CN117158320B - Construction method of eustoma grandiflorum multi-variety somatic embryo efficient regeneration system - Google Patents
Construction method of eustoma grandiflorum multi-variety somatic embryo efficient regeneration system Download PDFInfo
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Abstract
The invention discloses a construction method of an efficient regeneration system of eustoma grandiflorum multi-variety somatic embryos, which comprises the steps of seed disinfection and germination, embryogenic cell induction and proliferation, somatic embryo differentiation into buds, rooting and seedling formation of the differentiated buds, seedling hardening, field planting and the like. The method improves embryogenic callus induction efficiency and differentiation proliferation efficiency, can be used for secondary preservation and secondary induction, can realize rapid, stable, high-efficiency and low-cost seedling culture of eustoma grandiflorum, and simultaneously provides materials for molecular design breeding means for realizing ornamental characters such as flower color, flower style, flower fragrance and the like. Thereby accelerating the cultivation of the independent new variety of ornamental plants, prolonging the service time of the variety market and reducing the dependence on imported seedlings.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a construction method of a eustoma grandiflorum multi-variety somatic embryo efficient regeneration system.
Background
Eustoma grandiflorum (Eustoma grandiflorum), also known as radix Gentianae in grasslands, exquisite, etc., has wrinkled petals, unique flower shape, light and stagnant flower, and long flowering period. The rose-like shape of the flowers and the rich variety of colors are also called "thornless roses". Eustoma grandiflorum has a world cut flower market ranking of 4-10, is used as an emerging flower in the Yunnan flower industry, has a fourth production value, becomes a fourth largest cut flower after China rose, lily and carnation, and has higher market potential.
At present, the species source of eustoma grandiflorum in China mainly depends on foreign imported F1 generation hybrid varieties, and the species source is high in price and limited in supply. Because the selfing seeds of eustoma grandiflorum are seriously separated to reduce the yield and quality, hybrid seed production is usually adopted, but the hybrid parents are degenerated, most commercial varieties popular in the market are difficult to survive for more than 5 years in the market, and the commercial application is severely restricted. Therefore, research on efficient propagation and breeding techniques of eustoma grandiflorum is highly desirable.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a construction method of a high-efficiency regeneration system of eustoma grandiflorum multi-variety somatic embryos.
The invention aims at realizing the following technical scheme:
a construction method of an efficient regeneration system of eustoma grandiflorum multi-variety somatic embryos comprises the following steps:
(1) Seed disinfection and germination: collecting Eustoma grandiflorum seeds, adding a drop of Tween into a super clean bench with sodium hypochlorite solution of 2.5% active chlorine, soaking the seeds for 8-10 min, cleaning with sterile water, aseptically sowing on 1/2MS+30g/L sucrose+8g/L agar, and pH 5.8+ -1 culture medium, and germinating and growing in a culture room. The conditions of the culture room are 25+/-2 ℃, the photoperiod is 16 hours, the illumination intensity is 36 mu mol/m 2/s, and the growth is carried out for 30-40 days.
(2) Induction and proliferation of embryogenic cells: after growing for 30-40 days, observing the growth state of the plant, selecting the plant with about 10mm hypocotyl and healthy cotyledon without true leaf, cutting off hypocotyl and cotyledon, cutting off root cap, growing point and leaf edge, and inoculating to induction culture medium. The formula of the induction culture medium is as follows: MS+0.5-3 mg/L2, 4-D (i.e., 2, 4-dichlorophenoxyacetic acid) +30g/L sucrose+6 g/L agar, pH 5.8.+ -. 1. Culture conditions: culturing in dark for 30-40 days at 25+ -2deg.C.
(3) Somatic embryo differentiation into shoots: after induction culture for 30-40 days, taking callus on an induction culture medium, grinding the callus, transferring the callus to a somatic embryo differentiation culture medium, performing elongation germination in a dim light or dark environment, and culturing for about 20 days until cotyledon embryos are completely shaped, and then culturing by illumination. The formula of the culture medium is as follows: MS+0.5-1 mg/L6 BA (i.e. 6-benzylaminopurine) +30 g/L sucrose+8 g/L agar, pH 5.8.+ -. 1.
(4) Rooting and seedling formation of the differentiated buds: separating the regenerated buds of somatic embryo from parent, transferring to rooting culture medium containing auxin, and culturing in culture room until true leaves grow out and root system develops to form seedling. The rooting culture medium formula is 1/2MS+0-1mg/L IBA (i.e. indolebutyric acid) +30 g/L sucrose+8 g/L agar, the culture condition is room temperature of 25+/-2 ℃, the 16-hour photoperiod, 36 mu mol/m 2/s illumination intensity, and the growth is carried out for 30-40 days.
