CN114027189A

CN114027189A - Method for obtaining regeneration plants from mature embryos of common camellia oleifera through somatic embryogenesis

Info

Publication number: CN114027189A
Application number: CN202111293416.3A
Authority: CN
Inventors: 朱华国; 庞芳芹; 胡孝明; 成新琪; 汤欣欣; 乌兰
Original assignee: Huanggang Normal University
Current assignee: Huanggang Normal University
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2022-02-11
Anticipated expiration: 2041-11-03
Also published as: CN114027189B

Abstract

The invention discloses a method for obtaining a regeneration plant from a mature embryo of a common camellia oleifera through a somatic embryogenesis mode, which comprises the following steps: taking a common mature oil tea embryo in 10-month middle ten days to be disinfected as an explant; pre-culturing the explant, and then performing differentiation culture on a differentiation culture medium to obtain an embryonic callus; wherein 0.05-0.15 mg/L2, 4-D + 0.05-0.15 mg/L KT is added into the differentiation medium; transferring the embryonic callus to an embryoid development and plant regeneration culture medium for regeneration culture to obtain an embryoid with obvious cotyledon; transferring the embryoid with obvious cotyledon to a rooting culture medium for rooting culture to form a rooted complete plant, hardening and domesticating the complete plant, and transplanting the plant to a culture medium for culture. The differentiation rate of the embryonic callus and the embryoid reaches more than 25 percent, the regeneration plant rate reaches more than 40 percent, and a complete regeneration plant is obtained within 6 months.

Description

Method for obtaining regeneration plants from mature embryos of common camellia oleifera through somatic embryogenesis

Technical Field

The invention relates to the technical field of plant cell engineering, in particular to a method for obtaining a regeneration plant from a mature embryo of a common camellia oleifera through a somatic embryogenesis mode.

Background

The camellia oleifera is a general name of the tree species with higher oil content of seeds in the camellia plants, is also a unique woody oil plant species in China, and is also called four woody oil crops in the world together with olive, oil palm and coconut. The tea oil has unsaturated fatty acid content up to 90%, is similar to olive oil, but has vitamin E content twice higher than that of olive oil, contains specific physiologically active substances such as camellin, and has high nutritive value. Meanwhile, the camellia oleifera forest is a good ecological forest, the camellia oleifera is also an important economic crop for structure adjustment and accurate poverty relief of agriculture and forestry industries, and the development of the camellia oleifera industry has very high economic, social and ecological benefits. In addition, the edible oil consumption in China is huge, the current self-sufficiency rate is less than 40%, and the grain and oil safety in China is seriously influenced. The oil tea industry is developed, mountain land space is developed, the problem of land contention with grains is avoided, the use efficiency of national soil resources is improved, and the national grain and oil safety is guaranteed.

With the continuous improvement of production development and social demands, higher and higher requirements are put on the yield, quality, resistance and the like of the camellia oleifera varieties in production, and the solution of the problems cannot be met only by the traditional breeding means. Until now, certain progress is made in the aspect of oil tea tissue culture, but a genetic transformation technical system based on somatic embryogenesis is not established yet, and the genetic transformation technical system is related to low regeneration efficiency of plants obtained by oil tea in a somatic embryogenesis mode.

Therefore, there is a need to develop a method for obtaining regenerated plants by somatic embryogenesis using mature embryos of camellia oleifera with high plant regeneration efficiency.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for obtaining a regenerated plant from a mature embryo of a common oil tea in a somatic embryogenesis mode, wherein tea fruits on excellent single plants are selected from a mature oil tea forest in Hubei Macheng of the northern main planting area of the common oil tea to be used as materials for in vitro culture of the mature embryo, a regeneration system in the somatic embryogenesis mode is established, the differentiation rate of embryogenic callus and embryoid is up to more than 25%, the regeneration plant rate is up to more than 40%, and a complete regeneration plant is obtained within 6 months.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for obtaining a regeneration plant from a mature embryo of common camellia oleifera through a somatic embryogenesis mode, which comprises the following steps:

