CN109169280B - Preparation method of coix seed regenerated plant and culture medium used by preparation method - Google Patents
Preparation method of coix seed regenerated plant and culture medium used by preparation method 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
- A01H4/008—Methods for regeneration to complete plants
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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
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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
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Abstract
The invention discloses a preparation method of coix seed regenerated plants and a culture medium used by the method. The invention discloses a preparation method of coix seed regeneration plants, which comprises the following steps: 1) culturing coix sprout on callus induction culture medium to obtain embryonic callus; 2) culturing the embryonic callus on a differentiation culture medium to obtain a regeneration bud; 3) culturing the regeneration bud on a rooting culture medium to obtain a coix seed regeneration plant; the Coicis semen bud is the bud of Coicis semen seed 2-5 days after germination. The preparation method of the coix seed regeneration plant can obtain the coix seed in-vitro regeneration plant in a short time, can also carry out genetic transformation, solves the problems of low callus induction rate, difficult callus subculture, low differentiation capacity after subculture and the like of the prior coix seed callus, and has wider application prospect.
Description
Technical Field
The invention relates to a preparation method of coix seed regeneration plants and a culture medium used in the preparation method, belonging to the field of biotechnology.
Background
Coix Lacryma-jobi L is a plant taxonomic genus of Poaceae Coix, also called as grass beads, Sichuan valley, Coix Lacryma-jobi, six-millet, medicinal corn, Coix Lacryma-jobi L, and the like, native China and southeast Asia such as Laos, Burma and the like, and has at least 6000-10000 years of cultivation and domestication history in China. The traditional utilization mode of the coix is used as a health food and a medicinal plant, and medical famous works such as compendium of materia medica, ben cao jing shu and the like have records that the coix is used for treating diseases such as eczema, rheumatoid arthritis, flat wart and the like. In recent years, coix seed oil and coix seed ester extracted from coix have been used for treating diseases such as cancer, and among them, a conlaite injection and a coix seed dehumidifying and pain-relieving capsule produced from coix as a main raw material have been marketed.
Coix lacryma-jobi is a cross-pollinated crop of hermaphrodite plants, which is 2-fold, has 2 n-20 chromosomes and only a small part of materials are 4-fold, and 2 n-40 chromosomes. China, Yunnan, Sichuan, Guizhou, Guangxi and the like are one of the origins of the coix, and wild coix resources are very rich, are often distributed in humid zones such as river edges, pond edges, marshlands, uncultivated wastelands and the like, have rich forms and genetic variations, and have excellent moisture resistance and good disease resistance. In a natural state, wild coix seeds are propagated in two modes of seeds and roots, the coix seeds are carbon-4 plants, have high-yield biological foundation, are large in tillering, large in plant height and luxuriant in stem and leaf, and are used as excellent forage grass directly by farmers in Guangxi, Yunnan, Hunan and other places. In addition, in the minority regions such as Guangxi Liuzhou and Yunnan Xishuangbanna, the habit of picking wild coix seeds, roots, stems and the like as traditional Chinese medicines still exists so far, but the research of breeding novel pasture by utilizing wild coix is not seen.
Since 2000 years, the corn research institute of Sichuan agricultural university has pioneered the research of breeding perennial forage grass by using wild coix seed at home and abroad. The collection, arrangement and preservation of wild coix seed resources are widely carried out in the origin places of wild coix, namely Yunnan, Chuan, noble places in China and southeast Asia countries such as Laos, Burma, Vietnam and the like, a coix seed resource garden is established, and researches on the aspects of identification and evaluation of agronomic characters, reproductive characteristics, genetic characteristics, stress resistance, feeding characteristics, disease and insect resistance and the like of the coix are systematically carried out. A new variety of feeding-type coix, namely Daheishan coix, with excellent comprehensive properties, perennial and high nutritive value is bred, examined by the grass variety examination committee of Sichuan province in 2016 (12 months) and participated in the national pasture grass region test in 2017. On the basis, diploid Heishan Job's tears (2n is 20) and tetraploid aquatic Job's tears (2n is 40) are used as parents for distant hybridization to obtain a triploid Job's tears (2n is 30) with somatic cell chromosome number of 30, which shows outstanding stress resistance, vigorous growth, developed nutrition and better grass yield than parents, and the plant is named as Fengmu 88 and examined by the examining and deciding committee of Sichuan province grass varieties in 2017.