(5) Hardening seedlings: when the seedlings grow mature but the root system is not aged, the plants are taken out from the rooting culture medium, the culture medium is cleaned, and the plants are transferred into a matrix formed by mixing peat soil and perlite, and hardening seedlings are carried out in a cool and moist natural environment for 3-7 days.
(6) Planting: transplanting the strong seedlings after seedling hardening from the substrate to soil, and growing and flowering under normal cultivation operation.
Preferably, the eustoma grandiflorum seed in step (1) may be replaced with an explant of eustoma grandiflorum seedling.
Compared with the prior art, the invention has the beneficial effects that:
1. Although somatic embryo technology has many advantages, it has low induction efficiency and low seedling rate, so it has not been widely used in the market. The construction method of the eustoma grandiflorum multi-variety somatic cell regeneration system provides the optimal concentration of the induction hormone, and the eustoma grandiflorum can be quickly, stably and efficiently cultured, so that the culture period is shortened, and the method is applicable to a plurality of representative varieties with different flower types, flower colors and growing seasons on the market.
2. The method simplifies the plant regeneration process and saves a great amount of time for preparing the tissue culture material. And the mutation frequency of somatic cell cloning is low, so that somatic embryogenesis is more suitable for market application than organogenesis. In addition, mature somatic embryos can be stored in liquid nitrogen, can still grow into healthy plants after activation, and still maintain embryogenic properties at genetic and epigenetic levels, ensuring the sustainability of the material.
3. The embryogenic callus induced by the method can be multiplied for a plurality of times, and materials are supplied uninterruptedly; meanwhile, the regenerated plants can be used for efficiently inducing embryogenic cells again, seeds do not need to be used repeatedly, and the sustainability of the material is further ensured.
4. The method has high differentiation efficiency, so that embryogenic cells in embryogenic cell clusters are fully developed, and further, the somatic embryo regeneration efficiency and the budding efficiency are improved, and a foundation is provided for production application.
Drawings
FIG. 1 is a flow chart of a method for constructing a high-efficiency regeneration system for multiple varieties of somatic embryos of Eustoma grandiflorum described in example 1 (in the figure, 1A-Eustoma grandiflorum seeds germinate and grow; 1B-grow sterile seedlings for 30-40 days; 1C-explants dedifferentiate to form partial embryogenic cells; 1D-embryogenic callus proliferate; 1E-embryogenic callus differentiate into somatic embryos and germinate; 1F-regenerated buds mature and leave maternal tissue; 1G-Eustoma grandiflorum seedlings; 1H-flowering Eustoma grandiflorum).
FIG. 2 is a graph of morphological characteristics of eustoma grandiflorum somatic embryos of example 1 [ in the figure: 2A-embryogenic callus proliferation; differentiating the 2B-embryogenic callus into somatic embryos and germinating; embryo precursors from 2C-embryogenic callus differentiation: spherical embryo (arrow ①), torpedo embryo (arrow ②), cotyledon embryo (arrow ③); 2D-shoots detached from the mother body. Scale = 1cm ].
FIG. 3 is a photograph of a large number of regenerated seedlings produced efficiently in example 1.
FIG. 4 is a picture of a plant cultivated by field planting for 1 month in example 1.
FIG. 5 is a photograph of a plant flowering in example 1.
FIG. 6 is a comparative photograph showing the effect of mechanical rolling on differentiation efficiency in example 2.
FIG. 7 is a graph of callus induction versus bar graph for various varieties of eustoma grandiflorum using the method of the present invention in example 4.
FIG. 8 is a graph of germination rate versus bar graph for various varieties of eustoma grandiflorum using the method of the present invention in example 4.
FIG. 9 is a graph of regeneration induction coefficient versus scatter for different varieties of eustoma grandiflorum using the method of the present invention in example 4.
Description of the embodiments
The present invention will be further described with reference to examples, but the present invention is not limited to the examples.
