CN115812599B

CN115812599B - Efficient regeneration method using Japanese larch cotyledon as explant

Info

Publication number: CN115812599B
Application number: CN202211588219.9A
Authority: CN
Inventors: 朱强; 赖嘉俊
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2024-05-10
Anticipated expiration: 2042-12-12
Also published as: CN115812599A

Abstract

The invention discloses a method for inducing adventitious buds and obtaining regenerated plants by taking Japanese larch cotyledons as explants, which comprises the steps of sowing, sterilizing, inducing culture, proliferation culture, rooting culture, hardening off and transplanting. The invention utilizes the induction culture medium, the proliferation culture medium and the rooting culture medium containing different hormone ratios, so that the Japanese larch has high differentiation efficiency, high proliferation coefficient, high rooting rate, short plant growth demand time, strong adaptability after cultivation, strong and straight seedlings, good growth vigor, uniformity, great shortening of the seedling culture process and great cost saving. The invention establishes an adventitious bud induction system which takes Japanese larch cotyledon as an initial, successfully obtains regenerated plants, provides a technical means for rapid propagation of Japanese larch seedlings, and also provides a platform for establishing a Japanese larch transgenic system with high efficiency in the future.

Description

Efficient regeneration method using Japanese larch cotyledon as explant

Technical Field

The invention belongs to the technical field of plant cultivation and propagation, and particularly relates to a high-efficiency regeneration method taking Japanese larch cotyledon as an explant.

Background

Larch (Larch) is a Larch, belonging to the family pinaceae (Pinaceae), genus Larch (Larix). The genus larch in the world has 25 kinds of natural regions distributed in the temperate region, plain in the cold-warm zone and in the mountain climate region. In China, larix Gmelini is distributed in the area with the altitude of 300-1200 m in great and small Khingan, and has large accumulation and large forest area. The Japanese larch has the characteristics of strong adaptability, fast growth in early stage, fast forest formation, less plant diseases and insect pests and excellent material quality, is an important pulp material and building material tree species in northeast, northwest, north China and south Asian mountain areas, and is also a main afforestation tree species for returning and protecting forest projects.

The conventional breeding of Japanese larch is more influenced by natural conditions, the growth period is long, the genetic operation difficulty is high, the consumption of manpower and material resources is high, and the requirement of modern forest genetic breeding cannot be met. In recent years, with rapid development of genetic engineering breeding, research on genetic transformation of conifer plants is more and more, few researches on Japanese larch transgenes are reported in China at present, and no good Japanese larch regeneration system is an important reason, so that establishment of a high-efficiency stable conifer plant tissue culture system is a basic premise for genetic transformation. There have been many reports about tissue culture systems of Japanese larch, ji Liwang et al, which induce adventitious buds from current-year tender stem segments; wu Kexian and the like, taking the top buds of the side branches of the larch of the longwhite Japanese larch as explants to carry out in vitro culture to induce cluster buds, but the efficiency of inducing adventitious buds is low; wang Weida and the like, which take immature zygotic embryos as explants to induce adventitious buds, the plant regeneration and genetic improvement are restricted by low rooting rate.

The invention establishes a complete regeneration system of the Japanese larch by taking the cotyledon of the Japanese larch seedling as an explant, inducing adventitious buds, extending the adventitious buds to a certain extent, then successfully rooting and transplanting the cotyledon to a greenhouse, and lays a good foundation for the tissue culture and rapid propagation and genetic improvement of the Japanese larch in the later period. The number of adventitious buds formed by the Japanese larch leaf regeneration system is large, the genetic stability is good, and cotyledons have larger particle receiving areas, so that the method is suitable for genetic transformation and application research mediated by a gene gun and is also suitable for an agrobacterium-mediated genetic transformation system.

Disclosure of Invention

The invention aims to provide a high-efficiency regeneration method using Japanese larch cotyledon as an explant, which has the characteristics of convenient material taking, low cost, simple operation, high propagation speed, high propagation coefficient and the like, and can provide a high-efficiency regeneration system for a Japanese larch genetic transformation system.