The regeneration system of the coix is the premise and the basis for carrying out mutation breeding and functional gene verification through callus tissues. The predecessors made certain researches on explants, callus induction culture media, callus differentiation culture media, culture conditions and the like in the induction of a coix regeneration system, but still had the problems of low callus induction rate, difficult callus subculture, low differentiation capacity after subculture and the like, and the systematic researches were lacked. Particularly, in recent 30 years, studies on an in vitro coix regeneration system have hardly advanced and are in a state of being in a state of standing.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare the coix seed regeneration plant.
In order to solve the above technical problems, the present invention firstly provides a method for preparing a coix seed regenerated plant, comprising:
1) culturing coix sprout on callus induction culture medium to obtain embryonic callus;
2) culturing the embryonic callus on a differentiation culture medium to obtain a regeneration bud;
3) and culturing the regeneration bud on a rooting culture medium to obtain a coix seed regeneration plant.
In the above method, the coix seed bud can be a bud obtained after germination of the coix seed, i.e. a non-seed and non-root tissue obtained after germination of the coix seed.
The Coicis semen bud can be sterile bud. The aseptic bud can be obtained by culturing aseptic seeds of Coix lacryma-jobi in an aseptic environment. The sterile seeds can be obtained by sterilizing with 8% sodium hypochlorite solution. The effective chlorine content of the 8% sodium hypochlorite solution is 8%. The 8% sodium hypochlorite solution consists of a solvent and a solute, wherein the solvent is water, and the solute and the concentration of the solute in the 8% sodium hypochlorite solution are 8g/100 mL. The time for sterilizing the coix seed by using the sodium hypochlorite solution with the concentration of 8% can be 20 minutes.
The Coicis semen bud can be bud of Coicis semen seed 2-5 days after germination. Further, the coix seed sprout can be the sprout of coix seed on the 4 th day after germination. The length of the coix sprout can be 0.5 cm.
In the method, the callus induction culture medium consists of a solvent and solutes, wherein the solvent is an MS culture medium, and the solutes are NAA and 2, 4-D; the concentration of NAA in the callus induction culture medium is 1mg/L, and the concentration of 2,4-D is a1), a2) or a 3):
a1)1-4mg/L；
a2)1-3mg/L；
a3)1-2mg/L。
the differentiation culture medium consists of a solvent and a solute, wherein the solvent is an MS culture medium, and the solute is cefuroxime, 6-BA and kinetin; the concentrations of the cefuroxime, 6-BA and kinetin in the differentiation medium are respectively 250mg/L, 2mg/L and 2 mg/L.
The rooting medium consists of a solvent and a solute, wherein the solvent is 1/2MS medium, the solute is IBA, and the concentration of the IBA in the rooting medium is 0.5 mg/L; the 1/2MS culture medium is obtained by reducing the concentration of each solute in the MS culture medium by half.
The callus induction medium may have a pH of 5.8-6.0. The callus induction medium may have a pH of 5.8.
The differentiation medium may have a pH of 5.8-6.0. The differentiation medium may specifically have a pH of 5.8.
The rooting medium may have a pH of 5.8-6.0. The pH value of the rooting medium can be specifically 5.8.
In the above method, the conditions for the culturing in step 1) may be: the illumination intensity is 3000-. The conditions for the cultivation in step 1) may specifically be: the light intensity is 3000lx, and/or the temperature is 27 ℃, and/or the light cycle is 16h light/8 h dark.
The conditions for the cultivation in step 2) may be: the illumination intensity is 3000-. The conditions for culturing in step 2) may specifically be: the light intensity is 3000lx, and/or the temperature is 27 ℃, and/or the light cycle is 16h light/8 h dark.
The conditions for culturing in step 3) are as follows: the illumination intensity is 3000-. The conditions for culturing in step 3) may specifically be: the light intensity is 3000lx, and/or the temperature is 27 ℃, and/or the light cycle is 16h light/8 h dark.
