CN117178897B - Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants - Google Patents
Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants Download PDFInfo
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Abstract
The invention provides a method for rejuvenating mature somatic embryos of rubber trees and regenerating plants, and relates to the technical field of plant tissues and cell engineering. The method specifically comprises the following steps: selecting mature embryo with normal development, pale yellow color and moderate size and with 1-1.5cm cotyledon flattening, placing into 2,4-D solution containing 100mg/L, placing into a dark culture room at 23-27deg.C, shaking on a shaking table at 120-160rpm for 2-4D, taking out the mature embryo after shaking culture, cutting into embryo pieces of 3-5mm, and puncturing the epidermis of the embryo pieces with needle; inoculating the treated embryo blocks into a callus induction culture medium without 2,4-D, and performing callus induction, subculture, somatic embryo induction and plant induction to obtain the regenerated rubber tree plant. The method is not limited by sampling time and seasons, the materials are convenient to obtain, the materials are rich, the genetic stability of the female parent plant can be completely maintained, the proliferation coefficient is high, the tissue culture process is easy to control, and the method is particularly suitable for large-scale mass production and preparation.
Description
Technical Field
The invention belongs to the technical field of plant tissues and cell engineering, and particularly relates to a method for rejuvenating mature somatic embryos of rubber trees and regenerating plants.
Background
Rubber tree (Hevea brasiliensis muell. Arg.) belongs to the genus euphorbiaceae, is a perennial cross-pollinated arbor, and the produced natural rubber is an important industrial raw material and material. The regeneration plant obtained by the somatic embryogenesis way is an important method for the industrial production and genetic transformation of plant tissue culture seedlings.
In the prior art, regenerated plants obtained from rubber trees through a somatic embryogenesis way mainly adopt 1. Anther culture, 2. Inner bead culture and 3. Secondary somatic embryo circulation proliferation.
1. The anther culture technology of the rubber tree takes the male flowers of the rubber tree in a single-core stage as explants, peels off stamens, inoculates the stamens into an induced callus culture medium to induce callus, inoculates the callus into an induced embryo culture medium to induce embryogenesis, and finally inoculates the mature embryoid into a plant regeneration culture medium to induce complete plantlets. 2. The culture technique of the rubber tree inner beads is that the inner beads in young fruits 45-75 days after pollination of the rubber tree are used as explants to induce callus, and the callus is used for differentiating somatic embryos, and finally mature embryoids are used for differentiating plants. 3. The secondary embryo circulation proliferation is to cut the embryoid into 2-3mm small blocks with anther or inner bead as explant and induce secondary embryo, and the process circulates until enough embryoid is obtained and plant regeneration is induced.
The regeneration systems described above all have the following drawbacks: ① Anther, inner bead regeneration system: the source of the explant is easily limited by seasons and time, and the annual culture cannot be realized. The rubber tree has three flowering phases each year, namely spring flowers, summer flowers and autumn flowers, and the time for anther culture is limited each time. Generally, the natural pollination rate of the spring flowers of the rubber trees is higher than that of the summer flowers and the autumn flowers, so that the material taking time of the inner integument is generally 45-75d after the natural pollination of the spring flowers, namely, the optimal period for culturing the inner integument takes the samples from the middle 5 to the middle 6 of each year of Hainan China, and the shorter material taking time severely restricts the culturing of the inner integument. (Sun Aihua et al, culture of anthers of rubber trees, plant physiological communication, 2006, 42 (4): 785-789; huang Tian band, etc., rubber tree beads have been developed for life sciences research, 2011, 15 (2): 176-183). ② Secondary embryo circulation proliferation system: secondary embryos propagate in the room gradually accumulating hormones, possibly causing variation. ③ In the existing secondary embryo circulation proliferation process, the proliferation efficiency of the embryoid proliferation method is only 8 times (a method for rapidly propagating the self-root young clone of Brazilian rubber tree by utilizing embryoids, CN101138326B, hua Yuwei and the like), and the proliferation coefficient still needs to be improved.
Therefore, the establishment of a more efficient somatic embryogenesis technology system in the rubber tree is a technical problem to be solved urgently by those skilled in the art.
Disclosure of Invention
The invention aims to provide a method for rejuvenating mature somatic embryos of a rubber tree and regenerating plants, which takes mature embryos with flat cotyledons with normal development of about 1-1.5cm as explants, and optimizes the circulation and proliferation path of secondary somatic embryos by adding one-step 2,4-D shaking culture, dividing the mature embryos into small embryo blocks and puncturing the epidermis to be more beneficial to inducing callus. The method is not limited by sampling time and seasons, the materials are convenient to obtain, the proliferation coefficient is high, and the genetic stability of the female parent plant can be completely maintained.