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

a high-efficiency regeneration method using Japanese larch cotyledon as explant comprises the following steps:

1) Sowing: selecting mature and full seeds, soaking the seeds in tap water for 24h days, sowing the seeds in a matrix, germinating the seeds for 15-20 days, and selecting strong seedlings as materials for a next experiment;

2) And (3) disinfection: cleaning the seedlings obtained in the step 1) with tap water, removing roots, soaking in 75vol% alcohol for 30-60s, soaking in 0.1vol% mercuric chloride solution for 5-7 min, and washing with sterile water for 5 times;

3) Induction culture: taking cotyledon sections of the seedlings after the sterilization in the step 2) as explants, inoculating the explants to an induction medium, and culturing for 30 days to obtain adventitious buds; the formula of the induction culture medium is as follows: DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ;

4) Proliferation culture: cutting the adventitious buds obtained by culturing in the step 3), transferring the cut adventitious buds to a proliferation culture medium, and culturing for 60 days to obtain adventitious bud clusters; the proliferation culture medium comprises the following formula: DCR medium+1.5 mg/L6BA+0.001 mg/L TDZ+0.2g/L activated carbon;

5) Rooting culture: inoculating the adventitious bud cluster obtained by the culture in the step 4) to a rooting culture medium for culturing for 30-60 days to obtain a rooting seedling; the rooting culture medium comprises the following formula: DCR medium+0.05 mg/LNAA+0.2g/L activated carbon;

6) Hardening seedlings: transferring the rooting seedlings obtained by culturing in the step 5) to a seedling hardening chamber for hardening seedlings;

7) Transplanting: after hardening off the seedlings for 10 days, transplanting the rooting seedlings into a matrix, culturing for 1-2 weeks in a greenhouse, and then carrying out shading and astigmatism culture.

Wherein the matrix is prepared by mixing nutrient soil, vermiculite and perlite according to a volume ratio of 2:2:1.

Wherein, the conditions of induction culture, proliferation culture and rooting culture are as follows: the illumination rate is 80 mu mol.m ^-2·s^-1, the illumination time is 16 h/d, and the room temperature is 25+/-2 ℃.

Wherein, the step 6) specifically comprises the following steps: putting the rooting seedling obtained by culturing in the step 5) into tap water to perform seedling hardening in a semi-open bottle state, wherein the temperature of a seedling hardening room is 25 ℃, the illumination time is 16 h/d, the illumination intensity is 80 mu mol.m ^-2·s^-1, and the humidity is 65%.

Wherein, the step 7) specifically comprises the following steps: after hardening off the seedlings for 10 days, when the root system is changed from white to off-white, transplanting the rooting seedlings, firstly rinsing the rooting seedlings in clear water to remove the culture medium of the root during transplanting, then airing the surface moisture of the root system, transplanting the rooting seedlings into a container with a matrix, and culturing in a greenhouse for 1-2 weeks, wherein the greenhouse culturing conditions are as follows: the illumination time is 16 h/d, the illumination intensity is 2000 lux, the temperature is 21-26 ℃, then shading and astigmatism culture is carried out, water is sprayed every day, and the humidity is kept at 40-60%.

The application of the high-efficiency regeneration method in the in-vitro propagation of Japanese larch.

The application of the high-efficiency regeneration method in the establishment of a Japanese larch cultivation system.

The high-efficiency regeneration method is applied to the germplasm preservation of the excellent strain of Japanese larch.

Due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) The invention selects Japanese larch seedling cotyledon germinated in a greenhouse as a source of explants, and has the characteristics of simple material taking, no time and quantity limitation, and time and labor saving.

(2) The invention provides factors required by explants in the period of rooting and strengthening seedlings by using the culture mediums containing different hormone ratios so as to provide good rooting conditions and promote healthy and healthy seedlings, and the regenerated seedlings of the Japanese larch are obtained.

Drawings

Fig. 1: effect of different concentrations of Naphthalene Acetic Acid (NAA) on induction of adventitious buds of larch in japan.

Fig. 2: influence of different concentrations of activated carbon on the elongation of adventitious buds of larch.

Fig. 3: regeneration process using Japanese larch cotyledon as explant. Wherein A is a sterilized explant; b is an adventitious bud generated after the explant is inoculated into an induction culture medium for 15 d; c is adventitious buds generated after the explants are inoculated into an induction culture medium for 30 d; d is a single adventitious bud about 50 days after the adventitious bud is inoculated into the proliferation culture medium; e is the proliferation condition of adventitious buds in a culture dish; f is bud cluster cultured in a strong seedling culture medium; g is a rooted regenerated seedling; h is the transplanted regenerated seedling. The scale bar in the figure is 1cm.