The method may further comprise subculturing the embryogenic callus.
In the above method, the subculture may be performed on the callus induction medium or subculture medium; the subculture medium may be a medium obtained by adding copper sulfate or silver nitrate to the callus induction medium.
In the method, the addition amount of the copper sulfate or the silver nitrate in the subculture medium can be 0.85mg/L of the culture medium.
The conditions for the subculture may be: the illumination intensity is 3000-3500lx, the temperature is 25-28 ℃, and the illumination period is as follows: light for 16h and dark for 8 h. The conditions of the subculture may specifically be: the illumination intensity is 3000lx, the temperature is 27 ℃, and the illumination period is as follows: light for 16h and dark for 8 h.
The invention also provides the following methods of M1) or M2) or M3):
m1) method for inducing coixseed embryogenic callus, comprising: culturing the coix sprout on the callus induction culture medium to obtain coix embryonic callus;
m2) differentiation method of coixseed embryogenic callus, comprising: culturing the coix embryonic callus on the differentiation culture medium to obtain coix regeneration buds;
m3) the method for subculturing the embryonic callus of the coix, which comprises the following steps: and culturing the coix embryonic callus in the subculture medium to realize the subculture of the coix embryonic callus.
M1) can be cultured under the following conditions: the illumination intensity is 3000-. The conditions for the cultivation in step 1) may specifically be: the light intensity is 3000lx, and/or the temperature is 27 ℃, and/or the light cycle is 16h light/8 h dark.
M2) can be cultured under the following conditions: the illumination intensity is 3000-. The conditions for culturing in step 2) may specifically be: the light intensity is 3000lx, and/or the temperature is 27 ℃, and/or the light cycle is 16h light/8 h dark.
M3) can be cultured under the following conditions: the illumination intensity is 3000-3500lx, the temperature is 25-28 ℃, and the illumination period is as follows: light for 16h and dark for 8 h. The conditions of the subculture may specifically be: the illumination intensity is 3000lx, the temperature is 27 ℃, and the illumination period is as follows: light for 16h and dark for 8 h.
The invention also provides the following reagents of Y1) or Y2) or Y3) or Y4):
y1) culture medium, which is the callus induction culture medium, the differentiation culture medium, the rooting culture medium or the subculture medium;
y2) a complete medium consisting of the callus induction medium and the differentiation medium;
y3) a complete set of culture medium consisting of the callus induction medium, the differentiation medium and the rooting medium;
y4) set of culture medium consisting of the callus induction medium, the differentiation medium, the rooting medium and the subculture medium.
Y2), Y3) and Y4) can be used for the regeneration of coix plants.
The invention also provides the application of any one of the following X1) -X3):
x1) application of the reagent in preparing coix seed regeneration plants;
x2) the application of the reagent in genetic transformation of coix;
x3) the preparation method of said coix regenerated plant, or the induction method of said coix embryonic callus, or the differentiation method of said coix embryonic callus, or the application of said coix embryonic callus subculture method in the genetic transformation of coix.
In the present invention, each culture medium is a sterile culture medium.
The preparation method of the coix seed regeneration plant solves the problems of low callus induction rate, difficult callus subculture, low differentiation capacity after subculture and the like of the prior coix seed, can obtain the coix seed in-vitro regeneration plant in a short time, can perform genetic transformation, and has wider application prospect.
Drawings
FIG. 1 shows callus induction using Coix lacryma-jobi leaves. (A) And (B) are respectively the conditions of the 4 th d and the 7 th d after the coix seed leaves are inoculated.
FIG. 2 shows callus induction by root tip of Coix lacryma-jobi. (A) The whole root is enlarged to grow out callus, and (B) one end of the root is enlarged to grow out callus.
FIG. 3 shows callus induction using Coix lacryma-jobi stem. (A) Induction medium for 7d, and (B) induction medium for 20 d.
FIG. 4 shows the induction of callus by aerial root of Coix lacryma-jobi. (A) Aerial root culture for 10 days, and (B) aerial root culture for 15 days.
FIG. 5 shows callus induction using Coix lacryma-jobi bud. (A) Induction for 10 d; (B) induction for 15 d; (C) embryogenic callus was induced by shoots.