In order to achieve the above purpose, the invention provides a method for rejuvenating mature somatic embryos of rubber trees and regenerating plants, which specifically comprises the following steps:
(1) Embryo selection: selecting mature embryo bodies which are normal in development, light yellow in color and flat in cotyledons with the size of 1-1.5 cm;
(2) Preculture of embryo: placing the selected mature embryo body into a2, 4-D solution with the concentration of 100mg/L, and culturing in a darkroom to obtain a embryo with enhanced light transmittance and softer state;
(3) Callus induction: taking out the mature embryo body after preculture, cutting into pieces, scratching the epidermis of each embryo piece, placing the embryo piece in a callus induction culture medium, and performing dark culture to obtain yellow granular tissues with loose structures;
(4) Subculture of callus: inoculating the loose granular tissue obtained in the step (3) into a callus induction culture medium, and performing dark culture to obtain a yellowish granular tissue with a compact structure;
(5) Secondary embryo induction: inoculating the compact granular tissue obtained in the step (4) into an embryoid induction culture medium, and performing dark culture to obtain an embryoid;
(6) And (3) plant induction: inoculating the obtained mature embryoid into a plant induction culture medium, and culturing under illumination to obtain the regenerated rubber tree plant.
In a preferred embodiment, in the step (2), the mass to volume ratio of the mature embryo body and the 2,4-D solution is 1g: (20-30) ml.
In a preferred embodiment, in the step (2), the dark room culture conditions are: immersing and shaking on a shaking table at 120-160rpm in a darkroom at 23-27 ℃ for 2-4d. In the invention, 2,4-D solution with specific concentration is used for soaking and shaking into mature somatic embryos, so that explants for recovering the state of young tissues are obtained, and if the concentration is too high or too low, the number of the explants for inducing the callus is small, and the efficiency is low.
In a preferred embodiment, in step (3), selected rubber tree sterile culture embryos are developed for mature cotyledonary stage embryos.
In a preferred embodiment, in step (3), the diced embryo pieces have a size of 3-5mm. If the embryo is too small, the survival rate is lower in the later culture process, and the propagation coefficient is reduced; if the embryo is oversized, the wound tissue is not dedifferentiated to form callus in the later culture process, but the embryo continues to grow in the state of embryo, so that the material waste is also caused, and the size of the embryo after the cutting is designed to be 3-5mm in the invention.
In a preferred embodiment, in the step (3), the operation of puncturing the epidermis of each embryo piece may be performed by any means adopted by those skilled in the art, such as puncturing the epidermis of each embryo piece with a needle. Multiple wounds can be formed, and the epidermis of the embryo is scratched.
In a preferred embodiment, in the step (3) and the step (4), the callus induction medium is based on MS medium, and 0.5-1.5mg/L of naphthylacetic acid, 1-2mg/L of kinetin, 0.3g/L of asparagine, 0.1g/L of inositol, 50-70g/L of sucrose, 5% (V/V) of coconut water and 2.2g/L of Phytagel are added; the pH of the medium is adjusted to 5.8-6.2.2,4-D is used as an auxin to positively influence cell proliferation in tissue culture, and in the invention, as the embryo preculture step is additionally carried out, the embryo is immersed in a high-concentration 2,4-D solution for 2-4D, so that an original formation layer can be formed on the surface of an embryo block, and a high-pressure mould is manufactured, thereby being beneficial to embryo emergence.
In a preferred embodiment of the present invention,
In the step (3), the dark culture conditions are: dark culturing at 23-27deg.C for 25-35d;
in the step (4), the dark culture conditions are: dark culturing at 23-27deg.C for 20-30d.
In a preferred embodiment, in the step (5), the embryoid body induction medium is based on MS medium, and 2, 4-D0.05-0.07 mg/L, indoleacetic acid 0.5-1.5mg/L, kinetin 2-4mg/L, gibberellin 0.4-0.6mg/L, inositol 0.1g/L, sucrose 50-70g/L, coconut water 5% (V/V), activated carbon 0.1% (W/V), phytagel 2.2g/L, and distilled water to 1L are added; the pH of the medium is adjusted to 5.8-6.2.