Fig. 4: histochemical schematic of the formation process of adventitious buds of Japanese larch cotyledon. Wherein A is the longitudinal section of the cotyledon just inoculated; b is the longitudinal section of cotyledon inoculated for 10 days; c is meristem protrusion; D-E is the protruding longitudinal section of the bud primordium; f is the longitudinal section of the formed adventitious bud. The scale bar in the figure is 0.1mm.

Detailed Description

In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.

Example 1

The effect of Thidiazuron (TDZ) at different concentrations on the induction of adventitious buds of Larix Gmelini was examined as follows:

1) Sowing: selecting mature and full Japanese larch seeds, soaking the seeds in warm water for 24 hours, sowing the seeds on a cultivation substrate containing vermiculite, germinating the seeds for 15-20 days, and selecting strong seedlings as materials for a next experiment.

2) And (3) disinfection: cleaning the seedlings obtained in the step 1) with tap water, removing roots, soaking in 75vol% alcohol for 30-60s, soaking in 0.1vol% mercuric chloride solution for 5-7 min s, and washing with sterile water for 5 times each for 1min;

3) Induction culture: taking cotyledon sections of the sterilized seedlings as explants, inoculating the explants to an induction culture medium, culturing for 30 days under the conditions of illumination rate of 80 mu mol.m ^-2·s^-1, illumination time of 16 h/d and culture temperature of 25+/-2 ℃, and observing and recording the induction condition of adventitious buds; the formula of the induction culture medium is selected from any one of the following:

① DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ;

② DCR medium+0.75 mg/L6-BA+0.01 mg/L TDZ;

③ DCR medium+0.75 mg/L6-BA+0.05 mg/L TDZ.

Analysis shows that the TDZ concentration obviously affects the bud induction frequency, and when the TDZ is 0.001 mg/L, the induction rate of the cotyledon adventitious buds is highest and can reach 19.35%; with the increase of the concentration of TDZ, the induction rate shows a decreasing trend, and when the TDZ is 0.05 mg/L, the induction rate of adventitious buds is only 10.14%. As can be seen, the addition of 0.001 mg/L of TDZ had a good effect on the induction of adventitious buds of Larix Gmelini (Table 1).

TABLE 1 Effect of different concentrations of TDZ on the induction of adventitious buds of Larix Gmelini

Note that: the data are the mean ± error values of triplicate, different letters in the same column indicate significant differences at the 0.05 level, and the same letters indicate insignificant differences.

Example 2

The effect of Naphthalene Acetic Acid (NAA) at different concentrations on the induction of adventitious buds of Larix Gmelini was examined as follows:

2) And (3) disinfection: cleaning the seedlings obtained in the step 1) with tap water, removing roots, soaking in 75vol% alcohol for 30-60s, soaking in 0.1vol% mercuric chloride solution for 5-7 min s, and washing with sterile water for 5 times, wherein each time is 1 min.

① DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ;

② DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ+0.1mg/L NAA;

③ DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ+0.3mg/L NAA;

④ DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ+0.5mg/L NAA;

⑤ DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ+1mg/L NAA.

The observations indicate (FIG. 1) that the average adventitious bud induction rate reached maximum when NAA was not added; as the concentration of added NAA increases, the induction efficiency of adventitious buds of larch gradually decreases; when the concentration is increased to 0.3 mg/L, the adventitious bud induction efficiency is reduced to 6.5%; and when NAA concentration reached 1 mg/L, induction of adventitious buds began to be inhibited, and the status of adventitious buds began to worsen. It was demonstrated that low or no NAA added in combination with TDZ effectively promoted adventitious bud induction, whereas high NAA concentrations inhibited adventitious bud formation. Since NAA had a less pronounced effect on the induction of adventitious buds of cotyledons of Japanese larch seedlings, NAA was not added later in the experiment.

Example 3

The effect of varying concentrations of 6-benzyladenine (6-BA) in combination with Thidiazuron (TDZ) on the induction of adventitious buds of Larix Gmelini was examined as follows:

① DCR medium+0.75 mg/L6-BA+0.001 mg/L TDZ;

② DCR medium+0.75 mg/L6-BA+0.01 mg/L TDZ;

③ DCR medium+0.75 mg/L6-BA+0.05 mg/L TDZ;

④ DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ;

⑤ DCR medium+1.5 mg/L6-BA+0.01 mg/L TDZ;

⑥ DCR medium+1.5 mg/L6-BA+0.05 mg/L TDZ;

⑦ DCR medium+3 mg/L6-BA+0.001 mg/L TDZ;

⑧ DCR medium+3 mg/L6-BA+0.01 mg/L TDZ;

⑨ DCR medium+3 mg/L6-BA+0.05 mg/L TDZ.