FIG. 6 shows the effect of different anti-browning agents on callus culture. C0: culturing the obtained callus without adding an anti-browning agent; c1: adding 2mg/L vitamin C to culture to obtain callus; c2: adding 0.85mg/L copper sulfate to culture to obtain callus; c3: adding 0.85mg/L silver nitrate, and culturing to obtain callus.
FIG. 7 shows the differentiation of callus derived from Coix lacryma-jobi bud. a: inoculating the callus to D18 culture medium 7D, and starting to generate green spots; b: the callus inoculated on D18 medium for 20 days was differentiated to bud.
FIG. 8 shows the rooting and transplanting of the regenerated buds of Coix lacryma-jobi. (A) The callus is differentiated into buds; (B) rooting the regenerated buds; (C) hardening seedlings, opening the cover and culturing; (D) cleaning the regenerated seedlings after the root culture medium is removed before transplanting; (E) regenerated seedlings transplanted into pots.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 preparation of regenerated plant of Coix lacryma-jobi
In this example, seeds of Job's tears (Sichuan Singaporeaceae, Ltd.) were used as a research material to obtain sterile seedlings, and then different explants were used to induce regeneration of callus and plants. The method comprises the following specific steps:
first, induction of coix seed embryonic callus
1. Influence of different disinfectants on seed germination rate and contamination rate
The coix seed is externally provided with a layer of membranous involucre which carries a plurality of germs, and aseptic seedlings are difficult to obtain, so the inventor firstly researches the influence of different disinfectants on the germination rate and the contamination rate of the coix seed. The disinfectant is 0.1% mercuric chloride solution and 8% sodium hypochlorite solution, wherein the 0.1% mercuric chloride solution (0.1 g of mercuric chloride is weighed, and the volume is determined to 100ml by using ultrapure water to obtain the 0.1% mercuric chloride solution), and the effective chlorine content of the 8% sodium hypochlorite solution is 8% (the product of biological engineering (Shanghai) GmbH, 8g of sodium hypochlorite is weighed, and the volume is determined to 100ml to obtain the 8% sodium hypochlorite solution).
The coix seeds are disinfected by using a disinfectant, the disinfection time is shown in table 1, then the coix seeds are cleaned by using clear water, and the coix seeds are placed in an MS culture medium to be cultured for 1 week, and then the germination rate and the bacterial contamination rate are counted (table 1). The culture conditions used were: the illumination intensity is 3000lx, the temperature is 27 ℃, and the illumination period is as follows: light for 16h and dark for 8 h.
As can be seen from Table 1, 0.1% mercuric chloride solution and 8% sodium hypochlorite solution both have the sterilization effect on coix seeds, the sterilization effect is better and better along with the increase of the treatment time, but the germination rate of the seeds is lower and lower, which indicates that the disinfectant has more and more toxicity on the seeds along with the increase of the sterilization time. Under the same treatment time, the disinfection effect of the 0.1% mercuric chloride solution is better than that of the 8% sodium hypochlorite solution. However, considering that the mercuric chloride is a highly toxic article, the difference between the seed germination rate of the mercuric chloride solution with the concentration of 8 percent for 20min and the seed germination rate of the mercuric chloride solution with the concentration of 0.1 percent for 15min is not large. Therefore, the method for disinfecting the coix seed adopted in the subsequent experiment comprises the following steps: sterilizing with 8% sodium hypochlorite solution for 20 min.
TABLE 1 Disinfection Effect of different Disinfection reagents on Coix lacryma-jobi seeds
2. Selection of explants
Good embryogenic callus is the most critical step in the establishment of regeneration systems. The induction rates of the calli of different explants are very different on the same material, so that the inventor respectively induces the calli of the leaves, the root tips, the stems, the aerial roots and the buds of the coix lacryma-jobi, and counts the callus rate and the embryogenic callus rate to determine the most appropriate explant.
The used induction culture medium is a sterile culture medium obtained by adding 2,4-D into a minimal medium 1, the pH value is 5.8-6.0, the concentrations of 2,4-D in five induction culture media (respectively marked as A1, A2, A3, A4 and A5) are respectively 1mg/L, 2mg/L, 3mg/L, 4mg/L and 6mg/L, the used minimal medium 1 is a culture medium obtained by adding NAA into an MS solid culture medium, and the NAA content in each liter of the minimal medium 1 is 1 mg.