In a preferred embodiment, in the step (5), the dark culture conditions are: dark culture is carried out at 24-28 ℃ for 50-60d.
In a preferred embodiment, in the step (6), the plant induction medium is based on MS medium, and the plant induction medium is supplemented with 0.2-0.5mg/L of cytokinin, 2-3mg/L of gibberellin, 0.1-0.3mg/L of indoleacetic acid, 0.1g/L of inositol, 0.1% (W/V) of activated carbon, 30-50g/L of sucrose, 5% (V/V) of coconut water, 2.2g/L of Phytagel, and distilled water to 1L; the pH of the medium is adjusted to 5.8-6.2.
In a preferred embodiment, in the step (6), the light culture conditions are: at 24-28 ℃, a light source adopts a light source combination box, and the light source combination box is formed by white light and red light LED lamp beads according to the weight ratio of 7:1 quantity of the LED lamp beads are combined into 84 LED lamp beads, the total illumination intensity is 3500-3800Lux, and the LED lamp beads are cultured for 30-40d under the illumination condition that the illumination period is 12/12h (illumination/darkness).
Compared with the prior art, the method for rejuvenating mature somatic embryos and regenerating plants of the rubber trees has the following advantages:
1. in the invention, the source of the explant is not limited by sampling seasons, and the rubber tree test tube seedling can be produced continuously throughout the year, thereby overcoming the limitation of the sampling time and the seasons of the explant in the prior art.
2. The explant used in the invention has the advantages of convenient material acquisition, abundant materials, capability of completely maintaining the genetic stability of the female parent plant and high proliferation coefficient, and is especially suitable for large-scale cultivation.
3. The invention also improves a novel rubber tree embryogenesis step, provides a more efficient tissue culture system for large-scale breeding and transgenic breeding of the rubber tree from the root young clone, and has important significance for the development of the rubber tree industry and gene breeding in China.
Drawings
These and/or other aspects and advantages of the present invention will become more apparent and more readily appreciated from the following detailed description of the embodiments of the invention, taken in conjunction with the accompanying drawings, wherein:
FIG. 1 is a diagram of a blank used in step1 of the present invention;
FIG. 2 is a diagram of the embryo body after preculture according to step 2 of the present invention;
FIG. 3 is a schematic view of the epidermis of a needled punched embryo block according to the embodiment of the present invention, wherein the red arrows indicate the needled punching positions;
FIG. 4 shows calli induced in step 3 of the present example;
FIG. 5 shows spherical embryos induced in step 4 according to the present invention;
FIG. 6 shows a secondary embryo induced in step 5 of the present invention;
FIG. 7 shows regenerated plants obtained in step 6 of the present invention;
Detailed Description
Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc. used in the present invention are commercially available or may be prepared by existing methods;
In this example, the rubber tree embryo used is named: the commercial variety 73397 of the sterile rubber tree embryo is derived from the following sources: novel natural rubber planting material innovation base of China Tropical agricultural academy of sciences rubber institute.
Examples
(1) Embryo selection: selecting a commercial variety 73397 of sterile rubber tree embryo, and selecting a mature embryo body which is normal in development, light yellow in color and flat in cotyledon size of 1-1.5 cm;
(2) Preculture of embryo: the selected mature embryo body is prepared according to the mass-volume ratio of 1g:20ml is put into a2, 4-D solution with the concentration of 100mg/L, and is cultivated in a darkroom, namely, the darkroom at 25 ℃ is immersed and shaken for 3D on a shaking table at 140rpm, thus obtaining a somatic embryo with enhanced light transmittance and softer state;
(3) Callus induction: taking out the mature embryo body after preculture, cutting into embryo blocks with the thickness of 3-5mm, scratching the epidermis of each embryo block, placing the embryo blocks in a callus induction culture medium, and culturing for 30d in a darkroom with the temperature of 25 ℃ to obtain yellow granular tissues with loose structures;
(4) Subculture of callus: inoculating the loose granular tissue obtained in the step (3) into a callus induction culture medium, and culturing for 25 days in a darkroom at 25 ℃ to obtain a yellowish granular tissue with a compact structure;
the callus induction culture medium used in the steps (3) and (4) is based on an MS culture medium, and 1.5mg/L of naphthylacetic acid, 2mg/L of kinetin, 0.3g/L of asparagine, 0.1g/L of inositol, 70g/L of sucrose, 5% (V/V) of coconut water and 2.2g/L of Phytagel are added; the pH of the medium was adjusted to 5.8.
