CN115843682A - Method for inducing and regenerating tulip embryonic axis callus - Google Patents
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Abstract
The invention relates to a tulip hypocotyl callus induction and regeneration method, which comprises the following steps: 1. cleaning and disinfecting tulip seeds, and then inoculating the tulip seeds to 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 embryonic axis germinated from the tulip seeds as a callus induction explant, inoculating the callus induction explant onto a callus induction culture medium and carrying out dark culture, and 3, cutting the embryonic axis into small sections of 0.2-0.3 cm, inoculating the small sections onto an original induction culture medium and carrying out dark culture continuously; 4. after 15 days of induction culture, selecting the callus with vigorous growth for subculture to obtain the callus induced by the embryonic axis of tulip; 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 the hypocotyl, is derived from the embryogenic organ after the seed embryo germinates, and has high activity, strong meristematic capacity and large dedifferentiation potential.
Description
Technical Field
The invention relates to the technical field of plant rapid propagation, in particular to a method for inducing and regenerating tulip embryonic axis callus.
Background
Tulipa Gesneriana L.is a bulbous flower of Tulipa of Liliaceae. The tulip has unique flower shape and bright color, is deeply loved by people in the world, is widely applied to fresh cut flowers, pot culture and landscaping greening, and has high ornamental value and economic value. The propagation mode of the tulip comprises sowing propagation and bulb-separating propagation. Wherein, the sowing and breeding cycle is long, most of the cultivated species need 3 to 5 years from the seeds to the formation of flowering bulbs, the time cycle is long, and the breeding and reproduction expanding efficiency of tulip is restricted. The bulb is a main nutrition propagation organ of the tulip, generally, the seed bulb propagation coefficient of a commodity bulb is 1-6, which is greatly lower than the propagation efficiency of other annual or biennial herbaceous plants, so that most of cultivated species have the problems of low seed bulb propagation coefficient, seed bulb degradation 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 re-flower, and a large number of commodity balls need to be imported from foreign countries every year. How to improve the propagation efficiency of tulip is a technical bottleneck in the breeding and cultivation fields. In recent years, there have been many researches on tissue culture using scapes, axillary buds, stem segments, bulbs and the like as explants, and attempts have been made to establish an efficient tissue culture rapid propagation technical system to overcome the problem of low propagation efficiency of tulip. According to the report, the tulip tissue culture and rapid propagation technology at present mainly utilizes stems and flower stems to directly induce and regenerate bulblets, or utilizes seeds and scales to firstly dedifferentiate to obtain callus, and then induces the generation and expansion of the bulblets through the differentiation of regeneration buds. The said technology has different explant sampling parts and different explant material taking time, and callus inducing and regenerating efficiency.
The inventor of the technology establishes a tissue culture rapid propagation technical system (ZL 2020 1 0204397.1) of wild tulip by taking a seed embryo as an explant in the earlier stage, but the technical system has lower adaptability to tulip cultivated species, which indicates that different tissue culture rapid propagation technical modes may exist between the wild species and the cultivated species due to larger genetic background difference. In the wild tulip, although the callus can be directly induced by using the embryo, only one callus can be induced and generated during primary culture of one embryo, and the problems of limited explant number and low utilization rate exist, so that the application space of the method in the tissue culture and rapid propagation of the tulip is limited to a certain extent. Therefore, there is a need to establish a tissue culture rapid propagation technology which has relatively sufficient explant sources and is suitable for cultivars. The research and development of the technology have important significance for improving the propagation efficiency of tulip cultivars, shortening the breeding period and reducing the production cost. In addition, a tissue culture rapid propagation technical system with wide variety applicability is established, so that the problem of seedball degradation can be solved, the dependence on foreign seedballs is reduced, the breeding efficiency of tulip in China can be accelerated, the competitiveness of the tulip industry in China is fundamentally improved, and technical reserve is provided for realizing domestication of seedballs.