The callus induction rate is 100% of the number of explants from which callus was induced/total number of explants, and the embryogenic callus induction rate is 100% of the number of explants from which embryogenic callus was induced/total number of explants from which callus was induced.
Wherein the embryogenic callus is granular callus which grows slowly, is light yellow green and is rough; non-embryogenic callus is fast-growing, high-water-content, white or yellowish soft callus, or compact, opaque, non-segregating smooth or rough callus.
The callus induction culture conditions used hereinafter are all: the culture was carried out under light intensity of 3000lx and temperature of 27 ℃ in the dark.
2.1 Induction of embryogenic callus by leaves
Inoculating the sterilized coix seed to an MS solid culture medium for culture under the following culture conditions: the illumination intensity is 3000lx, the temperature is 27 ℃, and the illumination period is as follows: light for 16h and dark for 8 h. When the aseptic seedlings grow to the three-leaf one-heart stage, cutting the leaves into 0.5 cm-long small sections by using aseptic scissors and tweezers, inoculating the small sections onto 5 induction culture media with different 2,4-D concentrations, inoculating 100 leaf sections on each culture medium, inoculating 10 leaves on each culture dish, repeating the steps for 3 times, and counting the healing rate and the embryogenic callus rate after 20 days, wherein the results are shown in table 2.
As can be seen from Table 2, none of the leaves of Coix lacryma-jobi was dedifferentiated to form callus in the concentration range of 2,4-D used. In addition, callus was not induced by the use of more young leaves. The leaves of Coix lacryma-jobi start to turn yellow at the 4 th leaf after inoculation and start to brown and die after 7 days (figure 1), which shows that the leaves of Coix lacryma-jobi have no capacity of dedifferentiating to form callus and are not suitable for being used as explants for inducing embryogenic callus.
TABLE 2 Induction of Coix lacryma-jobi leaf callus
2.2 Induction of embryogenic callus by root tips
When the root of the aseptic seedling of the coix lacryma-jobi grows to 1-3cm, cutting the root with about 0.5cm by using aseptic scissors and tweezers, inoculating the root on 5 induction culture media with different 2,4-D concentrations, inoculating 100 segments of each culture medium, inoculating 10 segments of each culture dish, repeating the steps for 3 times, and counting the healing rate and the embryogenic callus rate after 20 days, wherein the healing rate and the embryogenic callus rate are shown in a table 3.
The results showed that, after 4 days of inoculation, milky white callus with moist surface was formed on the wound of the root of Coix lacryma-jobi, and some roots were enlarged and grown to form callus at only one end, and some were enlarged and grown callus at the whole length (FIG. 2). As can be seen from Table 3, the root tip of Coix lacryma-jobi can induce callus at 2,4-D concentration of 1-6 mg/L. The healing rate was the highest at 1mg/L and 2mg/L of 2,4-D, and was 96.67%, after which the rate showed a regular decline with increasing 2,4-D concentration, and the healing rate was the lowest at 6mg/L of 2,4-D, and was 11.67%. In addition, embryogenic callus was not directly obtained using the root tip.
TABLE 3 Induction of root tip callus of Coix lacryma-jobi
After 20 days, the callus is inoculated into the same induction culture medium for subculture, and the callus is gradually browned and died, which indicates that the root of the coix can only induce the callus, but the callus can not be subcultured.
2.3 Induction of embryogenic callus by Stem
When the aseptic seedlings grow to 8-10cm high, cutting stems into small sections of about 0.5cm by using aseptic scissors and tweezers, inoculating the small sections onto induction culture media with different 2,4-D concentrations, inoculating 100 sections of each culture medium, inoculating 10 sections of each culture dish, repeating the steps for 3 times, and counting the healing rate and the embryogenic callus rate after 30 days.
