阅读说明：本技术 一种高效的云锦杜鹃直接体细胞胚胎发生方法 (Efficient brocade rhododendron direct somatic embryogenesis method ) 是由 魏翔莺 张春英 郑秀霞 穆景利 于 2019-12-02 设计创作，主要内容包括：本发明公开了一种高效的云锦杜鹃直接体细胞胚胎发生方法,属于植物快速繁殖再生技术领域。本发明首次建立云锦杜鹃的高效体细胞胚胎发生系统,以云锦杜鹃实生苗叶片为外植体的体细胞直接胚胎发生再生植株,是一种高效的获得大量云锦杜鹃组培苗的无性繁殖方式。本发明完善了常绿杜鹃花亚属植物组培快繁体系,解决了传统的播种耗时长且容易产生变异和扦插繁殖系数很低的难题。该方法能够快速繁殖大量性状稳定的植株,快速建立优良单株的无性系,在云锦杜鹃种苗生产、种质创新和新品种选育方面均有重要的应用价值。(The invention discloses a high-efficiency rhododendron brocade direct somatic embryogenesis method, and belongs to the technical field of rapid plant propagation and regeneration. The invention establishes a high-efficiency somatic embryogenesis system of rhododendron micranthum for the first time, takes the leaves of the seedling of the rhododendron micranthum as explants for somatic cell direct embryogenesis to regenerate plants, and is an asexual propagation mode for efficiently obtaining a large amount of tissue culture seedlings of the rhododendron micranthum. The invention perfects the tissue culture and rapid propagation system of the rhododendron evergreen subgenus plant, and solves the problems of long time consumption, easy variation generation and low cuttage propagation coefficient in the traditional sowing. The method can rapidly propagate a large number of plants with stable characters, rapidly establish the clone of a good single plant, and have important application values in the aspects of brocade rhododendron seedling production, germplasm innovation and new variety breeding.)

1. A high-efficiency brocade rhododendron direct somatic embryogenesis method is characterized by comprising the following steps:

(1) preparing sterile seeding seedlings of rhododendron micranthum: the seeds of the rhododendron yunnanensis are sown in a culture medium after the surfaces of the seeds are disinfected, the temperature is 25 ℃, and the illumination intensity is 80mol.m^-2.s^-1Culturing in the tissue culture room for 2-3 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;

(2) induction of leaf somatic embryos: taking 2-4 young leaves at the top end of the rhododendron brocade seedling prepared in the step (1) in a super-clean workbench, cutting two ends of each leaf by using a blade, enabling the front side to face upwards, horizontally placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing the flat plate by using a sealing film, and culturing the flat plate in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in dark, wherein the light intensity is 80 lmol m^-2s^-1Culturing for 80-100 days; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: photographing and recording spherical embryos, heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos, and counting the somatic embryo incidence rate, the transformation rate and the average seedling number of each explant;

(3) transplanting and light water management: stripping the plantlets in the step (2) from the explants, transplanting the plantlets into a plug tray, spraying, culturing for 20 days, stopping spraying, and keeping good ventilation and shading treatment, wherein the humidity is 80% -90%; the water and fertilizer management is to pour water 2-3 times a week and pour 20-20-20 water-soluble fertilizer with the concentration of 200ppm once a week.

2. The efficient rhododendron yunnanensis direct somatic embryogenesis method according to claim 1, characterized in that: the seeding culture medium in the step (1) comprises the following components: 1/2 ER culture medium, 15g/L sucrose and 8g/L agar, and has pH of 5.0-5.2.

3. The efficient rhododendron yunnanensis direct somatic embryogenesis method according to claim 1, characterized in that: in the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: WPM +30g/L sucrose +8g/L agar, adjusting pH to 5.0-5.2, pressurizing at 121 deg.C for 30min, cooling to 50-60 deg.C, adding filter sterilized plant growth regulator: TDZ0.5mg/L and NAA0.2 mg/L, the culture medium was dispensed into petri dishes for solidification, 25ml per petri dish.

4. The efficient rhododendron yunnanensis direct somatic embryogenesis method according to claim 1, characterized in that the cultivation substrate is filled in the plug tray in the step (3), and the small plants are transplanted after being thoroughly poured with nutrient solution before being transplanted; the preparation method of the culture medium comprises the following steps: the sand is washed several times with the running water earlier, then the running water soaks three days, and the middle water that trades 3~5 times, and it is reserve to air-dry at last, presses 2 with grass carbon and the sand that is prepared for: 1, mixing and subpackaging in a standard plug tray with 96 holes; the formula of the nutrient solution is as follows: CaCl₂·2H₂O 0.05g/L, NaCl 0.025g/L, MgSO₄·7H₂O 0.15g/L,（NH₄）₂HPO₄0.25g/L, KH₂PO₄0.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B, 15g/L of glucose and 8g/L of agar, and adjusting the pH value to 5.0-5.2.