Disclosure of Invention
According to the problems in the prior art, the invention discloses a tulip hypocotyl callus induction and regeneration method, the method takes the hypocotyl of the germinated tulip seed as an explant, the explant can obtain a large amount of callus in a short time on a callus induction culture medium, and the regeneration bud is obtained after differentiation culture, thereby providing important technical support for proliferation and propagation and genetic transformation of tulip.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for inducing and regenerating tulip embryonic axis callus comprises the following steps:
step 1, cleaning and disinfecting tulip seeds, and then 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, selecting a part of hypocotyls generated by the germination of the tulip seeds as a callus induction explant, cutting the callus induction explant into small segments of 0.5cm, inoculating the small segments 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 embryonic axis into shorter segments of 0.2-0.3 cm, and inoculating the segments on the original induction culture medium for continuous dark culture;
step 4, selecting callus with light yellow color, compact structure and vigorous growth after 15 days of induction culture for subculture, and subculturing 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 disinfecting the tulip seeds comprises the following steps: the tulip seeds are placed in a solution added with 0.1 percent of Tween-20 and 2 to 3 drops of liquid detergent for soaking for 20min, then washed by running water for 2h, then soaked by sterile water for 8 to 10h, sterilized by 75 percent of ethanol for 1min in a super clean bench, washed by the sterile water for three times, sterilized by 2 percent of sodium hypochlorite solution for 20min, and washed by the sterile water for 5 to 6 times.
Further, in the step 1, the culture environment is 4 ℃ for dark culture for 40-60 days.
Further, in the step 2, when the embryonic axis of the tulip grows to 6-8 cm, the white part of the embryonic axis is selected as an explant for callus induction.
Further, the subculture 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.
Furthermore, the germination culture medium, the callus induction culture medium and the adventitious bud induction culture medium respectively comprise 30g/L of sucrose and 8g/L of agar, and the pH value is 5.7-5.8.
Further, the culture conditions in the step 5 are that the illumination time is 14-16 h/d, the illumination intensity is 1600-2000Lx, and the temperature is 23 +/-2 ℃.
The invention has the beneficial effects that:
1. the explant has sufficient source and strong dedifferentiation capability. The explant adopted by the invention is the hypocotyl, is derived from the embryogenic organ after the seed embryo germinates, and has high activity, strong meristematic capacity and large dedifferentiation potential. Compared with other explants, the callus induction is easier, the influence of environmental seasons is avoided, the obtaining and the storage are simple, and the explant source is sufficient.
2. The callus tissue has short induction period and high efficiency. 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 callus induction period compared with other explants and induction methods.
3. The method is suitable for multiple tulip cultivars. Due to the difference of genetic background, the tulip tissue culture rapid propagation technology is often different from variety to variety, and the same tissue culture technical system is difficult to be suitable for a plurality of varieties or strains. The method is suitable for tissue culture and rapid propagation of multiple tulip cultivars, expands the variety applicability and has wider application prospect.
The present invention will be described in detail below with reference to the drawings and examples.
Drawings
FIG. 1 is a technical process of the present invention, which takes the tissue culture rapid propagation and callus induction of tulip varieties 'Fan Landi', 'dow jones' and 'pure gold' as examples to compare the induction efficiency of the callus at different parts of hypocotyl after the seed embryo germinates.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
As shown in figure 1, the method for inducing and regenerating tulip embryonic axis callus specifically comprises the following steps:
(1) The tulip variety 'Fan Landi' ('Verandi') (variety 1) is selected as a material, has excellent ornamental property and resistance property, and is a cut flower and garden application variety which is popular in the market. Selecting the Fan Landi seeds with consistent shape and size, plump growth and good embryo.
(2) The tulip seeds are placed in a solution added with 2-3 drops of 0.1% of Tween-20 and liquid detergent for soaking for 20min, washed with running water for 2h, and soaked in sterile water for 8-10 h. In a clean bench, sterilizing with 75% ethanol 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) Inoculating the disinfected tulip seeds on a 1/2MS culture medium (pH 5.7-5.8), and carrying out dark culture for 60 days (4 ℃) until the seed embryos germinate and the length of the embryonic axis is about 6-8 cm.
(4) In a clean workbench, the tulip hypocotyl is placed on clean sterile filter paper, the white part and the green part of the hypocotyl are cut into small sections of about 0.5cm by a scalpel, and the small sections are inoculated on a callus induction culture medium (MS culture medium, 30g/L of sucrose, 8g/L of agar, 5.7-5.8 of pH, 2.4-D3.0 mg/L, 0.5mg/L of 6-BA and 0.3mg/L of KT) for pre-culture. Dark culture is carried out for 10 days (23 +/-2 ℃), and the expansion and elongation of the embryonic axis are 1 to 1.5 times of the original expansion and elongation.
(5) Cutting the elongated embryonic axis into small sections of about 0.2-0.3 cm, inoculating the small sections on the original culture medium, and continuously culturing the small sections in dark (23 +/-2 ℃). After 5 days, the generation of callus can be observed, and after 20 days of continuous culture, the callus can be seen to obviously expand and present milky white or yellow compact particles.