The results showed (Table 2) that the adventitious bud induction rate was lower when the 6-BA concentration was 0.75mg/L and 3 mg/L; the high efficiency when the concentration of 6-BA is 1.5mg/L shows that Japanese larch cotyledon has a certain tolerance to 6-BA, and the high and low values are unfavorable for the generation of adventitious buds; therefore, DCR medium supplemented with 0.001mg/L TDZ and 1.5 mg/L6-BA induced the highest efficiency of larch explants.

TABLE 2 Effect of combinations of different concentrations of 6-BA and TDZ on the induction of adventitious buds of Larix Gmelini

Example 4

The influence of the active carbon with different concentrations on the elongation of the adventitious bud of the larch of Japan is discussed, and the steps are as follows:

3) Induction culture: taking cotyledon sections of the sterilized seedlings as explants, inoculating the explants to an induction culture medium, culturing for 30 days under the conditions of illumination rate of 80 mu mol.m ^-2·s^-1, illumination time of 16 h/d and culture temperature of 25+/-2 ℃, and observing and recording the induction condition of adventitious buds; the formula of the induction culture medium is as follows: DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ.

4) Proliferation culture: cutting the adventitious bud obtained by culture, transferring to proliferation culture medium, culturing under the conditions of illumination rate of 80 μmol.m ^-2·s^-1, illumination time of 16 h/d and culture temperature of 25+ -2deg.C, observing and recording adventitious bud growth condition, and counting elongation after 60 days; the formula of the proliferation medium is selected from any one of the following:

① DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ;

② DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ+0.2g/L activated carbon;

③ DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ+0.5g/L activated carbon;

④ DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ+1g/L activated carbon.

The results showed (FIG. 2) that the elongation of the adventitious bud increased and then decreased with increasing amount of activated carbon, and the elongation reached the maximum at 0.2g/L of activated carbon. Thus, 0.2g/L of activated carbon was added to the medium in the subsequent experiments.

Example 5

1) Sowing: selecting mature and full Japanese larch seeds, soaking the seeds in warm water for 24 hours, sowing the seeds on a matrix prepared by mixing nutrient soil, vermiculite and perlite according to a ratio of 2:2:1 (v/v), germinating the seeds for 15-20 days, and selecting healthy and strong seedlings as materials for a next experiment.

3) Induction culture: taking cotyledon sections of the sterilized seedlings as explants, inoculating the explants to an induction culture medium, and culturing for 30 days under the conditions of illumination rate of 80 mu mol.m ^-2·s^-1, illumination time of 16 h/d and culture temperature of 25+/-2 ℃ to obtain adventitious buds; the formula of the induction culture medium is as follows: DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ.

4) Proliferation culture: cutting the adventitious buds obtained by culture, transferring to proliferation culture medium, and culturing for 60 days under the conditions of illumination rate of 80 mu mol.m ^-2·s^-1, illumination time of 16 h/d and culture temperature of 25+/-2 ℃ to obtain adventitious bud clusters; the proliferation culture medium comprises the following formula: DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ+0.2g/L activated carbon.

5) Rooting culture: inoculating the adventitious bud cluster obtained by culture to rooting culture medium, and culturing for 30-60 days under the conditions of illumination rate of 80 mu mol.m ^-2·s^-1, illumination time of 16 h/d and culture temperature of 25+/-2 ℃ to obtain rooting seedlings; the rooting culture medium comprises the following formula: DCR medium+0.05 mg/LNAA+0.2g/L activated charcoal. Under this condition, the rooting rate was 50%.

6) Hardening seedlings: putting the rooting seedling obtained by culture into tap water to perform seedling hardening in a semi-open bottle state, wherein the temperature of a seedling hardening room is 25 ℃, the illumination time is 16 h/d, the illumination intensity is 80 mu mol.m ^-2·s^-1, and the humidity is 65%.

7) Transplanting root seedlings: after hardening seedlings for 10 days, when the root system is changed from white to off-white, transplanting the rooting seedlings, firstly rinsing the rooting seedlings in clear water to remove culture medium of the root during transplanting, then airing the surface moisture of the root system, and then transplanting the rooting seedlings into a container filled with a matrix prepared by mixing nutrient soil, vermiculite and perlite according to a ratio of 2:2:1 (v/v), and culturing for 1-2 weeks in a greenhouse under the following culture conditions: the illumination time is 16 h/d, the illumination intensity is 2000 lux, the temperature is 21-26 ℃, then shading and astigmatism culture is carried out, water is sprayed every day, and the humidity is kept at 40-60%.