(5) Secondary embryo induction: inoculating the compact granular tissue obtained in the step (4) into embryoid induction culture medium, and culturing for 55d in a darkroom at 26 ℃ to obtain embryoid;
Wherein, the embryoid induction culture medium is based on MS culture medium, added with 2,4-D0.05mg/L, 1mg/L indoleacetic acid, 2mg/L kinetin, 0.6mg/L gibberellin, 0.1g/L inositol, 50g/L sucrose, 5% (V/V) coconut water, 0.1% (W/V) activated carbon and 2.2g/L Phytagel, and distilled water is added to 1L; the pH of the medium was adjusted to 5.8.
(6) And (3) plant induction: inoculating the obtained mature embryoid into a plant induction culture medium, and carrying out illumination culture to obtain a rubber tree regenerated plant;
Wherein, the illumination culture conditions are as follows: in the environment of 26 ℃, a light source combination box is taken as a light source, and the light source combination box is formed by mixing white light and red light LED lamp beads according to the following ratio of 7:1 quantity of the LED lamp beads which are 84 in total, wherein the total illumination intensity is 3600Lux, and the illumination period is 12/12h (illumination/darkness) for 35d of cultivation under illumination conditions;
the plant induction culture medium is based on MS culture medium, and is added with 0.5mg/L cytokinin, 3mg/L gibberellin, 0.3mg/L indoleacetic acid, 0.1g/L inositol, 0.1% (W/V) active carbon, 50g/L sucrose, 5% (V/V) coconut water, 2.2g/L Phytagel and distilled water to 1L; the pH of the medium was adjusted to 5.8.
In this example, the callus occurrence rate was 71.43%, the number of embryo pieces regenerated was 30.+ -. 7, and the proliferation factor of regenerated embryos was 30 times.
Comparative example
1. Influence of rubber embryo on embryo mass induction callus in different development periods
The classification is carried out according to the shape of the rubber tree embryo, and the classification can be divided into a five-period primordial embryo stage, a spherical embryo stage, a heart-shaped embryo stage, a torpedo embryo stage and a cotyledon embryo stage. Because the embryo existence time of the primordial embryo stage, the spherical embryo stage and the heart-shaped embryo stage is short, the difficulty in obtaining materials is not easy compared with other stages. Therefore, the influence of the rubber embryo on the induction of callus on the embryo mass was tested by selecting torpedo embryo stage and cotyledon embryo stage, and the results are shown in Table 1.
TABLE 1 influence of rubber embryos at different developmental stages on embryo mass-induced callus
| Type of climatic period | Number of explants inoculated | Number of explants to induce callus | Callus rate (%) |
| Torpedo embryo stage | 23 | 9 | 34.62 |
| Cotyledon embryo stage | 45 | 38 | 84.44 |
As can be seen from Table 1, the embryos in cotyledon embryo stage were propagated as somatic embryos and higher callus occurrence rate was obtained than in other stages. In addition, the cotyledon embryo stage belongs to the last stage of the growth of the rubber tree embryo, and the rubber tree embryo at the stage has the characteristics of easy availability, light yellow color, flat cotyledon and the like, and is more suitable for cultivation.
2. Influence of different preculture time on embryogenic mass induced callus
The embryo with normal development is selected, the color is light yellow, and the cotyledon with moderate size is about 1cm to 1.5 cm. The mass volume ratio is 1g:25ml of the selected mature embryo was placed in a 2,4-D solution containing 100mg/L and placed on a shaking table at room temperature (25 ℃) of 140rpm in a dark culture chamber to be immersed for 1D, 3D, 5D. The results are shown in Table 2.
TABLE 2 influence of different preculture times on embryogenic mass-induced callus
As can be seen from Table 2, the induction rate of the preculture 3d callus was the highest and reached more than 70%. As embryoid is soaked in high-concentration 2,4-D solution and is soaked on a shaking table for 2-4D, a primary formation layer is formed on the surface of the embryoid, if the pre-culture time is too short (such as 1D), the primary formation layer does not appear, and if the pre-culture time is too long (such as 5D), the primary formation layer is differentiated into a spherical embryo to develop, and the callus occurrence rate is reduced.
3. Different ratios of the preculture solutions affect the embryogenic mass-induced callus
The embryo with normal development is selected, the color is light yellow, and the cotyledon with moderate size is about 1 cm to 1.5 cm. The mass volume ratio is 1g:25ml of the selected mature embryo was placed in a shaker containing 100 mg/L2, 4-D solution, 100mg/L IAA solution, 50 mg/L2, 4-D+50mg/L IAA solution at room temperature (25 ℃) of 140rpm in a dark culture room and immersed for 3D. The results are shown in Table 3.
