CN112106663B - Method for establishing waxberry seedling clone - Google Patents
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Abstract
The invention discloses a method for establishing a waxberry seedling clone, belonging to the technical field of plant breeding. The invention comprises the following steps: selecting waxberry fruits in a hard core period, removing pulp and shells of the kernels, taking out seeds, and sterilizing; removing seed coats of the seeds and keeping kernels; inoculating the kernels on a basic culture medium for pre-culture; then transferring the kernels which expand and turn green on the basic culture medium to a differentiation induction culture medium for culture to obtain differentiated materials; inoculating the differentiated material into an adventitious bud induction proliferation culture medium for culture to obtain differentiated adventitious buds; finally, the differentiated adventitious buds are separated one by one and inoculated in a rooting culture medium for culturing and rooting, and the waxberry vegetative clone is obtained. Compared with the prior art that the seed seedling is adopted, the method adopts the kernels of immature seeds as explants, greatly shortens the seedling culture period, can obtain multiple seedlings from one kernel, and is favorable for accelerating the process of new variety breeding and variety updating of the waxberries or obtaining a large number of excellent stocks for grafting.
Description
Technical Field
The invention belongs to the technical field of plant breeding, and particularly relates to a method for establishing a waxberry seedling clone.
Background
The waxberry (Moreal rubra Bieb. et Zucc) is a special fruit tree species originally produced in China and mainly distributed in the south of Yangtze river. The fruit is sour and sweet, and has unique flavor. In recent years, the growing area of the waxberries is rapidly developed and the benefit is remarkable, and the waxberries become an important income source for farmers in partial mountainous areas. But the popularization and cultivation of the waxberries have the situation of single variety type. The varieties popularized for a long time are all local varieties or are grafted and propagated from a preferred single plant.
The methods for grafting the waxberry onto the rootstock and breeding the waxberry in the prior art are mainly carried out in a mode of cultivating kernels to obtain seedlings, but mature waxberry shells are hard, the emergence rate is low (50-60%), the emergence rate of a cultivated variety with good quality is low, the seedling culture period is long, and the method is not beneficial to accelerating the breeding of new waxberry varieties and the process of variety updating, so that a method for obtaining the waxberry seedling clone more quickly and efficiently is needed.
The plant tissue culture and the plant rapid propagation technical system formed on the basis are important platforms for developing high-efficiency breeding and breeding of production product species for fruit trees. The method has the greatest characteristic that clones with consistent genetic backgrounds can be formed in a short time, a large amount of materials are provided for subsequent selection tests, meanwhile, the method can be stored indoors, and a large amount of land resources are saved. Most of reports about tissue culture of waxberries use stem segments as culture materials, and kernel as culture materials are not reported so far.
Disclosure of Invention
Aiming at the defects in the existing breeding and breeding technology, the invention aims to solve the technical problem of providing a method for establishing a waxberry true vegetative clone.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for establishing a waxberry seedling clone comprises the following steps:
1) selecting immature waxberry fruits in a hard core period, removing pulp and fruit core shells, taking out seeds, and sterilizing for later use;
2) removing seed coats from the seeds and keeping kernels;
3) inoculating the kernels on a basic culture medium for pre-culture;
4) transferring the kernels which expand and turn green on a basic culture medium to a differentiation induction culture medium for culture to obtain differentiated materials, wherein the differentiation induction culture medium comprises: a basic culture medium, indoleacetic acid, TDZ and gibberellin;
5) inoculating the differentiated material into an adventitious bud induction proliferation culture medium for culture to obtain differentiated adventitious buds, wherein the adventitious bud induction proliferation culture medium comprises: a basic culture medium, indolebutyric acid, 6-BA and gibberellin;
6) separating the differentiated adventitious buds one by one, inoculating the adventitious buds to a rooting culture medium for culturing and rooting to obtain the waxberry vegetative clone, wherein the rooting culture medium comprises: minimal medium + indolebutyric acid + gibberellin.
