阅读说明：本技术 一种利用水杨酸促进杉木体胚发生的方法 (Method for promoting fir somatic embryogenesis by using salicylic acid ) 是由 陈金慧 陆叶 郭玉琳 施季森 黄世清 成铁龙 胡向阳 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种利用水杨酸促进杉木体胚发生的方法,属于植物组培技术领域。本发明所提供的方法具体为：以DCR为基本培养基,附加有浓度不超过8mg/L的水杨酸作为杉木体胚诱导培养基；通过杉木未成熟球果获得未成熟种胚,将其放在在愈伤诱导培养基上培养25～30天获得杉木胚性胚柄团；将所得杉木胚性胚柄团放入杉木体胚诱导培养基中,于培养箱中进行暗培养,诱导促进杉木体胚发生,23℃条件下,培养60～100天,即可得到比对照数目最高可达2倍以上的体胚,大大提高了杉木体胚的诱导效率,具有很好的实用性。(The invention discloses a method for promoting fir somatic embryogenesis by using salicylic acid, and belongs to the technical field of plant tissue culture. The method provided by the invention specifically comprises the following steps: DCR is used as a basic culture medium, and salicylic acid with the concentration not more than 8mg/L is added to be used as a fir somatic embryo induction culture medium; obtaining an immature embryo through immature cones of the China fir, and culturing the immature embryo on a callus induction culture medium for 25-30 days to obtain an embryonic suspensor mass of the China fir; the obtained fir embryonic suspensor masses are placed in a fir somatic embryo induction culture medium, dark culture is carried out in an incubator, induction promotes fir somatic embryo generation, and the somatic embryos with the highest contrast number of more than 2 times can be obtained after culture for 60-100 days at the temperature of 23 ℃, so that the induction efficiency of the fir somatic embryos is greatly improved, and the method has good practicability.)

1. A method for promoting fir somatic embryogenesis by using salicylic acid is characterized in that a fir embryonic suspensor mass is added into a somatic embryo induction culture medium and is subjected to dark culture in an incubator to induce and promote fir somatic embryogenesis; wherein, the somatic embryo induction culture medium: DCR is used as a basic culture medium, and salicylic acid with the concentration not more than 8mg/L is added.

2. The method of claim 1, wherein the somatic embryo induction medium further comprises: 1mg/L VC, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2-4 g/L Ac, 2.8g/L agar, 0.2g/L asparagine, 0.2g/L proline, 100-200 g/L PEG, 1-5 mg/L GA, 2-8 mg/L LABA.

3. The method for promoting fir somatic embryogenesis according to claim 2, wherein the concentration of salicylic acid is 2-8 mg/L.

4. The method for promoting fir somatic embryogenesis by using salicylic acid according to any one of claims 1-3, wherein the cultivation temperature in the dark cultivation is 23 ℃ and the cultivation time is 60-100 days.

5. The method for promoting fir somatic embryogenesis by using salicylic acid as claimed in any one of claims 1-3, wherein the concentration of salicylic acid is 6-8 mg/L.

6. The method for promoting fir somatic embryogenesis by using salicylic acid according to any one of claims 1-3, wherein the method for obtaining fir embryonal suspensor mass is as follows: taking immature cones of the fir to obtain fir seeds, sterilizing the fir seeds in an ultra-clean workbench, removing seed coats under a microscope, cutting off endosperm, taking out seed embryos, putting the seed embryos into a callus induction culture medium, and culturing at a dark temperature of 23 ℃ for 25-30 days to obtain embryonic suspensor masses of the fir; wherein the callus induction culture medium is DCR culture medium added with 0.5mg/L KT, 0.5mg/L BA, 1mg/L VC, 1-2 mg/L2, 4-D, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2.5g/LAc and 2.3g/L agar.

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for promoting fir somatic embryogenesis by using salicylic acid.

Background

China fir (Cunninghina lancelata) belongs to China fir genus, is one of the important wood species in China, has fast growth, straight and round dry shape, light, soft and delicate material, easy processing, beautiful texture, special fragrance, no warping and cracking, moth resistance and corrosion resistance, and is an excellent raw material for industries such as buildings, furniture, bridges, shipbuilding, paper making, wood fiber and the like. At present, fir seedlings are propagated mainly through three approaches: seed, cuttage and traditional tissue culture [ warm submachine, fir embryogenic tissue induction and subculture research based on immature zygotic embryos [ D ]. fujian agriculture and forestry university, 2011 ], but the yield of improved seeds, the sowing quality of seeds are unstable, the supply of advanced improved seeds above the 2 nd generation is insufficient, the sowing quality of seeds is poor, particularly, the seeds are too astringent and have more grains, which usually account for 40-60% of the weight [ zheng renhua. 63-65. ]; the cutting seedling raising is limited by the number of the scion of the mother tree, the field space, the rooting rate and the like, and the scale of the cutting seedling raising is difficult to meet the large demand of the modern forestry production on the fir seedlings; in addition, the induction of the traditional tissue culture roots is difficult, the quantity of the induced roots is small and weak, and the quality of the seedlings and the success rate of afforestation are influenced to a certain extent. Therefore, a high-efficiency embryo generating system of the immature embryo of the fir is established, which is beneficial to accelerating the industrialized development of the fast-growing excellent seedlings of the fir.