TABLE 3 influence of different ratios of preculture solutions on embryogenic mass-induced callus generation
As shown in Table 3, it was found that the callus induction rate was highest in the late stage of callus induction by using 100 mg/L2, 4-D solution as the preculture solution, and it was probably one of the reasons that IAA was a hormone which was relatively easily decomposed, and decomposed during shaking, resulting in a decrease in the concentration of hormone, thereby affecting the effect. Thus, the protocol of the present invention selects 100 mg/L2, 4-D solution as the pre-incubation solution.
4. Influence of different embryo sizes on inducing embryo callus
The embryo with normal development is selected, the color is light yellow, and the cotyledon with moderate size is about 1 cm to 1.5 cm. The mass volume ratio is 1g:25ml of the selected mature embryo is put into a 2,4-D solution containing 100mg/L, placed on a shaking table with a dark culture room at 140rpm and at room temperature (25 ℃) for 3D, taken out and cut into embryo blocks with the sizes of 1-3mm, 3-5mm and 5-7mm, and the embryo blocks are needled to break the epidermis. The results are shown in Table 4.
TABLE 4 influence of different embryo sizes on the induction of embryo callus
| Embryo block size | Number of explants inoculated | Callus numbers with embryogenesis | Incidence of callus (%) |
| 1-3mm | 36 | 15 | 41.67 |
| 3-5mm | 38 | 29 | 76.32 |
| 5-7mm | 32 | 6 | 18.75 |
As can be seen from Table 4, the callus induction rate of embryo size of 3-5mm is highest and can reach more than 70%. The survival rate of the embryo blocks with the size of 1-3mm is lower in the later culture process, and the embryo blocks with the size of 5-7mm are oversized, so that tissues at the wound part do not dedifferentiate to form callus in the later culture process, but continue to grow in the embryo state.
5. Influence of different callus induction media on induction of callus:
The embryo with normal development is selected, the color is light yellow, and the cotyledon with moderate size is about 1 cm to 1.5 cm. The mass volume ratio is 1g:25ml of the selected mature embryo was put into a solution containing 100mg/L of 2,4-D, immersed in a shaking table at room temperature (25 ℃) of 140rpm in a dark culture room for 3D, the precultured mature embryo was taken out, cut into pieces and needle punched through the epidermis of the embryo piece, and placed in the induced callus medium (A) described in example 1, and 15mg/L of 2,4-D was added to the induced callus medium (B) on the basis of (A) for 30D, and the results are shown in Table 5.
TABLE 5 influence of different callus induction media on the induction of callus
| Type of culture medium | Average incidence rate of callus is 100% | 5% Significant level | 1% Significant level |
| A | 50.00±4.57 | a | A |
| B | 27.25±4.06 | b | B |
As can be seen from Table 5, A, B had significant differences in the incidence of callus induced by the two media, the A media had significantly higher callus incidence than that of B media, and the A media induced callus growth amounts were significantly higher than that of B media. As can be seen, although A, B induced callus in both media, the B media induced callus on the basis of 2,4-D addition was less effective than medium A without 2,4-D addition.
6. Increase the proliferation efficiency of the secondary embryo circulation path in the pre-culture process:
the embryo with normal development is selected, the color is light yellow, and the cotyledon with moderate size is about 1 cm to 1.5 cm. The mass volume ratio is 1g:25ml of the selected mature embryo is put into a solution containing 100mg/L of 2,4-D, the solution is placed on a shaking table with a dark culture room at 140rpm and at room temperature (25 ℃) for 3D, the mature embryo after preculture is taken out, the mature embryo is cut into pieces and needled to break the epidermis of the embryo piece, the embryo piece is placed into an induced callus culture medium for dark culture for 30D, the obtained loose granular tissue is inoculated into a secondary culture medium for dark culture for 25D, and the granular tissue with light yellow color and compact structure is obtained; inoculating in embryoid induction culture, and dark culturing at 26deg.C for 55d to obtain embryoid. The culture medium and culture conditions used were identical to those of the examples, and the above experiment was repeated three times, three sets of the experiment were repeated each time, and the proliferation efficiency was evaluated, and the results are shown in Table 6, and embryoid bodies are shown in FIG. 6.