Further, the method for establishing the waxberry vegetative clone comprises the following steps:
1) selecting immature waxberry fruits in a hard core period, removing pulp and fruit core shells, taking out seeds, and sterilizing for later use;
2) removing seed coats of the seeds under the aseptic condition, and keeping kernels;
3) inoculating the kernels on a basic culture medium for pre-culture;
4) transferring the kernels which expand and turn green on a basic culture medium to a differentiation induction culture medium for culture to obtain differentiated materials, wherein the differentiation induction culture medium comprises: a basic culture medium, 0.5mg/L indoleacetic acid, 0.5mg/L TDZ and 0.5mg/L gibberellin;
5) inoculating the differentiated material into an adventitious bud induction proliferation culture medium for culturing to obtain differentiated adventitious buds, and subculturing once every 30 days, wherein the adventitious bud induction proliferation culture medium comprises: a basic culture medium, 0.5mg/L indolebutyric acid, 0.5 mg/L6-BA and 0.3mg/L gibberellin;
6) separating the differentiated adventitious buds one by one, inoculating the adventitious buds to a rooting culture medium for culturing and rooting to obtain the waxberry vegetative clone, wherein the rooting culture medium comprises: the minimal medium is added with 0.5mg/L indolebutyric acid and 0.5mg/L gibberellin.
Further, the method for establishing the waxberry vegetative clone comprises the following steps:
1) selecting immature waxberry fruits in a hard core period, removing pulp and fruit core shells, taking out seeds, and sterilizing for later use;
2) removing seed coats of the seeds under the aseptic condition, and keeping complete kernels;
3) inoculating the kernel on a basic culture medium for pre-culture for 21 days;
4) transferring the kernels which expand and turn green on a basic culture medium to a differentiation induction culture medium for culturing for 40 days to obtain differentiated materials, wherein the differentiation induction culture medium comprises: a basic culture medium, 0.5mg/L indoleacetic acid, 0.5mg/L TDZ and 0.5mg/L gibberellin;
5) inoculating the differentiated material into an adventitious bud induction proliferation culture medium for culturing for 60 days to obtain differentiated adventitious buds, and subculturing once every 30 days, wherein the adventitious bud induction culture medium comprises: a basic culture medium, 0.5mg/L indolebutyric acid, 0.5 mg/L6-BA and 0.3mg/L gibberellin;
6) separating the differentiated adventitious buds one by one, selecting adventitious buds with the length of 1.5-2cm, inoculating the adventitious buds to a rooting culture medium, and culturing for 30 days to obtain the waxberry vegetative clone, wherein the rooting culture medium comprises: the minimal medium is added with 0.5mg/L indolebutyric acid and 0.5mg/L gibberellin.
Further, in step 1), the sterilization specifically comprises: washing with small amount of detergent, rinsing with running water for 2 hr, soaking in 70% ethanol for 30s under shaking in a clean bench, soaking in 0.1% mercuric chloride under shaking for 8-10min, and rinsing with sterile water for 5 times.
Further, in step 3), the minimal medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +30g/L sucrose +5g/L agar.
Further, in step 4), the minimal medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +30g/L sucrose +5g/L agar.
Further, in step 5), the minimal medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +40g/L sucrose +5g/L agar.
Further, in step 6), the minimal medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +20g/L sucrose +5g/L agar.
Further, the culture conditions in steps 3) to 6) are as follows: the illumination intensity is 2000lx, the illumination time is 16 hours/day, the culture temperature is 26 ℃, and the pH value of the culture medium is 5.7-5.8.
Compared with the prior art, the invention has the beneficial effects that:
compared with the existing waxberry tissue culture technology, the method for establishing the waxberry vegetative clone takes kernels in immature hard-core waxberry fruits as explants, and the waxberry vegetative clone is quickly established through the steps of pre-culture, differentiation induction culture, proliferation induction culture, rooting culture and the like, so that the method is convenient and efficient; compared with the traditional method for breeding seedlings by adopting seeds, the method for breeding the waxberry seedlings by adopting the seeds in the unripe waxberry fruits can be carried out without waiting for the waxberry fruits to be completely mature, the seedling breeding period is greatly shortened, the waxberry seedling clone can be obtained more quickly and efficiently, multiple seedlings can be obtained from one seed, and the method is favorable for accelerating the breeding of new waxberry varieties and the process of variety updating or quickly obtaining a large number of excellent stocks for grafting.