As can be seen from FIG. 3, the stem of Coix lacryma-jobi was inoculated in the induction medium for 7 days, and the inside of part of the stem was elongated, and after 20 days, the elongated part began to expand to grow callus, and the part of the stem end which was not elongated also expanded to grow callus. The callus induced by the stem was yellowish, coarse granular, and the inside of the callus was a solid undifferentiated shoot (fig. 3). Within the concentration gradient of 1-3 mg/L2,4-D, the healing rate is gradually increased along with the increase of the concentration of 2, 4-D; within the concentration gradient of 3-6mg/L2,4-D, the callus rate gradually decreased with the increase of 2,4-D concentration, wherein the induction rate of the callus of the coix stem was the largest at the concentration of 3 mg/L2,4-D, and the callus rate was 26.0% (Table 4).
The callus is transferred to the same induction culture medium for subculture, most of the callus is gradually browned and died, few of the callus is gradually enlarged, tender stems which are not dedifferentiated are found inside by cutting with a scalpel, only the surface part of the callus is callus, the callus is peeled off and continuously cultured, and the callus is gradually browned and died, which shows that the stem of the coix lacryma-jobi cannot induce the callus which can be subcultured. In addition, embryogenic callus was not directly obtained from the stems.
TABLE 4 Induction of Coix lacryma-jobi Stem callus
2.4 Induction of embryogenic callus by aerial rooting
Taking 20D aseptic seedlings of the coix lacryma-jobi, cutting off the main roots, inducing the growth of the aerial roots, cutting the aerial roots into small sections of about 0.5cm, inoculating the small sections to induction culture mediums with different 2,4-D concentrations, inoculating 100 sections to each culture medium, inoculating 10 sections to each culture dish, repeating the steps for 3 times, and counting the healing rate and the embryogenic callus rate after 30D.
The results showed that, similar to the major root of Job's tears, after the aerial root was cultured on the induction medium for 7 days, one end of the root expanded to form a pale yellow callus with wet surface, and after 20 days, the callus did not grow into pale yellow, and the surface of the root appeared to be fine granular structure, and the root was gently sandwiched with tweezers and scattered, and the interior was white and transparent (FIG. 4).
As can be seen from Table 5, in the concentration range of 2,4-D used in the present invention, aerial roots of Coix lacryma-jobi all induced callus, but the callus rate was greatly different in different media, the callus rate was the highest at a concentration of 1 mg/L2,4-D in A1 medium and 52%, and the callus rate was the lowest at a concentration of 5 mg/L2,4-D in A5 medium and 8%.
TABLE 5 Induction of Coix lacryma-jobi aerial root callus
Subculturing the callus, and finding that after subculturing for 5d, the callus begins to brown, gradually dies and loses the subculture capability. In addition, embryogenic callus was not directly obtained using aerial roots.
2.5 Induction of embryogenic callus by shoots
And (3) taking buds about 0.5cm after coix seeds germinate (4 days after germination), inoculating the buds to induction culture media with different 2,4-D concentrations, inoculating 100 buds to each culture medium, inoculating 10 sections of buds to each culture dish, repeating the steps for 3 days, and counting the healing rate and the embryogenic callus rate after 30 days.
As can be seen from Table 7, all 5 induction media used induced callus. The healing rate shows the regular change of the decline with the rise of the concentration of 2,4-D, wherein the healing rate is the highest and is 79.33 percent when the concentration of 2,4-D in the A1 culture medium is 1 mg/L; in the A5 culture medium, when the concentration of 2,4-D is 1mg/L and 6mg/L, the healing rate is the lowest and is 46 percent. The five induction culture mediums can induce embryogenic callus except that the A5 culture medium can not induce embryogenic callus, and the A1, A2, A3 and A4 culture mediums can induce embryogenic callus, wherein the embryogenic callus induction rate of the A1 culture medium is 18.67%. The resulting callus is shown in FIG. 5.
The A1, A2, A3 and A4 callus induction culture media specifically comprise a solvent and a solute, the solvent is an MS culture medium, the solute is NAA and 2,4-D, the concentration of NAA in A1, A2, A3 and A4 callus induction culture media is 1mg/L, the concentration of 2,4-D in A1, A2, A3 and A4 callus induction culture media is 1mg/L, 2mg/L, 3mg/L and 4mg/L respectively, and the pH value is 5.8-6.0.