In conclusion, a plurality of groups of experiments can show that the technical scheme provided by the invention can stably and efficiently obtain the rubber tree plants, is not limited by sampling time and seasons, is convenient to obtain materials and high in proliferation coefficient, and provides a more efficient tissue culture system for the large-scale breeding of the rubber tree self-rooted young clone and transgenic breeding.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (6)
1. The method for rejuvenating and regenerating the mature somatic embryos of the rubber trees is characterized by comprising the following steps of:
(1) Embryo selection: selecting mature embryo bodies which are normal in development, light yellow in color and flat in cotyledons with the size of 1-1.5 cm;
(2) Preculture of embryo: placing the selected mature embryo body into a2, 4-D solution with the concentration of 100mg/L, and culturing in a darkroom to obtain a embryo with enhanced light transmittance and softer state;
(3) Callus induction: taking out the mature embryo body after preculture, cutting into pieces, scratching the epidermis of each embryo piece, placing the embryo piece in a callus induction culture medium, and performing dark culture to obtain yellow granular tissues with loose structures;
(4) Subculture of callus: inoculating the loose granular tissue obtained in the step (3) into a callus induction culture medium, and performing dark culture to obtain a yellowish granular tissue with a compact structure;
(5) Secondary embryo induction: inoculating the compact granular tissue obtained in the step (4) into an embryoid induction culture medium, and performing dark culture to obtain an embryoid;
(6) And (3) plant induction: inoculating the obtained mature embryoid into a plant induction culture medium, and carrying out illumination culture to obtain a rubber tree regenerated plant;
in the step (2), the culture conditions of the darkroom are as follows: immersing and shaking on a shaking table at 120-160rpm in a darkroom at 23-27 ℃ for 2-4d;
in the step (3), the size of the blanks after dicing is 3-5 mm;
in the step (3) and the step (4), the callus induction culture medium is based on an MS culture medium, and 0.5-1.5mg/L of naphthylacetic acid, 1-2mg/L of kinetin, 0.3g/L of asparagine, 0.1g/L of inositol, 50-70g/L of sucrose, 5% (V/V) of coconut water and 2.2g/L of Phytagel are added; regulating the pH of the culture medium to 5.8-6.2;
In the step (5), the embryoid induction medium is based on an MS medium, and is additionally provided with 0.05-0.07mg/L of 2,4-D, 0.5-1.5mg/L of indoleacetic acid, 2-4mg/L of kinetin, 0.4-0.6mg/L of gibberellin, 0.1g/L of inositol, 50-70g/L of sucrose, 5% (V/V) of coconut water, 0.1% (W/V) of activated carbon, 2.2g/L of Phytagel and 1L of distilled water; the pH of the medium is adjusted to 5.8-6.2.
2. The method for rejuvenation and plant regeneration according to claim 1, wherein in the step (2), the mass-to-volume ratio of the mature embryo to the 2,4-D solution is 1g: (20-30) ml.
3. The method for rejuvenation and plant regeneration of mature somatic embryos of rubber tree according to claim 1, wherein,
In the step (3), the dark culture conditions are: dark culturing at 23-27deg.C for 25-35d;
in the step (4), the dark culture conditions are: dark culturing at 23-27deg.C for 20-30d.
4. The method for rejuvenation and plant regeneration according to claim 1, wherein in the step (5), the dark culture conditions are as follows: dark culture is carried out at 24-28 ℃ for 50-60d.
5. The method for rejuvenating and regenerating a mature somatic embryo of rubber tree according to claim 1, wherein in the step (6), the plant induction medium is an MS medium-based medium, 0.2-0.5mg/L of additional kinetin, 2-3mg/L of gibberellin, 0.1-0.3mg/L of indoleacetic acid, 0.1g/L of inositol, 0.1% (W/V) of activated carbon, 30-50g/L of sucrose, 5% (V/V) of coconut water, 2.2g/L of phytagel and distilled water to 1L are supplemented; the pH of the medium is adjusted to 5.8-6.2.
6. The method for rejuvenation and plant regeneration according to claim 1, wherein in the step (6), the light culture conditions are as follows: at 24-28 ℃, a light source adopts a light source combination box, and the light source combination box is formed by white light and red light LED lamp beads according to the weight ratio of 7:1 quantity of the LED lamp beads are combined into 84 LED lamp beads, the total illumination intensity is 3500-3800 Lux, and the LED lamp beads are cultured for 30-40d under the illumination condition that the illumination period is 12/12h (illumination/darkness).
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