taking a common mature oil tea embryo in 10-month middle ten days to be disinfected as an explant;

pre-culturing the explant, and then performing differentiation culture on a differentiation culture medium to obtain an embryonic callus; wherein 0.05-0.15 mg/L2, 4-D + 0.05-0.15 mg/L KT is added into the differentiation medium;

transferring the embryonic callus to an embryoid development and plant regeneration culture medium for regeneration culture to obtain an embryoid with obvious cotyledon;

transferring the embryoid with obvious cotyledon to rooting culture medium for rooting culture to form rooted complete plant.

And (4) hardening and domesticating the rooted complete plant, and transplanting the acclimated complete plant into a culture medium for culture.

Further, the medium used for the preculture is MSB hormone-free medium.

Further, the pre-culture time is 25-35 d.

Furthermore, the differentiation medium is MSB medium added with 0.1 mg/L2, 4-D +0.1mg/L KT.

Furthermore, the time of the differentiation culture is 50-70 d, and the subculture is performed for 1-2 times.

Further, the medium for embryoid body development and plant regeneration is the hormone-free MSB medium.

Furthermore, the regeneration culture time is 50-70 d, and the intermediate subculture is carried out for 1-2 times.

Further, the rooting medium is 1/2MSB medium without hormone.

Further, the rooting culture time is 25-35 d.

Further, the conditions of the differentiation culture, the regeneration culture and the rooting culture all comprise: culturing in a constant-temperature illumination incubator with the temperature of 28 +/-1 ℃ and the temperature of 2000 and 3000lux, wherein the illumination time is 14-16 h/d.

Further, the method for sterilizing mature embryos of common camellia oleifera in 10 th of the month as explants comprises the following steps:

removing peel and seed coat of Camellia oleifera fruit from mature embryo of Camellia oleifera in 10-month middle ten days by hand, surface sterilizing in 75% ethanol for 30s, and adding 0.1% HgCl₂Sterilizing for 10min, washing with sterile water for 4-5 times to obtain sterile explant.

Further, the MSB medium is specifically composed as follows: 1.9g/L potassium nitrate, 1.65g/L ammonium sulfate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate heptahydrate, 0.44g/L calcium chloride dihydrate, 22.3mg/L manganese sulfate tetrahydrate, 6.2mg/L boric acid, 8.6mg/L zinc sulfate heptahydrate, 0.83mg/L potassium iodide, 0.25mg/L sodium molybdate dihydrate, 0.025mg/L copper sulfate pentahydrate, 0.025mg/L cobalt chloride hexahydrate, 37.3mg/L sodium ethylenediaminetetraacetate, 27.8mg/L ferrous sulfate heptahydrate, 2mg/L glycine, 1mg/L nicotinic acid, 10mg/L thiamine hydrochloride, 1mg/L pyridoxine hydrochloride, and 100mg/L inositol.

The pre-culture medium, the differentiation medium, the embryoid development and plant regeneration medium and the rooting medium have pH ranges of 5.8-6.2, and preferably the pH is 6.0.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. selecting excellent single-plant camellia fruits from mature camellia oleifera forests in Hubei Macheng of main northern region of common camellia oleifera as materials, performing in-vitro culture of mature embryos, establishing a regeneration system in a somatic embryogenesis mode, achieving the differentiation rate of embryogenic callus and embryoid of more than 25%, the regeneration plant rate of more than 40%, and obtaining complete regeneration plants within 6 months.

