CN110896859B - Method for promoting fir somatic embryogenesis by using salicylic acid - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
- A01H4/006—Encapsulated embryos for plant reproduction, e.g. artificial seeds
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- Developmental Biology & Embryology (AREA)
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Abstract
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.
Description
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.
Example 1
Preparing a mother solution: salicylic acid is easy to dissolve in water, 1.0g of salicylic acid powder is weighed, dissolved in a beaker by using ultrapure water, the volume is fixed to 1L, salicylic acid mother liquor of 1mg/mL is prepared, and finally the salicylic acid mother liquor is transferred to a refrigerator of 4 ℃ and stored by using a brown bottle for later use.
Embryogenic embryonal suspensor mass induction and proliferation medium: the induction of the embryonic suspensor mass of the cedar is the process of inducing the cedar seeds to form callus, and the formula of the culture medium is that 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/L Ac and 2.3g/L glehnia littoralis are added into a DCR culture medium.
Somatic embryo induction medium: the induction of fir somatic embryo is the process of callus induction of fir to form somatic embryo. The formula of the culture medium is that 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 and 0.2g/L proline are added into a DCR basic culture medium. 100-200 g/L PEG, 1-5 mg/L GA, 2-8 mg/L ABA.
Embryonic suspensor mass induction and proliferation: sterilizing Chinese fir seeds in a clean bench, removing seed coats under a microscope, cutting endosperm to take out embryo, putting the embryo in a callus induction culture medium, and enabling the embryo to contact with the culture medium as much as possible, thereby facilitating the formation of callus. The callus subculture period of the fir is 25-30 days, and the culture temperature is 23 ℃.
And (3) somatic embryo induction: adding the embryogenic suspensor masses A and B into salicylic acid somatic embryo culture media with different concentrations (0mg/L, 2mg/L, 4 mg/L, 6mg/L and 8mg/L), inoculating 6 callus blocks into each dish by taking a somatic embryo material induced by the medium without adding exogenous salicylic acid (0mg/L) as a control, repeating the culture in at least 3 dishes, and culturing in dark at the culture temperature of 23 ℃.
Data statistics and analysis are carried out by adopting One Way ANOVA and a multiple comparison test method, analysis software is SPSS 16.0, the significance level is set to be alpha-0.05, and analysis results of the SPSS software are plotted by adopting GraphPad.
(1) Somatic embryogenesis of embryogenic suspensor masses A
As shown in FIG. 1, at 60d, cotyledon embryos are generated at each concentration (0, 2, 4, 6, 8mg/L), and the average number of mature cotyledon embryos per dish of SA at 0-8 mg/L is 6, 8, 10.3, 13.3, 10.3 in sequence; the number of somatic embryos is the largest when the concentration is 6mg/L, which is about 2.21 times of that of a control group, and the difference with the control group is obvious; the somatic embryo content of each dish of the rest concentrations is higher than that of the control group, but no significant difference exists; at 100d, the number of mature cotyledon embryos of each concentration is gradually increased and reaches the maximum value, and the average number of mature cotyledon embryos in each dish of the control group is 18; the number of 6mg/L somatic embryos is about 36.3, which is about 2.01 times of that of a control group, and the difference with the control group is obvious; the number of cotyledon embryos under the rest concentration is not obviously different from that of the control group, which shows that the SA of 6mg/L is beneficial to embryogenesis of embryonal suspensor mass A. As can be seen in FIG. 2, at 30d, 6mg/L salicylic acid treatment had produced cotyledonary embryos (FIG. 2d), while the control group was still in the columnar embryo stage (FIG. 2d), indicating that salicylic acid promoted somatic embryo growth; at 45 days, cotyledon embryos are generated under the treatment of 0-8 mg/L salicylic acid. In conclusion, 6mg/L salicylic acid promotes embryogenesis of embryonal suspensor mass A.
(2) Somatic embryogenesis of embryogenic suspensor masses B
As can be seen from FIG. 3, at 60d, mature cotyledon embryos are generated at each concentration of 0-8 mg/LSA, and the number of the mature cotyledon embryos gradually increases with the increase of the concentration; when the SA concentration is 8mg/L, the number of mature cotyledon embryos is the largest, and the number of mature cotyledon embryos is obviously different from that of a control group; at 100 days, the cotyledon embryos are mature, the number of the mature cotyledon embryos is increased, when the SA concentration is 8mg/L, the number of the cotyledon embryos is the largest, the average number of the mature cotyledon embryos in each vessel is 60.3, the average number of the cotyledon embryos in each vessel in the control group is 30.3, which is 1.99 times that of the control group, and the result shows that the 8mg/L salicylic acid has the best effect of promoting the somatic embryogenesis of the embryogenic embryonal suspensor group B in the SA concentration of 0-8 mg/L. As shown in FIG. 4, at 0d, the embryonal suspensor mass is transparent in color (FIG. 4 a); at 10d, the embryo heads became dense (FIG. 4 b); at 20d, the embryo heads turned green, the embryonic axes elongated and turned red (FIG. 4c) toward columnar embryos; at 30d, a columnar embryo is produced, the suspensor continues to elongate and become thin (fig. 4d), and the content of the columnar embryo is higher compared with that of the control group (fig. 4 d); at 45d, different concentrations of salicylic acid treated hypocotyl B produced cotyledonary embryos (FIGS. 4 e-4 i).
Claims (3)
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: taking DCR as a basic culture medium, adding: 6mg/L salicylic acid, 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 and 2-8 mg/LABA.
2. The method for promoting fir somatic embryogenesis according to claim 1, wherein the cultivation temperature in dark cultivation is 23 ℃ and the cultivation time is 60-100 days.
3. The method for promoting fir somatic embryogenesis by using salicylic acid as claimed in claim 1, 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.
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