CN112470933A - Method for inducing embryogenic callus and proliferating by using immature embryo of sequoia intermedia - Google Patents
Method for inducing embryogenic callus and proliferating by using immature embryo of sequoia intermedia Download PDFInfo
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for inducing embryogenic callus and proliferating immature embryos of sequoia intermedia, belonging to the technical field of forest cell engineering. The method comprises the steps of collecting immature cones of the Chinese fir, taking seeds which develop to early cotyledon embryos, separating immature zygotic embryos, carrying out induction culture on embryogenic callus containing embryogenic embryonal stalk cell masses, transferring the obtained embryogenic callus into a maintenance and proliferation solid culture medium, and carrying out subculture to obtain a large amount of proliferated embryogenic callus. The method is not limited by seasons and external environments, can efficiently induce embryogenic callus, obtains a large amount of white and compact embryogenic callus in a maintenance and multiplication culture medium in a short period, provides an efficient way for the rapid propagation of excellent strains of the taxus chinensis, is not only beneficial to the preservation of excellent germplasm resources, provides an early foundation for subsequent regeneration plants and genetic transformation receptor systems, but also can realize the aim of propagating high-quality seedlings in large quantities, and greatly meets the market demand.
Description
Technical Field
The invention belongs to the technical field of forest cell engineering, and particularly relates to a method for inducing embryogenic callus and proliferating immature embryos of sequoia intermedia.
Background
The genus, genus (Taxodium Rich) belongs to the family of the cedaceae (Taxodiaceae), and is mainly divided into 3 species, larch (Taxodium distichum Rich), juniper (t.acendens bronn), and mexico larch (t.mcronatum Tenore). The larch belongs to tall trees, and is widely used for wetland forestation, wind prevention and soil fixation and gully filling in countries such as Europe and America due to deep root penetration, huge root system and fast base expansion. The taxus chinensis (Taxodium 'Zhongshanshashan') is also called hybrid larch, and is a general name of excellent clone obtained by interspecific hybridization of 3 trees of larch, Mexican larch and pond fir. The sequoia intermedia has the main advantages of the species of the sequoia: the tree is a semi-evergreen tall tree, the trunk is tall and straight, the tree shape is beautiful, branches and leaves are luxuriant, the crown is compact and round, the leaf fall period is short, the growth is fast, the wind resistance is high, the saline-alkali resistance is high, the flooding resistance is high, the yield is high, the ornamental value is high, and the like. At present, the accumulated afforestation demonstration area of more than ten coastal and inland provinces (cities) in Jiangsu, Zhejiang, Chongqing, Yunnan and the like exceeds 1.9 multiplied by 104ha, future timberland, energy forest, carbon forest, leisure forest and water source in ChinaThe method has great application potential in the aspects of building conservation forests and the like, building coastal protection forests, greening highways and urban and rural areas, farmland forest networks, forestation on mudflats and the like.
Although the sequoia intermedia can naturally fruit, the emergence rate is extremely low, the breeding period is long, the improved variety breeding mainly adopts the traditional cuttage breeding technology at present, but the limitation of natural environment and seasonality is large, and the long-term continuous production is very difficult. At present, although some reports about adventitious bud proliferation of the China fir exist, the China fir has low proliferation rate and difficult rooting and is not enough to meet the huge demand of the seedling market in a short time.
The forest somatic embryogenesis technology is the technology with the highest forest propagation efficiency and the most application prospect at present. The forest somatic embryogenesis technology is that a somatic cell is induced to turn to embryonic cell development under artificial conditions to obtain an embryonic cell, then the somatic cell embryo is induced to form a whole regeneration plant through artificial control conditions, and in principle, one embryonic cell (group) develops into one seedling, so that the forest somatic embryogenesis technology has infinite characteristics. In the forest somatic embryogenesis technology, induction of embryogenic callus and maintenance and proliferation of embryogenic cells are key initial steps of the somatic embryogenesis system. Therefore, it is necessary to establish a highly efficient and stable method for inducing, maintaining and proliferating embryogenic callus.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for inducing embryogenic callus and proliferating immature embryos of sequoia intermedia.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for inducing embryogenic callus and proliferating by using immature embryos of sequoia intermedia comprises the following steps:
1) collecting immature cones of the sequoia intermedia, taking seeds which develop to early cotyledon embryos, and separating immature zygotic embryos under the aseptic condition after surface sterilization;
2) under the aseptic condition, inoculating the immature zygotic embryo into an induction solid culture medium for induction culture to obtain an embryogenic callus containing an embryogenic suspensor cell mass;
3) transferring the embryogenic callus obtained in the step 2) into a maintaining and proliferating solid culture medium under an aseptic condition, and performing subculture to obtain a large amount of proliferating embryogenic callus.