2. The tissue culture object of the invention is mature embryo, which is convenient to obtain than immature embryo and can better meet the tissue culture requirement.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a picture of the results of callus differentiation and embryoid generation of Camellia oleifera;

FIG. 2 is a picture of the development of oil tea embryoid bodies and plant regeneration results; FIG. 2A is a diagram showing the result of embryoid body development and plant regeneration; 2B is a picture of the rooting culture result of the regeneration plant;

FIG. 3 shows rooting and domestication transplanting of Camellia oleifera regenerated plants; wherein FIG. 3A is a picture of normal regenerated plant results; FIG. 3B is a picture of the results of normal plants after transplantation.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

because mature embryos are weak in differentiation capacity and low in regeneration plant rate, the mature embryos are not adopted in the prior art, but immature embryos are adopted to obtain regeneration plants;

however, because the immature embryos are limited by the development period and are easily affected by disinfection, the mature embryos are convenient to obtain compared with the immature embryos and can better meet the requirement of tissue culture, and therefore, a method for obtaining regeneration plants from the mature embryos of the common oil tea by a somatic embryogenesis mode is urgently needed to be developed;

according to an exemplary embodiment of the present invention, there is provided a method for obtaining a regenerated plant from a mature embryo of Camellia oleifera Abel by somatic embryogenesis, the method comprising:

Mature embryos are used as explants and sequentially undergo differentiation culture, regeneration culture and rooting culture, wherein 0.05-0.15 mg/L2, 4-D + 0.05-0.15 mg/L KT is added into a differentiation culture medium; a regeneration system in a somatic embryogenesis mode is established, the differentiation rate of the embryogenic callus and the embryoid is more than 25%, the plant regeneration rate is more than 40%, and a complete regeneration plant is obtained within 6 months.

In the technical scheme, if the concentration of the 2,4-D is less than 0.05mg/L or more than 0.15mg/L, the callus is not favorable to be formed. The KT concentration is not favorable for embryoid differentiation if the KT concentration is less than 0.05mg/L or more than 0.15 mg/L.

The method for obtaining a regenerated plant from a mature embryo of common camellia oleifera according to the present application by somatic embryogenesis will be described in detail below with reference to examples, comparative examples and experimental data.

Example 1

The method for obtaining the regeneration plant from the mature embryo of the common camellia oleifera provided by the embodiment in a somatic embryogenesis mode comprises the following steps:

and step S1, selecting excellent single-plant tea fruits from mature tea-oil trees in the river town of Hua shan village in Macheng city, Hubei province in 10 th of the middle ten days, storing the tea-oil trees in a refrigerator at 4 ℃, and completing disinfection and inoculation within 3 days. Peeling peel and seed coat of Camellia oleifera fruit by hand under aseptic condition of superclean bench, surface sterilizing in 75% alcohol for 30s, and sterilizing with 0.1% HgCl₂The seeds were sterilized for 10 minutes and rinsed with sterile water 4 times.

And S2, inoculating the mature embryos to a pre-culture medium under aseptic conditions, inoculating 2-3 mature embryos to each bottle, and pre-culturing the triangular bottles inoculated with the mature embryos in a constant-temperature incubator at 28 ℃ under the condition of illumination for 16 hours/day and illumination intensity of 2000 lux. The pre-culture time is 25-35 d. The culture medium adopted by the pre-culture is MSB culture medium without hormone;

then, the mature embryos without pollution are subcultured to a differentiation medium (0.1 mg/L2, 4-D +0.1mg/L KT is added to the MSB medium, and the pH is adjusted to 5.8) for differentiation regulation. After 1 subculture, explants of embryogenic callus and embryoid bodies will appear, as shown in FIG. 1; the differentiation medium is MSB medium added with 0.1 mg/L2, 4-D +0.1mg/L KT.