Drawings
FIG. 1 is a process diagram of obtaining a vegetative clone of red bayberry plant using red bayberry kernel as explant according to the present invention;
FIG. 2 is a diagram of differentiated materials obtained by differentiation induction;
FIG. 3 is a diagram of adventitious buds obtained by induction of adventitious bud proliferation;
FIG. 4 is a graph of adventitious bud rooting.
Detailed Description
The invention is further described with reference to specific examples.
Example 1:
as shown in fig. 1, a method for establishing a clone of red bayberry includes the following steps:
1) selecting immature fruits of waxberries in a hard core period, removing pulp and fruit cores and shells, taking out seeds, cleaning with a small amount of detergent, slightly and slowly washing with running water for 2h, then shaking and soaking in 70% ethanol for 30s in an ultraclean workbench, then shaking and soaking in 0.1% mercuric chloride for 8-10min, washing with sterile water for 5 times, placing on sterilized dry filter paper, removing seed coats under sterile conditions, and keeping complete kernels.
2) Inoculating the treated kernels on a basic culture medium for culture, wherein the basic culture medium comprises WPM, 100mg/L ammonium sulfate, 40mg/L calcium chloride, 30g/L sucrose and 5g/L agar. The culture conditions were: the illumination intensity is 2000lx, the illumination time is 16 hours/day, the culture temperature is about 26 ℃, the pH value of the culture medium is 5.7-5.8, and the culture is carried out for 21 days.
3) Inoculating the green-changed and expanded material on a differentiation induction culture medium, wherein the illumination intensity is 2000lx, the illumination time is 16 hours/day, the culture temperature is about 26 ℃, the pH value of the culture medium is 5.7-5.8, and the culture is carried out for 40 days. The differentiation-inducing medium formulation is shown in table 1.
TABLE 1 formulation of differentiation induction Medium
| Numbering | Formulation of |
| A1 | Minimal medium +0.5mg/L indoleacetic acid +0.5mg/L TDZ |
| A2 | Minimal medium +0.5mg/L indoleacetic acid +0.5mg/L TDZ +0.5mg/L gibberellin |
| A3 | Minimal medium +0.2mg/L indoleacetic acid +0.2mg/L TDZ |
| A4 | Minimal medium +0.2mg/L indoleacetic acid +0.2mg/L TDZ +0.2mg/L gibberellin |
| A5 | Minimal medium +1.0mg/L indoleacetic acid +1.0mg/L TDZ |
| A6 | Minimal medium +1.0mg/L indoleacetic acid +1.0mg/L TDZ +1.0mg/L gibberellin |
In the table, the minimal medium was WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +30g/L sucrose +5g/L agar. After 40 days, the number of new buds of the differentiated material obtained by inducing differentiation in each culture medium was counted, and as shown in table 2 and fig. 2, it can be seen that the culture effect of the A2 differentiation induction culture medium was the best, the differentiation number was 44, and the average number of new buds was 5.1.
TABLE 2 differentiation induction Medium Effect
4) Inoculating the differentiated material on an adventitious bud proliferation induction culture medium, wherein the illumination intensity is 2000lx, the illumination time is 16 hours/day, the culture temperature is about 26 ℃, the pH value of the culture medium is 5.7-5.8, and the culture is carried out for 60 days (1 subculture in 30 days). The differentiation-inducing medium formulation is shown in Table 3.