Further, in the step 1), collecting immature cones of the sequoia sylvestris from the middle and last ten days of 8 months to the middle and last 9 months, taking seeds which grow to early cotyledon embryos, performing surface sterilization on the seeds by using 75% ethanol and 2.5% sodium hypochlorite (soaking the seeds in 75% ethanol for 45-60 seconds, then sterilizing the seeds by using 2.5% sodium hypochlorite solution for 18-20 minutes, and finally washing the seeds by using sterile water for 3-4 times), and separating the immature zygotic embryos under the aseptic condition.
Further, in the step 1), immature cones of the sequoia intermedia are collected in the middle and last ten days of 8 months, and seeds which develop to early cotyledon embryos are taken.
Further, in the step 2), the formula of the induction solid culture medium is that the modified DCR minimal medium is added with NAA, 6-BA, L-Glu, Pro, Inositol, Maltose and agar; in step 3), the formulation of the maintenance and propagation solid medium was 3/4 modified DCR minimal medium supplemented with NAA, 6-BA, KT, Pro, Inositol, CH, Maltose and agar.
Further, the improved DCR minimal medium is based on the DCR minimal medium, and the contents of the following components are adjusted under the condition that other components are unchanged: NH (NH)4NO3 400mg/L，KNO3 340mg/L，MgSO4·7H2O 180mg/L，KH2PO4170mg/L，CaCl2·2H2O 85mg/L，Ca(NO3)2·4H2O 556mg/L。
Further, in step 2), the formula of the induction solid medium is as follows: modified DCR minimal medium + NAA 2.0-4.0 mg/L +6-BA 0.5-1.5 mg/L + L-Glu 0.3g/L + Pro 0.2g/L + Inositol 0.2g/L + Maltose 25g/L + agar 8.5 g/L.
Further, in step 3), the formula of the solid culture medium for maintaining and proliferating is as follows: 3/4 modified DCR minimal medium + NAA 1.0-2.0 mg/L +6-BA 0.2-0.5 mg/L + KT 1.0mg/L + Pro 0.2g/L + Inositol 0.2g/L + CH 0.5g/L + Maltose 30g/L + agar 8.5 g/L.
Further, in step 2), the conditions for induction culture are as follows: and (4) culturing for 20-25 days under a dark condition and at the temperature of 23 ℃.
Further, in step 3), the conditions of subculture are as follows: and (3) controlling the temperature to be 23 ℃ under a dark condition, and carrying out subculture once every 18-20 days.
Has the advantages that: compared with the prior art, the invention has the advantages that:
aiming at the problems that the adventitious buds of the sequoia intermedia are difficult to proliferate and root, the plant regeneration rate is low, the cuttage propagation is difficult to continuously produce for a long time, the production application requirements are difficult to meet, and the like, the invention provides a method for inducing embryonic callus by using immature embryos of the sequoia intermedia, which is not limited by seasons and external environments, can efficiently induce the embryonic callus, and obtain a large amount of white and compact embryonic callus in a maintenance and proliferation culture medium in a short period, provides an efficient way for the rapid propagation of excellent strains of the sequoia intermedia, is not only favorable for the preservation of excellent germplasm resources, provides an early foundation for subsequent regenerated plants and genetic transformation receptor systems, but also can realize the goal of propagating high-quality seedlings in large quantities, and greatly meets the market demands.
Drawings
FIG. 1 is a diagram of immature zygotic embryo of Taxus chinensis;
FIG. 2 is a diagram of primary embryogenic callus induced from immature embryos;
FIG. 3 is a diagram of embryogenic callus in proliferation and maintenance culture;
FIG. 4 is a micrograph of embryogenic callus with an arrow indicating the embryogenic suspensor cell mass.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. In the following examples, unless otherwise specified, all experimental procedures were carried out according to conventional methods.
The modified DCR minimal medium formulation used in the following examples was: NH (NH)4NO3 400mg/L，KNO3 340mg/L，MgSO4·7H2O 180mg/L，KH2PO4 170mg/L，CaCl2·2H2O 85mg/L，Ca(NO3)2·4H2O 556mg/L，KI 0.83mg/L，H3BO3 6.2mg/L，MnSO4·4H2O 22.3mg/L，ZnSO4·4H2O 6.9mg/L，Na2MoO4·2H2O 0.25mg/L，CoCl2·6H2O 0.025mg/L，CuSO4·5H2O 0.25mg/L，NiCl2 0.025mg/L，FeSO4·7H2O 27.80mg/L，Na2-EDTA·2H2O37.30 mg/L, inositol 200.00mg/L, nicotinic acid 0.50mg/L, glycine 2.00mg/L, thiamine hydrochloride 1mg/L, pyridoxine hydrochloride 0.5 mg/L.