Somatic embryogenesis belongs to the asexual propagation technology, and the produced seedlings have small genetic background difference and are regular. Compared with cuttage and traditional tissue culture, the method has the advantages of larger scale and lower cost. However, the somatic embryogenesis of Taxus chinensis, a gymnosperm, has a completely different pathway and mechanism from the somatic embryogenesis of angiosperm, and it is difficult to convert mature seeds and plants developed from the seeds, vegetative cells into embryonic cells. However, in conifer species, some of the nucellar cells are in an undifferentiated stage during the development of seeds after fertilization of the female gametophyte, and under appropriate culture conditions, the blastogenesis polyblast property can be utilized to induce somatic embryo formation. Gupta et al (1985, 1995) reported that using this biological property, somatic embryos of Douglas fir (Pseudotsuga menziesii) and slash pine (Pinussiliotii) were induced, which can be roughly divided into four steps: the induction, the maintenance and the proliferation of an Embryonic Suspensor Mass (ESM), the maturation, the germination and the plant regeneration of a somatic embryo prove that the technical system has the prospect of achieving industrialization.

However, although there are reports of using immature embryos of fir to induce somatic embryogenesis, only individual somatic embryos can be formed on explants, and the problems of low induction rate and small quantity exist, so that the method is difficult to apply in production. Therefore, a high-efficiency embryo generating system of the immature embryo of the fir is established, which is beneficial to accelerating the industrialized development of the fast-growing excellent seedlings of the fir. Studies have shown that salicylic acid has different effects on somatic embryogenesis of different plants, and the mechanism of the salicylic acid is yet to be further explored, but no report of the salicylic acid applied to somatic embryogenesis of China fir is found at present.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for promoting the somatic embryogenesis of a fir by using salicylic acid, which can improve the induction efficiency of the somatic embryos of the fir.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a method for promoting fir somatic embryogenesis by using salicylic acid comprises: adding the embryonic suspensor mass of the fir into a somatic embryo induction culture medium, and performing dark culture in an incubator to induce and promote the somatic embryogenesis of the fir; wherein, the somatic embryo induction culture medium: DCR is used as a basic culture medium, and salicylic acid with the concentration not more than 8mg/L is added.

The somatic embryo induction medium further comprises: 1mg/L VC, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2-4 g/L Ac, 2.8g/L agar, 0.2g/L asparagine, 0.2g/L proline, 100-200 g/L PEG, 1-5 mg/L GA, 2-8 mg/L LABA.

The concentration of the salicylic acid is 2-8 mg/L.

The concentration of the salicylic acid is 6 mg/L.

The culture temperature of the dark culture is 23 ℃, and the culture time is 60-100 days.

The concentration of the salicylic acid is 6-8 mg/L.

The method for obtaining the embryonic suspensor mass of the fir comprises the following steps: taking immature cones of the fir to obtain fir seeds, sterilizing the fir seeds in an ultra-clean workbench, removing seed coats under a microscope, cutting off endosperm, taking out seed embryos, putting the seed embryos into a callus induction culture medium, and culturing at a dark temperature of 23 ℃ for 25-30 days to obtain embryonic suspensor masses of the fir; wherein the callus induction culture medium is DCR culture medium added with 0.5mg/L KT, 0.5mg/L BA, 1mg/L VC, 1-2 mg/L2, 4-D, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2.5g/LAc and 2.3g/L agar.

Has the advantages that: compared with the prior art, the invention has the advantages that: according to the method for promoting the fir somatic embryo generation by using the salicylic acid, the DCR is used as a basic culture medium, the salicylic acid with the concentration not more than 8mg/L is added, the fir embryonal suspensor mass is inoculated on the somatic embryo induction culture medium, dark culture is carried out in an incubator, the culture temperature is 23 ℃, induction promotion is carried out on the fir somatic embryo generation, and the somatic embryo with the contrast number being more than 2 times can be obtained after 60-100 days of culture, so that the induction efficiency of the fir somatic embryo is greatly improved, a high-efficiency fir immature embryo generation system is established, the method is favorable for accelerating the industrialized development of the fast-growing excellent seedlings of the firs, and has good practicability.

Drawings

FIG. 1 is a statistical chart of the number of mature cotyledon embryos of fir A treated with salicylic acid at different concentrations;

FIG. 2 is a diagram showing the development state of embryos of A-type fir treated by salicylic acid at different concentrations and different culture times under a stereoscope; a to d are embryonic suspensor development processes of adding 6mg/L salicylic acid for 0, 10, 20 and 30 days, e to i are embryonic suspensor development conditions of adding 0, 2, 4, 6 and 8mg/L salicylic acid when d is 45 days respectively, scales from a to d are 2mm, and scales from e to f are 5 mm;

FIG. 3 is a statistical chart of the number of mature cotyledon embryos B generated under salicylic acid treatment at different concentrations;

FIG. 4 is a diagram showing the development state of somatic embryo B of fir treated with salicylic acid at different concentrations and different culture times under a stereoscope; FIGS. a, b, c and d represent the process of somatic embryogenesis with 8mg/L salicylic acid added at 0d, 10d, 20d and 30d, respectively; e. f, g, h and i respectively represent the occurrence of fir somatic embryo B treated by 0, 2, 4, 6 and 8mg/L salicylic acid at 45d, the scales from a to d are 2mm, and the scales from e to f are 5 mm.

Detailed Description

The invention is further described with reference to specific examples.

The starting material used in the following examples was immature cones randomly harvested from the fujian Yangtze forest farm and marked, and embryogenic embryonal suspensor masses (marked A, B, randomly from two different fir trees) induced by immature zygotic embryos were used as starting material.