The regeneration process is shown in fig. 3. Inoculating Japanese larch cotyledon (figure 3A) on an adventitious bud induction culture medium for induction culture for 15-20d, starting to expand cotyledons, starting to closely arrange cells around cell walls, starting to gradually generate small protrusions on the surfaces of left and right cotyledons during culture for 21d, wherein the small protrusions are signals of adventitious bud initiation, and gradually forming adventitious buds during culture for 30d (figures 3B-3C); when the adventitious buds are grown for about 60 days, the adventitious buds are elongated to a certain length, and adventitious bud clusters are obtained (shown in fig. 3D-3E); moving the obtained adventitious bud cluster to a rooting culture medium, promoting rooting and continuing elongation (shown in figures 3F-3G); seedlings were removed from the flasks at the time of rooting culture for 30-60d, acclimatized, transplanted to soil, and continued to grow (FIG. 3H).

According to paraffin section results (FIG. 4), it was found that adventitious buds of Japanese larch cotyledon occurred without callus stage, and that adventitious buds and the mother body remained in vascular communication, belonging to direct organogenesis pathway (FIGS. 4B to 4D). Under the stimulation of hormone, the cells on the surface and subsurface layers of cotyledons rapidly divide to form meristematic tissues, and the processes start to protrude (fig. 4C), and the cells on the two sides of the meristematic tissues also start to accelerate the division to form budding primordia (fig. 4D) under the stimulation of hormone, further form macroscopic adventitious buds (fig. 4E), transfer the adventitious buds reaching a certain height into rooting culture medium, and observe the budding primordia after 30D inoculation.

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A high-efficiency regeneration method taking Japanese larch cotyledon as an explant is characterized by comprising the following steps of: the method comprises the following steps:

1) Sowing: selecting mature and full seeds, soaking the seeds in tap water for 24 hours, sowing the seeds in a matrix, germinating the seeds for 15-20 days, and selecting strong seedlings as materials for a next experiment;

2) And (3) disinfection: cleaning the seedlings obtained in the step 1) with tap water, removing roots, soaking in 75vol% alcohol for 30-60 s, soaking in 0.1vol% mercuric chloride solution for 5-7 min, and washing with sterile water for 5 times;

4) Proliferation culture: cutting the adventitious buds obtained by culturing in the step 3), transferring the cut adventitious buds to a proliferation culture medium, and culturing for 60 days to obtain adventitious bud clusters; the proliferation culture medium comprises the following formula: DCR medium+1.5 mg/L6-BA+0.001 mg/L TDZ+0.2g/L activated carbon;

7) Transplanting: transplanting the rooting seedlings into a matrix after hardening seedlings for 10 days, culturing for 1-2 weeks in a greenhouse, and then carrying out shading and light scattering culture

Culturing;

The matrix is prepared by mixing nutrient soil, vermiculite and perlite according to a volume ratio of 2:2:1, mixing to obtain the product;

The conditions of induction culture, proliferation culture and rooting culture are as follows: the illumination speed is 80 mu mol.m ^-2·s^-1, the illumination time is 16h/d, and the room temperature is 25+/-2 ℃;

The step 6) is specifically as follows: putting the rooting seedling obtained by culturing in the step 5) into tap water to perform seedling hardening in a semi-open bottle state, wherein the temperature of a seedling hardening room is 25 ℃, the illumination time is 16h/d, the illumination intensity is 80 mu mol.m ^-2·s^-1, and the humidity is 65%;

The step 7) specifically comprises the following steps: after hardening off the seedlings for 10 days, when the root system is changed from white to off-white, transplanting the rooting seedlings, firstly rinsing the rooting seedlings in clear water to remove the culture medium of the root during transplanting, then airing the surface moisture of the root system, transplanting the rooting seedlings into a container filled with a matrix, and culturing for 1-2 weeks in a greenhouse under the following conditions: the illumination time is 16h/d, the illumination intensity is 2000lux, the temperature is 21-26 ℃, then shading and astigmatism culture is carried out, water is sprayed every day, and the humidity is kept at 40% -60%.

2. Use of the efficient regeneration method according to claim 1 in the in vitro propagation of larch in japan.

3. Use of the efficient regeneration method according to claim 1 for establishing a cultivation system of larch.

4. Use of the efficient regeneration method according to claim 1 for germplasm preservation of an excellent strain of larch in japan.