Technical Field

The invention belongs to the technical field of rapid propagation and regeneration of plants, and particularly relates to a high-efficiency brocade rhododendron direct somatic embryogenesis method.

Background

The rhododendron micranthum serves as an important parent of the plant of the Ericaceae, has high ornamental value, is extremely cold-resistant and has fragrance. The traditional propagation mode is seed sowing or stem cutting, but the breeding period is long and the efficiency is low, so the regeneration of plants through isolated culture is a method for efficiently and quickly propagating a large number of plants. The rhododendron micranthum is a woody plant, and the plant body contains more polyphenols which can block cell division, so that the establishment of a regeneration system is difficult, and a scheme for regeneration through somatic embryogenesis is not established.

The embryonic cells have strong capacity of receiving exogenous DNA, are ideal genetic transformation competent cells, most somatic embryos developed from the embryonic cells with the characteristics of egg cells are of single cell origin, and transgenic plants obtained by transformation have few chimeras, and are the most ideal genetic transformation receptor system. Therefore, the establishment of the somatic embryogenesis system has important application value in the aspects of brocade rhododendron seedling production, germplasm innovation and new variety breeding.

Disclosure of Invention

The invention aims to provide a high-efficiency rhododendron brocade direct somatic embryogenesis method aiming at the problems that the conventional seed propagation and cutting propagation time of rhododendron brocade is slow and variation is easy to generate.

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

a high-efficiency brocade rhododendron direct somatic embryogenesis method comprises the following steps:

(1) preparing sterile seeding seedlings of rhododendron micranthum: the seeds of the rhododendron yunnanensis are sown in a culture medium after the surfaces of the seeds are disinfected, the temperature is 25 ℃, and the illumination intensity is 80mol.m^-2.s^-1Culturing in the tissue culture room for 2-3 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;

(2) induction of leaf somatic embryos: placing 2-4 young leaves at the top end of the rhododendron brocade seedling prepared in the step (1) on wet sterile filter paper in a super-clean workbench, cutting two ends of each leaf with a blade, enabling the front side to face upwards, flatly placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing with a sealing film, and culturing in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in dark, wherein the light intensity is 80 lmol m^{- 2}s^-1Culturing ofThe time is 80-100 d; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: shooting and recording spherical embryos, heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos, and counting the somatic embryo incidence rate, the transformation rate and the average seedling number of each explant;

(3) transplanting and light water management: peeling off the plantlets obtained in the step (2) from the explants, transplanting the plantlets into a plug tray, performing spray culture, stopping spraying after 20 days with the humidity of 80-90%, and keeping good ventilation and shading treatment; the water and fertilizer management is to pour water 2-3 times a week and pour 20-20-20 water-soluble fertilizer (with the concentration of 200 ppm) once a week.

The culture medium in the step (1) comprises the following components: 1/2 ER culture medium, 15g/L sucrose and 8g/L agar, and has pH of 5.0-5.2.

In the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: woody plant culture medium WPM +30g/L sucrose +8g/L agar, pH value is adjusted to 5.0-5.2, high pressure is carried out for 30min at 121 ℃, and after cooling to 50-60 ℃, plant growth regulator which is filtered and sterilized is added: TDZ (thidiazuron) 0.5mg/L and NAA (naphthylacetic acid) 0.2 mg/L, the medium was dispensed into petri dishes and solidified for use (25 ml per petri dish).

The formula of the ER culture medium is as follows: NH (NH)₄NO₃400 mg/L； (NH₄)₂SO₄132 mg/L，H₃BO₃6.2 mg/L，CaCl₂332.2 mg/L，CoCl₂. 6H₂O 0.025mg/L，CuSO₄. 5H₂O 0.025mg/L，DTPA 39.34 mg/L，FeSO₄.7H₂O 27.8 mg/L，MgSO₄180.8 mg/L，MnSO₄. H₂O 16.9 mg/L，Na₂MoO₄.2H₂O0.25 mg/L，C₆H₁₂O₆100 mg/L，KNO₃202 mg/L，KH₂PO₄408 mg/L， C₁₂H₁₇ClN₄OS . HCl 0.4mg/L，ZnSO₄. 7H₂O8.6 mg/L, agar 8g/L, sucrose 30g/L, and pH 5.0-5.2.