Example 1
1-5, A method for constructing a eustoma grandiflorum multi-variety somatic embryo efficient regeneration system comprises the following steps:
(1) Seed disinfection and germination: collecting eustoma grandiflorum seeds, diluting sodium hypochlorite solution (active chlorine >5.5% diluted to 2.5% active chlorine) with sterile water in an ultra-clean workbench, adding a drop of Tween into the diluted sodium hypochlorite solution, soaking the seeds for 10 minutes, pouring out the disinfectant after disinfection, and cleaning with sterile water for 3-5 times until cleaning. The seeds are sterilized and then sown on a solid medium of 1/2MS+30g/L sucrose+agar 8g/L and pH5.8+ -1, and placed in a culture chamber for germination and growth (FIG. 1-1A). The conditions of the culture room are 25+/-2 ℃, the photoperiod is 16 hours, the illumination intensity is 36 mu mol/m 2/s, and the growth is carried out for 30 days.
(2) Induction and proliferation of embryogenic cells: after 30 days of growth, tender sterile plants (shown in figures 1-1B) are grown, and plants with good growth state, namely, the lower embryo with the axial length of about 10mm, are selected, and cotyledons grow healthily without true leaves. The seedlings were placed on an ultra clean bench and hypocotyls and cotyledons were cut off with a surgical knife and forceps. Root crowns are cut off when hypocotyls are cut off, growing points and leaf edges are cut off when cotyledons are cut off, a proper amount of wound is manufactured to facilitate callus formation, and the cut explants are inoculated to an induction culture medium. The formula of the culture medium is as follows: MS+0.5-3 mg/L2, 4-D+30 g/L sucrose+6 g/L agar, pH 5.8+ -1. Culture conditions: culturing in dark for 30-40 days at 25+ -2deg.C.
(3) Somatic embryo differentiation into shoots: chlorophyll and the like began to fade around 7 days, and dedifferentiate to form a part of embryogenic cells (FIG. 1-1C). After 40 days of induction culture, embryogenic callus proliferated in large amounts and had dense small projections on the surface (FIGS. 1-1D, FIGS. 2-2A), i.e., spherical embryos. Callus on induction medium is taken in super clean bench, crushed and transferred to somatic embryo differentiation medium, and the callus is elongated and germinated in weak light or dark environment for 20 days to form embryo precursors with heart shape, torpedo shape and cotyledon shape, and after the cotyledon embryo is complete, healthy cotyledon embryo is separated from the embryo, and the embryo is transferred to light culture to promote photosynthesis and growth. The formula of the culture medium is as follows: MS+0.5-1 mg/L6 BA+30 g/L sucrose+8 g/L agar, pH5.8.+ -. 1.
(4) Rooting and seedling formation of the differentiated buds: the well-developed somatic embryo regeneration buds are separated from the maternal tissue blocks on an ultra-clean bench (figures 1-1F and 2-2D), are directly transferred into rooting culture medium containing auxin, are placed in a culture room until true leaves grow, root systems develop and seedlings grow (figures 1-1G) (unlike the common callus regeneration or adventitious bud regeneration, somatic embryos induced by culture with eustoma grandiflorum cotyledon explants exhibit loose golden cream-like embryogenic callus, a large number of buds in the embryogenic callus differentiate and fall off from the maternal, the fallen buds develop into root primordia and cotyledon morphology), the culture medium formula is 1/2MS+0-1mg/L IBA+30G/L sucrose, the culture condition is room temperature of 25+/-2 ℃,16 hours photoperiod, 36 mu mol/m 2/s illumination intensity and the growth is carried out for 30 days. FIG. 3 shows a large number of regenerated seedlings produced efficiently using the method of the present invention.
(5) Hardening seedlings: when the seedlings grow mature but the root system is not aged, taking out plants from the culture medium, washing the culture medium remained at the root by using clear water, transferring the seedlings into a matrix formed by mixing peat soil and perlite according to the mass ratio of 8:2, and hardening the seedlings in a cool and moist natural environment for 3-7 days.
(6) Planting: the strong seedlings after seedling hardening are transplanted to soil from the substrate, and grow and bloom under normal cultivation operation (fig. 1-1H), fig. 4 is a plant cultivated for 1 month by field planting, and fig. 5 is a flowering plant.
The specific parameters of this example are shown in table 1.
Example 2 mechanical Rolling to increase somatic embryo differentiation efficiency
In this example, callus on the induction medium of example 1 was induced and cultured for 40 days, and was divided into two equal parts at random, one part was directly transferred onto the somatic embryo differentiation medium without rolling, and the other part was mechanically moderately rolled and then transferred onto the somatic embryo differentiation medium for differentiation. The medium and procedure for the differentiation culture were the same as in example 1.
As shown in FIG. 6, only a small amount of primordium was developed into buds without rolling for direct differentiation culture, and the differentiation efficiency was low (FIGS. 6-6A); after moderate rolling, differentiation culture is carried out, all the primordium grows into buds, and the differentiation efficiency is greatly improved (figures 6-6B).