The MSB basic culture medium comprises the following components: 1.9g/L potassium nitrate, 1.65g/L ammonium sulfate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate heptahydrate, 0.44g/L calcium chloride dihydrate, 22.3mg/L manganese sulfate tetrahydrate, 6.2mg/L boric acid, 8.6mg/L zinc sulfate heptahydrate, 0.83mg/L potassium iodide, 0.25mg/L sodium molybdate dihydrate, 0.025mg/L copper sulfate pentahydrate, 0.025mg/L cobalt chloride hexahydrate, 37.3mg/L sodium ethylenediaminetetraacetate, 27.8mg/L ferrous sulfate heptahydrate, 2mg/L glycine, 1mg/L nicotinic acid, 10mg/L thiamine hydrochloride, 1mg/L pyridoxine hydrochloride, and 100mg/L inositol. 3% (W/V) sucrose, 0.9% (W/V) agar was added. Sterilizing with high pressure steam at 1.2kg/cm²The temperature is 121 ℃, and the sterilization time is 15 min.

And step S3, transferring the explants with embryogenic callus and embryoid to an embryoid development and plant regeneration culture medium (MSB culture medium without hormone and pH adjusted to 5.8) for continuous culture.

Step S4, obtaining embryoid with obvious cotyledon (as shown in figure 2), transferring to rooting medium (1/2MSB, pH adjusted to 5.8) for culturing. Complete regeneration plants can be obtained within 6 months, as shown in figure 3;

and step S5, domesticating, and transferring to a nutrition pot for continuous growth. The above treatment was carried out in 8 flasks, each flask was inoculated with 3 embryos and repeated 3 times, with a subculture cycle of 30 days.

Example 2

then, the mature embryos without pollution are subcultured to a differentiation medium (0.1 mg/L2, 4-D +0.1mg/L KT is added to the MSB medium, and the pH is adjusted to 5.8) for differentiation regulation. After 1 subculture, explants with embryogenic callus and embryoid bodies appear; the differentiation medium is MSB medium added with 0.05 mg/L2, 4-D +0.05mg/L KT.

The MSB basic culture medium comprises the following components: 1.9g/L of potassium nitrate, 1.65g/L of ammonium sulfate, 0.17g/L of monopotassium phosphate, 0.37g/L of magnesium sulfate heptahydrate, 0.44g/L of calcium chloride dihydrate,22.3mg/L of manganese sulfate tetrahydrate, 6.2mg/L of boric acid, 8.6mg/L of zinc sulfate heptahydrate, 0.83mg/L of potassium iodide, 0.25mg/L of sodium molybdate dihydrate, 0.025mg/L of copper sulfate pentahydrate, 0.025mg/L of cobalt chloride hexahydrate, 37.3mg/L of sodium ethylene diamine tetracetate, 27.8mg/L of ferrous sulfate heptahydrate, 2mg/L of glycine, 1mg/L of nicotinic acid, 10mg/L of thiamine hydrochloride, 1mg/L of pyridoxine hydrochloride and 100mg/L of inositol. 3% (W/V) sucrose, 0.9% (W/V) agar was added. Sterilizing with high pressure steam at 1.2kg/cm²The temperature is 121 ℃, and the sterilization time is 15 min.

And step S4, after obtaining embryoid with obvious cotyledon, transferring to rooting medium (1/2MSB, pH is adjusted to 5.8) for culturing. The complete regeneration plant can be obtained within 6 months,

Example 3

then, the mature embryos without pollution are subcultured to a differentiation medium (0.1 mg/L2, 4-D +0.1mg/L KT is added to the MSB medium, and the pH is adjusted to 5.8) for differentiation regulation. After 1 subculture, explants with embryogenic callus and embryoid bodies appear; the differentiation medium is MSB medium added with 0.15 mg/L2, 4-D +0.15mg/L KT.

Comparative example 1

In this comparative example, the differentiation medium was MSB medium supplemented with only 0.1mg/L of 2,4-D, and the rest of the procedure was the same as in example 1.

Comparative example 2

In this comparative example, the differentiation medium was MSB medium supplemented with 0.1mg/L KT alone, and the rest of the procedure was the same as in example 1.

Comparative example 3

In this comparative example, the concentration of 2,4-D in the differentiation medium was changed to 2mg/L and the concentration of KT was changed to 1mg/L, and the rest of the procedure was the same as in example 1.