TABLE 3 formulation of adventitious bud Induction proliferation Medium
| Numbering | Formulation of |
| B1 | Minimal medium +0.5mg/L indolebutyric acid +0.5 mg/L6-BA |
| B2 | Minimal medium +0.5mg/L indolebutyric acid +0.5 mg/L6-BA +0.3mg/L gibberellin |
| B3 | Minimal medium +0.2mg/L indolebutyric acid +0.5mg/L TDZ |
| B4 | Minimal medium +0.2mg/L indolebutyric acid +0.5mg/L TDZ +0.3mg/L gibberellin |
| B5 | Minimal medium +0.5mg/L indoleacetic acid +0.5 mg/L6-BA |
| B6 | Minimal medium, 0.5mg/L indoleacetic acid, 0.5 mg/L6-BA and 0.3mg/L gibberellin |
| B7 | Minimal medium +0.2mg/L indoleacetic acid +0.5mg/L TDZ |
| B8 | Minimal medium +0.2mg/L indoleacetic acid +0.5mg/L TDZ +0.3mg/L gibberellin |
| B9 | Minimal medium +1.0mg/L indolebutyric acid +0.5 mg/L6-BA +0.5mg/L gibberellin |
| B10 | Minimal medium +1.0mg/L indolebutyric acid +0.5mg/L TDZ +0.5mg/L gibberellin |
In the table, the minimal medium was WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +40g/L sucrose +5g/L agar. After 60 days, the number of adventitious bud-induced proliferations and the proliferation fold of each culture medium were counted, and as shown in table 4 and fig. 3, it was found that the culture effect of the B2 differentiation-induced culture medium was the best, the number of proliferations occurred was 42, and the proliferation fold was 6.9 fold.
TABLE 4 Effect of adventitious bud Induction proliferation Medium
5) The differentiated adventitious buds are separated one by one, the adventitious buds with the length of 1.5-2cm are selected and inoculated in a rooting culture medium, the illumination intensity is 2000lx, the illumination time is 16 hours/day, the culture temperature is about 26 ℃, the pH value of the culture medium is 5.7-5.8, and the culture is carried out for 30 days (1 subculture in 30 days). The rooting medium formulation is shown in table 5.
TABLE 5 formulation of rooting medium
| Numbering | Formulation of |
| C1 | Minimal medium, 0.5mg/L indolebutyric acid and 0.5mg/L gibberellin |
| C2 | Minimal medium +0.5mg/L indolebutyric acid +0.2 mg/L6-BA +0.5mg/L gibberellin |
| C3 | Minimal medium +1.0mg/L indolebutyric acid +0.5mg/L gibberellin |
| C4 | Minimal medium +1.0mg/L indolebutyric acid +0.2mg/L TDZ +0.3mg/L gibberellin |
In the table, the minimal medium was WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +20g/L sucrose +5g/L agar. After 30 days, the rooting condition of each culture medium is counted, and as shown in table 6 and fig. 4, it can be seen that the average rooting numbers of the culture media C1 and C3 are more than those of other culture media and are not greatly different from each other, while the rooting number of the C1 culture medium is far more than that of the C3 culture medium and other culture media, so that the C1 culture medium is most suitable for rooting adventitious buds.
TABLE 6 Effect of rooting Medium
| Serial number | Inoculation amount (strain) | Number of rooted plants | Average number of roots |
| C1 | 50 | 36 | 2.8 |
| C2 | 50 | 22 | 2.3 |
| C3 | 50 | 26 | 3.0 |
| C4 | 50 | 19 | 1.9 |
Claims (4)
1. A method for establishing a waxberry seedling clone is characterized by comprising the following steps:
1) selecting immature waxberry fruits in a hard core period, removing pulp and fruit core shells, taking out seeds, and sterilizing for later use;
2) removing seed coats of the seeds under the aseptic condition, and keeping kernels;
3) inoculating the kernels on a basic culture medium for pre-culture;
4) transferring the kernels which expand and turn green on a basic culture medium to a differentiation induction culture medium for culture to obtain differentiated materials, wherein the differentiation induction culture medium comprises: a basic culture medium, 0.5mg/L indoleacetic acid, 0.5mg/L TDZ and 0.5mg/L gibberellin;
5) inoculating the differentiated material into an adventitious bud induction proliferation culture medium for culturing to obtain differentiated adventitious buds, and subculturing once every 30 days, wherein the adventitious bud induction proliferation culture medium comprises: a basic culture medium, 0.5mg/L indolebutyric acid, 0.5 mg/L6-BA and 0.3mg/L gibberellin;
6) separating the differentiated adventitious buds one by one, inoculating the adventitious buds to a rooting culture medium for culturing and rooting to obtain the waxberry vegetative clone, wherein the rooting culture medium comprises: a basic culture medium, 0.5mg/L indolebutyric acid and 0.5mg/L gibberellin;
wherein, in the step 3) and the step 4, the basic culture medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +30g/L sucrose +5g/L agar;
in step 5), the basic culture medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +40g/L sucrose +5g/L agar;
in step 6), the minimal medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +20g/L sucrose +5g/L agar.