Example 1
(1) Preparation of immature zygotic embryo of Taxus chinensis
Collecting immature cones of the Sequoia strobilacea every 7 days from late 8 th to middle 9 th, and rapidly storing at 4 deg.C. Taking out the zygotic embryos, observing under a dissecting mirror, taking seeds which grow to early cotyledon embryos, soaking the seeds in 75% ethanol for 45-60 seconds, sterilizing the seeds for 18-20 minutes by using 2.5% sodium hypochlorite solution, washing the seeds for 3-4 times by using sterile water, peeling off hard seed coats outside the seeds by using a sterile scalpel and a tweezers piece under the dissecting mirror under the sterile condition, taking out immature zygotic embryos, inoculating the immature zygotic embryos into a solid embryogenic callus induction culture medium, and inoculating 8-10 seeds into each culture dish. The immature zygotic embryo of Taxus chinensis is shown in FIG. 1.
(2) Induction of embryogenic callus
Under aseptic conditions, inoculating the immature zygotic embryos into an induction solid culture medium, culturing for 20-25 days, controlling the temperature in the dark at 23 ℃, and inducing to obtain embryogenic callus, as shown in figure 2. Wherein the induction solid culture medium is: modified DCR minimal medium + NAA2.0mg/L +6-BA 0.5mg/L + L-Glu 0.3g/L, Pro 0.2.2 g/L + Inositol 0.2g/L + Maltose 25g/L + agar 8.5g/L, pH 5.6.
Wherein NAA is naphthylacetic acid, 6-BA is 6-benzylaminopurine, L-Glu is L-glutamine, Pro is proline, Inositol is Inositol, and Maltose is Maltose.
The L-glutamine can effectively inhibit the browning of the callus and the culture medium and obviously improve the growth of the callus.
Proline can be used as amino acid to supplement nutrients, and can improve the resistance of the tissue and the survival rate of the callus.
Inositol is mainly involved in physiological activities such as carbohydrate and phospholipid metabolism and ion balance, and can promote the growth of callus and the formation of embryoid under proper conditions.
Maltose provides a carbon source for the induction solid medium.
The states of embryogenic callus induced by immature embryos at different collection times from late 8 th month to middle 9 th month are shown in table 1, and seeds collected in late 8 th month are suitable for inducing embryogenic callus, the induction rate is more than 87%, and the callus state is good. In 9 months, although callus can be induced due to mature embryo and high differentiation state, the callus is poor in state and is not suitable for being used as an initial explant.
TABLE 1 statistics of immature embryo induced embryogenic callus status at different harvest times
(3) Embryonic callus maintenance and proliferation stage
And (3) under an aseptic condition, transferring the embryogenic callus obtained by induction in the step (2) into a maintenance and proliferation solid culture medium, controlling the temperature at 23 ℃ under a dark condition, and subculturing once every 18-20 days to obtain a large amount of proliferation embryogenic callus. Wherein the maintenance and proliferation culture medium is: 3/4 modified DCR minimal medium + NAA 1.5mg/L +6-BA 0.4mg/L + KT 1.0mg/L + Pro 0.2g/L + Inositol 0.2g/L + CH 0.5g/L + Maltose 30g/L + agar 8.5 g/L.
Wherein NAA is naphthylacetic acid, 6-BA is 6-benzylaminopurine, KT is 6-furfurylaminopurine, Pro is proline, Inositol is Inositol, CH is hydrolyzed casein, and Maltose is Maltose.
The 6-furfuryl amino purine has the functions of promoting cell division and tissue differentiation, and may be used in tissue culture, promoting cell division and inducing callus differentiation.
The hydrolyzed casein can be used as amino acid to supplement nutrients on one hand, and has good promotion effect on the formation of embryoid on the other hand.
Example 2
Essentially the same as in example 1, except that the solid medium induced in step (2) was: modified DCR minimal medium + NAA 3.0mg/L +6-BA 1.0mg/L + L-Glu 0.3g/L, Pro 0.2.2 g/L + Inositol 0.2g/L + Maltose 25g/L + agar 8.5g/L, pH 5.6.
The maintenance and proliferation culture medium in the step (3) is as follows: 3/4 modified DCR minimal medium + NAA2.0mg/L +6-BA 0.2mg/L + KT 1.0mg/L + Pro 0.2g/L + Inositol 0.2g/L + CH 0.5g/L + Maltose 30g/L + agar 8.5g/L, pH 5.8. The other steps and control conditions are the same as those in the embodiment 1, and the experimental results show that the induction effect of the embodiment 1 can also be achieved.