Experimental example 1

Statistics of the results of the regeneration plants obtained from the mature embryos of common camellia oleifera in examples 1 to 3 and comparative examples 1 to 2 by means of somatic embryogenesis are shown in table 1. Wherein, the differentiation rate of the embryonic callus and the embryoid is counted after the differentiation culture is carried out for 60 days, and the embryogenic rate is counted after the differentiation regulation culture is carried out for 60 days.

Embryogenic callus and embryoid differentiation rate (%) — the number of mature embryos forming embryogenic callus and embryoid/the number of inoculated mature embryos × 100%;

the regenerated plant rate (%). is the number of regenerated plants/number of inoculated mature embryos × 100%.

TABLE 1

As can be seen from the data in table 1,

in comparative example 1, the differentiation medium was MSB medium supplemented with only 0.1mg/L of 2,4-D, embryogenic callus and embryoid had higher differentiation rates, but no regenerated plant was obtained;

in comparative example 2, the differentiation medium was MSB medium supplemented with only 0.1mg/L KT, the differentiation rates of embryogenic callus and embryoid were low, and no regenerated plant was obtained;

in the comparative example 3, the concentrations of 2,4-D and KT are higher, the differentiation rates of embryogenic callus and embryoid are higher, but no regeneration plant is obtained;

example 1-example 3, tissue culture of mature embryos of Camellia oleifera Abel, 0.1 mg/L2, 4-D +0.1mg/L KT-treated embryogenic callus and embryoid differentiation rates were 25.22% -28.33%, respectively; the regeneration plant rate is 41.26% -48.67%.

The above results indicate that the differentiation medium of the present invention, in which 2,4-D and KT are either absent or at concentrations outside the range of the examples of the present invention, does not achieve a satisfactory effect.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method for obtaining a regeneration plant from a mature embryo of common camellia oleifera through a somatic embryogenesis mode is characterized by comprising the following steps:

2. The method for obtaining a regenerated plant from a mature embryo of camellia oleifera as claimed in claim 1, wherein the culture medium used for the pre-culture is MSB hormone-free medium.

3. The method for obtaining a regenerated plant from a mature embryo of common camellia oleifera as claimed in claim 1 by means of somatic embryogenesis, wherein the pre-culture time is 25-35 d.

4. The method for obtaining a regenerated plant from a mature embryo of camellia oleifera as claimed in claim 1, wherein the differentiation medium is MSB medium supplemented with 0.05-0.15 mg/L2, 4-D + 0.05-0.15 mg/L KT.

5. The method for obtaining the regeneration plant of the mature embryo of common camellia oleifera as claimed in claim 1, wherein the differentiation culture time is 50-70 d, and 1-2 times of subculture is performed.

6. The method for obtaining a regenerated plant from a common camellia oleifera mature embryo by means of somatic embryogenesis according to claim 1, wherein the medium for embryoid body development and plant regeneration is a hormone-free MSB medium.

7. The method for obtaining the regeneration plant of the mature embryo of common camellia oleifera as claimed in claim 1, wherein the regeneration culture time is 50-70 d, and 1-2 times of intermediate subculture.

8. The method for obtaining regenerated plants from mature embryos of camellia oleifera as claimed in claim 1, wherein the rooting medium is 1/2MSB hormone-free medium.

9. The method for obtaining the regeneration plant of the mature embryo of common camellia oleifera as claimed in claim 1, wherein the rooting culture time is 25-35 days.

10. The method for obtaining regenerated plants from mature embryos of camellia oleifera as claimed in claim 1 by somatic embryogenesis, wherein the conditions of the differentiation culture, the regeneration culture and the rooting culture all comprise: culturing in a constant-temperature illumination incubator with the temperature of 28 +/-1 ℃ and the temperature of 2000 and 3000lux, wherein the illumination time is 14-16 h/d.