In the process of somatic embryo differentiation, each embryogenic callus contains a large number of embryogenic cells, and when the callus is directly transferred into a differentiation medium for differentiation, a large number of somatic embryos cannot normally develop and stay in the stage of spherical embryo due to lack of growth space and nutrient supply, and cannot normally develop into cotyledon embryos. According to the invention, loose embryogenic callus is dispersed before embryogenic cells are differentiated by mechanical rolling and then inoculated into a differentiation medium, so that the differentiation efficiency of somatic embryos is improved, the emergence rate is improved, and a necessary condition is provided for further mass propagation cultivation.
Example 3 regeneration plant Secondary efficient Induction of embryogenic callus
The embryogenic callus which is successfully induced can be multiplied for a plurality of times, and materials are supplied continuously (shown in figures 1-1D), but the embryogenic cells can lose embryogenesis due to the plurality of times of multiplication, the embryogenic cells can be induced again by the regenerated plants with high efficiency, the seeds do not need to be used repeatedly, and the sustainability of the materials is further ensured.
In this example, hypocotyls and cotyledons were excised from well-developed somatic embryo regenerated buds or regenerated plants (FIGS. 1 to 1F and 1 to 1G) on an ultra clean bench with a surgical knife and forceps. Root crowns are cut off when hypocotyls are cut off, growing points and leaf edges are cut off when cotyledons are cut off, a proper amount of wound is manufactured to facilitate callus formation, and the cut explants are inoculated to an induction culture medium. After an induction period (30-40 days) normal embryogenic calli were formed. That is, the whole production technical process can be completed without the participation of seeds (fig. 1-1A) in the process shown in fig. 1, and meanwhile, embryogenesis of embryogenic callus is obtained again, so that the sustainability of materials and production is ensured.
Example 4 Eustoma grandiflorum Multi-variety Induction efficiency test
In this example, a number of commercially available eustoma grandiflorum varieties were selected, and the system stability and versatility of the commercially available varieties were tested by the method described in example 1, with the selected varieties being representative in flower color, flower type, season of planting, and variety resistance.
(1) Tender plants grown by culturing according to the method described in the step (1) of example 1 were taken, and embryogenic cells were induced and proliferated for a plurality of selected varieties according to the method described in the step (2) of example 1, and the results are shown in table 2, where callus induction rate=number of callus-forming plants/total number of inoculations×100%.
According to the data in Table 2, all varieties can be stably induced to produce embryogenic callus by using the embryogenic cell induction and proliferation method of the present invention. The callus induction rate of most varieties is close to 100 percent except that the induction rate of somatic embryos of few varieties is about 60-80 percent (shown in figure 7).
(2) The embryogenic calli of all varieties in item (1) above were subjected to differentiation culture according to the method described in step (3) of example 1, and the results are shown in table 3, with germination rate = number of germinated calli/total inoculated calli x 100%.
According to the data in Table 3, it was shown that all varieties were able to stably differentiate somatic embryos and mature buds using the somatic embryo differentiation culture method of the present invention. The differentiation efficiency of few varieties is lower, but can reach more than 50%, and the differentiation efficiency of most varieties is nearly 100% (shown in figure 8).
(3) Induction coefficient = total budding number/number of budding calli inoculated, the results are shown in table 4.
According to the data in Table 4, it is shown that plant regeneration can be efficiently performed by somatic embryo route using the regeneration system disclosed in the present invention, all varieties are stably regenerated and the induction coefficient of most varieties can reach more than 20 (as shown in FIG. 9).
In the embodiment, cotyledons and hypocotyls of a plurality of eustoma grandiflorum seedlings are used as explants, embryo cells are induced by using 2,4-D, and loose golden yellow creamy embryogenic callus can be induced after culturing for 30-40 days. Embryogenic callus was placed in 6-BA-added medium for culture, and after about 30 days, a large number of somatic embryos were observed. The isolated somatic embryos root after 30 days of culture in rooting medium and form plants with a morphology similar to that of the seed germinated seedlings. The results show that all varieties can stably induce embryogenic callus and form somatic embryos.