Example 3
Essentially the same as in example 1, except that the solid medium induced in step (2) was: modified DCR minimal medium + NAA 4.0mg/L +6-BA 1.5mg/L + L-Glu 0.3g/L, Pro 0.2.2 g/L + Inositol 0.2g/L + Maltose 25g/L + agar 8.5g/L, pH 5.6.
The maintenance and proliferation culture medium in the step (3) is as follows: 3/4 modified DCR minimal medium + NAA 1.0mg/L +6-BA 0.5mg/L + KT 1.0mg/L + Pro 0.2g/L + Inositol 0.2g/L + CH 0.5g/L + Maltose 30g/L + agar 8.5g/L, pH 5.8. The other steps and control conditions are the same as those in the embodiment 1, and the experimental results show that the induction effect of the embodiment 1 can also be achieved.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to yield one or more new ranges of values, which ranges of values are to be considered as specifically disclosed herein.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. A method for inducing embryogenic callus and proliferating by utilizing immature embryos of sequoia intermedia is characterized by comprising the following steps of:
1) collecting immature cones of the sequoia intermedia, taking seeds which develop to early cotyledon embryos, and separating immature zygotic embryos under the aseptic condition after surface sterilization;
2) under the aseptic condition, inoculating the immature zygotic embryo into an induction solid culture medium for induction culture to obtain an embryogenic callus containing an embryogenic suspensor cell mass;
3) transferring the embryogenic callus obtained in the step 2) into a maintaining and proliferating solid culture medium under an aseptic condition, and performing subculture to obtain a large amount of proliferating embryogenic callus.
2. The method for inducing embryogenic callus and proliferating immature embryo of sequoia intermedia according to claim 1, wherein in step 1), immature cones of sequoia intermedia are collected from the middle and late 8 months to the middle 9 months, seeds of cotyledon embryos that have developed to the early stage are harvested, surface sterilized with 75% ethanol and 2.5% sodium hypochlorite, and then the immature zygotic embryos are isolated under aseptic conditions.
3. The method for inducing embryogenic callus and proliferating by using immature embryo of Taxus cuspidata according to claim 1, wherein in step 1), immature cones of Taxus cuspidata are collected in late 8 months, and seeds of embryos that have developed to early cotyledon are collected.
4. The method for inducing embryogenic callus and propagation by using immature embryo of Taxus cuspidata according to claim 1, wherein in step 2), the formulation of the inducing solid medium is modified DCR minimal medium supplemented with NAA, 6-BA, L-Glu, Pro, Inositol, Maltose and agar; in step 3), the formulation of the maintenance and propagation solid medium is 3/4 modified DCR minimal medium supplemented with NAA, 6-BA, KT, Pro, Inositol, CH, Maltose and agar.
5. The method of claim 4, wherein the modified DCR minimal medium is DCR minimal medium, and the content of the following components is adjusted under the condition that other components are not changed: NH (NH)4NO3 400mg/L，KNO3 340mg/L，MgSO4·7H2O 180mg/L，KH2PO4170mg/L，CaCl2·2H2O 85mg/L，Ca(NO3)2·4H2O 556mg/L。
6. The method for inducing embryogenic callus and proliferating by using immature embryos of Taxus cuspidata according to claim 4, wherein the formula of the solid induction medium in the step 2) is as follows: modified DCR minimal medium + NAA 2.0-4.0 mg/L +6-BA 0.5-1.5 mg/L + L-Glu 0.3g/L + Pro 0.2g/L + Inosito 10.2g/L + Maltose 25g/L + agar 8.5 g/L.
7. The method for inducing embryogenic callus and proliferating using immature embryo of Taxus cuspidata according to claim 4, wherein the formula of the solid medium for maintaining and proliferating in step 3) is: 3/4 modified DCR minimal medium + NAA 1.0-2.0 mg/L +6-BA 0.2-0.5 mg/L + KT 1.0mg/L + Pro 0.2g/L + Inositol 0.2g/L + CH 0.5g/L + Maltose 30g/L + agar 8.5 g/L.
8. The method for inducing embryogenic callus and proliferating by using immature embryo of Taxus cuspidata according to claim 1, wherein the conditions for induction culture in step 2) are as follows: and (4) culturing for 20-25 days under a dark condition and at the temperature of 23 ℃.
9. The method for inducing embryogenic callus and proliferating by using immature embryo of Taxus cuspidata according to claim 1, wherein the condition of subculture in step 3) is: and (3) controlling the temperature to be 23 ℃ under a dark condition, and carrying out subculture once every 18-20 days.
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