The woody plant culture medium WPM comprises the following components in percentage by weight: NH (NH)₄NO₃400 mg/L，H₃BO₃6.2 mg/L，CaCl₂72.47 mg/L，Ca(NO₃)₂. 4H₂O 556 mg/L，CuSO₄0.16 mg/L，C₁₀H₁₄N₂Na₂O₈.2H₂O 37.3 mg/L，FeSO₄.7H₂O 27.85 mg/L，MgSO₄180.7 mg/L，MnSO₄. H2O 22.3 mg/L，Na₂MoO₄. 2H₂O0.25 mg/L，KH₂PO₄170 mg/L，K₂SO₄990 mg/L，ZnSO₄.7H₂8.6mg/L of agar 8g/L, 30g/L of cane sugar and 5.0-5.2 of PH.

Filling a culture medium in the hole tray in the step (3), and transplanting after completely pouring the culture medium with a nutrient solution; the preparation method of the culture medium comprises the following steps: the sand is washed several times with the running water earlier, then the running water soaks three days, and the middle water that trades 3~5 times, and it is reserve to air-dry at last, presses 2 with grass carbon and the sand that is prepared for: 1, mixing and subpackaging in a standard plug tray with 96 holes; the formula of the nutrient solution is as follows: CaCl₂·2H₂O 0.05g/L, NaCl 0.025g/L, MgSO₄·7H₂O 0.15g/L,（NH₄）₂HPO₄0.25g/L, KH₂PO₄0.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B, 15g/L of glucose and 20g/L of agar, and adjusting the pH value to 5.0-5.2.

The invention has the advantages that:

1. establishes a high-efficiency brocade rhododendron somatic embryogenesis system, and has high propagation coefficient and stable character. Solves the problems of separation of seed propagation characters and difficult cuttage rooting.

2. The invention perfects the rhododendron tissue culture rapid propagation system, the survival rate of the tissue culture seedlings after transplantation reaches more than 90%, and the seedlings do not need to undergo the rooting stage in the tissue culture bottle, thereby greatly shortening the tissue culture period, saving time and space, greatly reducing cost and having important significance for improving the tissue culture industrialization level.

Drawings

FIG. 1 shows four stages of somatic embryogenesis of Rhododendron micranthum. A: a spherical tire stage; b: a heart-shaped embryo stage; c: torpedo shaped embryo stage; d: cotyledonary embryo stage.

FIG. 2 somatic embryos are transplanted to the greenhouse after seedling establishment. A: single leaf edge production of somatic embryos; b: somatic embryos generated by a single leaf form seedlings to generate plantlets; c.: single plantlet stripped off after somatic embryo seedling formation; d: transplanting the plantlets after the somatic embryos become seedlings to the plug tray.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.

Culture medium:

the formula of the ER culture medium is as follows: NH (NH)₄NO₃400 mg/L； (NH₄)₂SO₄132 mg/L，H₃BO₃6.2 mg/L，CaCl₂332.2 mg/L，CoCl₂. 6H₂O 0.025mg/L，CuSO₄. 5H₂O 0.025mg/L，DTPA 39.34 mg/L，FeSO₄.7H₂O 27.8 mg/L，MgSO₄180.8 mg/L，MnSO₄. H₂O 16.9 mg/L，Na₂MoO₄. 2H₂O 0.25mg/L，C₆H₁₂O₆100 mg/L，KNO₃202 mg/L，KH₂PO₄408 mg/L， C₁₂H₁₇ClN₄OS . HCl 0.4 mg/L，ZnSO₄. 7H₂O8.6 mg/L, agar 8g/L, sucrose 30g/L, and pH 5.0-5.2.

The WPM culture medium comprises the following components: NH (NH)₄NO₃400 mg/L，H₃BO₃6.2 mg/L，CaCl₂72.47 mg/L，Ca(NO₃)₂. 4H₂O 556 mg/L，CuSO₄0.16 mg/L，C₁₀H₁₄N₂Na₂O₈.2H₂O 37.3 mg/L， FeSO₄.7H₂O27.85 mg/L，MgSO₄180.7 mg/L，MnSO₄. H2O 22.3 mg/L，Na₂MoO₄. 2H₂O 0.25 mg/L，KH₂PO₄170 mg/L，K₂SO₄990 mg/L，ZnSO₄.7H₂8.6mg/L of agar 8g/L, 30g/L of cane sugar and 5.0-5.2 of PH.