth after 15 days of induction culture for subculture, and carrying out subculture once every 30 days to obtain tulip hypocotyl-induced callus;
and 5, transferring the callus induced by the tulip hypocotyl into an adventitious bud induction culture medium to induce and generate adventitious buds.
2. The method for inducing and regenerating tulip hypocotyl callus according to claim 1, wherein in step 1, the specific method for cleaning and sterilizing tulip seeds comprises: soaking tulip seed in solution with 0.1% Tween-20 and 2-3 drops of detergent for 20min, washing with water for 2h, soaking with sterile water for 8-10 h, sterilizing with 75% ethanol in an ultra clean bench for 1min, washing with sterile water for three times, sterilizing with 2% sodium hypochlorite solution for 20min, and washing with sterile water for 5-6 times.
3. The method for inducing and regenerating a tulip hypocotyl callus according to claim 1, wherein in the step 1, the culture environment is dark culture at 4 ℃ for 40-60 days.
4. The method for inducing and regenerating tulip hypocotyl callus according to claim 1, wherein the secondary medium is MS medium and comprises 1.0mg/L dichlorophenoxyacetic acid, 0.5 mg/L6-benzylaminopurine and 0.1mg/L kinetin.
5. The method for inducing and regenerating tulip hypocotyl callus according to claim 1, wherein the germination medium, the callus induction medium and the adventitious bud induction medium each comprise 30g/L sucrose and 8g/L agar, and the pH value is 5.7-5.8.
6. The method for inducing and regenerating tulip hypocotyl callus according to claim 1, wherein the culturing conditions in step 5 are that the light irradiation time is 14-16 h/d, the light irradiation intensity is 1600-2000Lx, and the temperature is 23+ -2 ℃.
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