2. The method for establishing a vegetative clone of red bayberry of claim 1, comprising the steps of:
1) selecting immature waxberry fruits in a hard core period, removing pulp and fruit core shells, taking out seeds, and sterilizing for later use;
2) removing seed coats of the seeds under the aseptic condition, and keeping kernels;
3) inoculating the kernel on a basic culture medium for pre-culture for 21 days;
4) transferring the kernels which expand and turn green on a basic culture medium to a differentiation induction culture medium for culturing for 40 days to obtain differentiated materials, wherein the differentiation induction culture medium comprises: a basic culture medium, 0.5mg/L indoleacetic acid, 0.5mg/L TDZ and 0.5mg/L gibberellin;
5) inoculating the differentiated material into an adventitious bud induction proliferation culture medium to culture for 60 days to obtain differentiated adventitious buds, and subculturing once every 30 days, wherein the adventitious bud induction proliferation culture medium comprises: a basic culture medium, 0.5mg/L indolebutyric acid, 0.5 mg/L6-BA and 0.3mg/L gibberellin;
6) separating the differentiated adventitious buds one by one, selecting adventitious buds with the length of 1.5-2cm, inoculating the adventitious buds to a rooting culture medium, and culturing for 30 days to obtain the waxberry vegetative clone, wherein the rooting culture medium comprises: a basic culture medium, 0.5mg/L indolebutyric acid and 0.5mg/L gibberellin;
wherein, in the step 3) and the step 4, the basic culture medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +30g/L sucrose +5g/L agar;
in step 5), the basic culture medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +40g/L sucrose +5g/L agar;
in step 6), the minimal medium is: WPM +100mg/L ammonium sulfate +40mg/L calcium chloride +20g/L sucrose +5g/L agar.
3. The method for establishing a clone of red bayberry plant according to any one of claims 1-2, wherein the sterilization and disinfection in step 1) is specifically: cleaning with detergent, slowly washing with running water for 2 hr, soaking in 70% ethanol for 30s in a clean bench under shaking, soaking in 0.1% mercuric chloride for 8-10min under shaking, and washing with sterile water for 5 times.
4. The method for establishing a clone of red bayberry fruit according to any one of claims 1-2, wherein the culture conditions in steps 3) -6) are as follows: the illumination intensity is 2000lx, the illumination time is 16 hours/day, the culture temperature is 26 ℃, and the pH value of the culture medium is 5.7-5.8.
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| JP2006141252A (en) * | 2004-11-17 | 2006-06-08 | Nippon Paper Industries Co Ltd | Method for producing plant body |
| CN102119661A (en) * | 2010-12-24 | 2011-07-13 | 浙江省农业科学院 | Tissue culture method of myrica rubra |
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| JP2006141252A (en) * | 2004-11-17 | 2006-06-08 | Nippon Paper Industries Co Ltd | Method for producing plant body |
| CN102119661A (en) * | 2010-12-24 | 2011-07-13 | 浙江省农业科学院 | Tissue culture method of myrica rubra |
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| 云南野生杨梅快繁体系的建立;孔梅等;《浙江农业科学》;20151231;摘要，材料与方法 * |
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