Claims (5)
1. The construction method of the eustoma grandiflorum multi-variety somatic embryo efficient regeneration system is characterized by comprising the following steps of:
(1) Seed disinfection and germination: sterilizing Eustoma grandiflorum seeds, sowing on a solid culture medium of 1/2MS+30g/L sucrose+agar 8g/L and pH 5.8+ -1, and culturing in a culture room under illumination for germination and growth for 30-40 days;
(2) Induction and proliferation of embryogenic cells: after 30 days of sowing, selecting a plant with about 10mm hypocotyl and healthy cotyledons without true leaves, cutting the hypocotyl and the cotyledons, inoculating the plant into an induction culture medium, wherein the formula of the induction culture medium is as follows: MS+0.5-3 mg/L2, 4-D+30 g/L sucrose+6 g/L agar, pH5.8+ -1, cultured in dark for 30-40 days;
(3) Somatic embryo differentiation into shoots: after induction culture for 30-40 days, taking callus on an induction culture medium, grinding the callus, transferring the callus to a somatic embryo differentiation culture medium, and carrying out elongation germination in a dim light or dark environment until cotyledon embryos are complete, and then transferring the callus to illumination culture, wherein the culture medium comprises the following formula: MS+0.5-1 mg/L6-BA+30 g/L sucrose+8 g/L agar, pH 5.8+ -1;
(4) Rooting and seedling formation of the differentiated buds: separating the regenerated buds of the somatic embryos which are well developed from the parent, transferring the regenerated buds into a rooting culture medium, placing the rooting culture medium in a culture room for illumination culture until true leaves grow out, root systems develop and seedlings grow, wherein the rooting culture medium formula is 1/2MS+0-1mg/L IBA+30 g/L sucrose+8 g/L agar, and growing for 30-40 days;
(5) Hardening seedlings: when the seedlings grow mature but the root system is not aged, taking out plants from a rooting culture medium, cleaning the culture medium, transferring the plants into a matrix formed by mixing peat soil and perlite, and hardening seedlings in a cool and moist natural environment;
(6) Planting: transplanting the hardened seedlings from the substrate to soil, and culturing normally until the seedlings grow and bloom.
2. The method for constructing a high-efficiency regeneration system for eustoma grandiflorum multi-variety somatic embryos according to claim 1, wherein the culture temperature in the steps (1) to (4) is 25+/-2 ℃.
3. The method for constructing a high-efficiency regeneration system for eustoma grandiflorum multi-variety somatic embryos according to claim 1, wherein the photoperiod of the illumination culture in the step (1), the step (3) and the step (4) is 16 hours, and the illumination intensity is 36 mu mol/m 2/s.
4. The method for constructing a high-efficiency regeneration system for multiple varieties of somatic embryos of eustoma grandiflorum according to claim 1, wherein the disinfection step in step (1) is as follows: the eustoma grandiflorum seeds are placed in an ultra-clean workbench, one drop of Tween is added into the eustoma grandiflorum seeds by using a sodium hypochlorite solution of 2.5% active chlorine, the seeds are soaked for 8-10 minutes, and then the eustoma grandiflorum seeds are washed by using sterile water.
5. The method of constructing a high-efficiency regeneration system for multiple eustoma grandiflorum somatic embryos according to claim 1, wherein after hypocotyls and cotyledons are cut in the step (2), root crowns, growing points and leaf edges are cut off, and then inoculated into an induction medium.
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Citations (4)
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|---|---|---|---|---|
| JPH07155081A (en) * | 1993-12-08 | 1995-06-20 | Hokko Chem Ind Co Ltd | Production of seed and seedling of dwarfed eustoma resselianum g. don |
| CN101836587A (en) * | 2010-03-29 | 2010-09-22 | 云南省农业科学院花卉研究所 | Method for obtaining eustoma regeneration plant by anther culture |
| CN102485897A (en) * | 2010-12-06 | 2012-06-06 | 华中农业大学 | Method for changing petal colors by using cotton gene GbF3H |
| CN103635572A (en) * | 2011-07-01 | 2014-03-12 | 株式会社资生堂 | Plant cell differentiation promoter |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07155081A (en) * | 1993-12-08 | 1995-06-20 | Hokko Chem Ind Co Ltd | Production of seed and seedling of dwarfed eustoma resselianum g. don |
| CN101836587A (en) * | 2010-03-29 | 2010-09-22 | 云南省农业科学院花卉研究所 | Method for obtaining eustoma regeneration plant by anther culture |
| CN102485897A (en) * | 2010-12-06 | 2012-06-06 | 华中农业大学 | Method for changing petal colors by using cotton gene GbF3H |
| CN103635572A (en) * | 2011-07-01 | 2014-03-12 | 株式会社资生堂 | Plant cell differentiation promoter |
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