(6) The callus with good growth is inoculated to a subculture medium (MS culture medium, 30g/L of sucrose, 8g/L of agar, pH 5.7-5.8,2.4-D1.0 mg/L,6-BA 0.5mg/L and KT 0.1 mg/L). Subculture was carried out for 30 days as one cycle.
(7) Selecting callus with good growth, inoculating the callus on adventitious bud induction culture medium (MS culture medium, 30g/L of cane sugar, 8g/L of agar, pH 5.7-5.8, 0.5mg/L of NAA, 1.0mg/L of 6-BA), and performing adventitious bud induction culture (culture conditions: illumination time is 14-16 h/d, illumination intensity is 1600-2000Lx, and temperature is 23 +/-2 ℃). The callus induction rate and regeneration effect are shown in Table 1.
Example 2:
an induction and regeneration method of tulip embryonic axis callus comprises the following specific implementation steps:
(1) The tulip seeds with consistent shape and size, full growth and good seed embryo are selected by taking seeds of a tulip variety 'Dow Jones' (variety 2) as materials.
(2) - (7) same as in example 1.
Example 3:
an induction and regeneration method of tulip embryonic axis callus comprises the following specific implementation steps:
(1) The method is characterized in that seeds of tulip varieties 'pure Gold' (variety 3) are used as materials, and tulip seeds which are consistent in shape and size, full in growth and good in 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, and the same as in example 1.
As can be seen from Table 1, white hypocotyls of tulip from different varieties have high callus induction rate, the induction rate is 74-95%, and the callus has good state, strong multiplication capacity and difficult browning; the green hypocotyl part has lower callus induction rate only in partial varieties; in the cluster bud induction stage, the regeneration rate of the callus induced by the white hypocotyl is higher, the number of the induced cluster buds is large, the regeneration efficiency is between 33 and 45 percent, and the callus induced by the green hypocotyl cannot be regenerated.
TABLE 1 different varieties of hypocotyl callus induction rate and regeneration rate of cluster buds
The foregoing is illustrative of the best mode of the invention and details not described herein are within the common general knowledge of a person of ordinary skill in the art. The scope of the present invention is defined by the appended claims, and any equivalent modifications based on the technical teaching of the present invention are also within the scope of the present invention.
Claims (7)
1. A tulip embryonic axis callus induction and regeneration method is characterized by comprising the following steps:
step 1, cleaning and disinfecting tulip seeds, and then 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, selecting a part of hypocotyls generated by the germination of the tulip seeds as a callus induction explant, cutting the callus induction explant into small segments of 0.5cm, inoculating the small segments 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 embryonic axis into shorter segments of 0.2-0.3 cm, and inoculating the segments on the original induction culture medium for continuous dark culture;
step 4, selecting callus with light yellow color, compact structure and vigorous growth after 15 days of induction culture for subculture, and subculturing 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 embryonic axis callus as claimed in claim 1, wherein the specific method for washing and disinfecting tulip seeds in step 1 is as follows: the tulip seeds are placed in a solution added with 0.1 percent of Tween-20 and 2 to 3 drops of liquid detergent for soaking for 20min, then washed by running water for 2h, then soaked by sterile water for 8 to 10h, sterilized by 75 percent of ethanol for 1min in a super clean bench, washed by the sterile water for three times, sterilized by 2 percent of sodium hypochlorite solution for 20min, and washed by the sterile water for 5 to 6 times.
3. The method for inducing and regenerating tulip embryonic axis callus according to claim 1, wherein in step 1, the culture environment is 4 ℃ dark culture for 40-60 days.
4. The method for inducing and regenerating tulip embryonic axis callus as claimed in claim 1, wherein in step 2, white part of the embryonic axis is selected as explant for callus induction when the tulip embryonic axis grows to 6-8 cm.
5. The method for inducing and regenerating tulip embryonic axis callus as claimed in claim 1, wherein the subculture medium is MS medium and comprises 1.0mg/L of dichlorophenoxyacetic acid, 0.5mg/L of 6-benzylaminopurine and 0.1mg/L of kinetin.
6. The method for inducing and regenerating tulip hypocotyl callus as claimed in claim 1, wherein said germination medium, callus induction medium and adventitious bud induction medium each comprise 30g/L sucrose and 8g/L agar, and pH is 5.7-5.8.
7. The method for inducing and regenerating tulip hypocotyl callus as claimed in claim 1, wherein the culturing conditions in step 5 are 14-16 h/d of light intensity 1600-2000Lx and 23 ± 2 ℃.
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