TABLE 6 Induction of callus of Coix lacryma-jobi bud
Second, the Effect of anti-browning agent on the subculture of embryogenic callus
The inventors studied the effect of different anti-browning agents (vitamin C, copper sulfate and silver nitrate) on the embryogenic callus of Coix lacryma-jobi. The media used were as follows:
c0 medium: a1 induction medium in step one.
C1 medium: the culture medium obtained by adding vitamin C to C0 culture medium, wherein the addition amount of vitamin C is 2mg/L culture medium.
C2 medium: the culture medium obtained by adding copper sulfate to the C0 culture medium was added in an amount of 0.85 mg/L.
C3 medium: the culture medium was prepared by adding silver nitrate to C0 culture medium, and the amount of silver nitrate added was 0.85mg/L culture medium.
And (3) culturing the embryogenic callus (showing light yellow color and soft texture) obtained in the first step in the four culture media respectively, wherein the inoculation amount of the callus on each culture medium is 10, the length of the inoculated callus is 0.5cm, and after culturing for 20 days, observing the growth condition of the callus, and counting the survival number and the survival length (namely the length in the initial length direction). The culture conditions used were: the culture was carried out under light at 3000lx and at 27 ℃ in the dark.
As can be seen from Table 7 and FIG. 6, vitamin C has no obvious effect on inhibiting callus browning of Coix lacryma-jobi and improving callus state (C1 in FIG. 6), while copper sulfate and silver nitrate obviously increase survival rate of callus and effectively improve callus state (C2 and C3 in FIG. 6). The coix callus is dark in color before subculture, and is glossy and in the form of light yellow granules after being transferred into a subculture medium added with silver nitrate. Therefore, silver nitrate or copper sulfate can be added into the subculture medium, and the effects of reducing callus browning and improving callus state are achieved.
TABLE 7 Effect of different anti-browning agents on callus subculture
Third, redifferentiation of callus
Inoculating embryogenic callus induced by coix sprout to different differentiation culture media, inoculating 100 callus to each differentiation culture medium, inoculating 10 callus to each culture dish, and counting the survival number of callus, the length of callus, the number of callus at green point, the number of callus at bud and the growth condition of callus after 20 days.
The culture conditions used were: the illumination intensity is 3000lx, the temperature is 27 ℃, and the light cycle is 12 h/12 h in light/dark.
The culture medium used was D1-D18 differentiation medium, each differentiation medium was obtained by adding hormone to minimal medium 2, pH was 5.8-6.0, and the type and amount of hormone added to each differentiation medium are shown in Table 8. The basic culture medium 2 is a sterile culture medium obtained by adding the cefuroxime to the MS solid culture medium, and the content of the cefuroxime in each liter of the differentiation culture medium is 250 mg/L.
TABLE 8 callus differentiation Medium
Note: KT in Table 8 is kinetin.
As shown in Table 9, the embryogenic calli induced from coix sprouts on 17 differentiation media other than D10 differentiation media differentiated green spots and gradually expanded into granules, but only D18 differentiation media differentiated sprouts (FIG. 7). The differentiation of the coixenogenic callus is greatly influenced by different hormones and concentration combinations, and the D18 differentiation medium can be used for the differentiation of the coixenogenic callus. The D18 differentiation medium is composed of solvent and solute, the solvent is MS medium, the solute and its concentration in D18 differentiation medium are respectively cefamycin 250mg/L, 6-BA 2mg/L and kinetin 2mg/L, pH value is 5.8-6.0.
TABLE 9 differentiation of Coix lacryma-jobi bud callus on different differentiation media
Fourth, the coix seed regenerated bud takes root and smelts the seedling
Transferring to rooting culture medium for culturing when the bud in the differentiation culture medium is 2-3cm high under conditions of illumination intensity of 3000lx, temperature of 27 deg.C, and photoperiod of 16 h/8 h in darkness for 7d to root, growing into complete plant, hardening, and transplanting to soil to obtain Coicis semen regenerated plant (FIG. 8).
The rooting culture medium is obtained by adding IBA into 1/2MS culture medium, wherein the concentration of IBA in the culture medium is 0.5mg/L, and the pH value is 5.8-6.0. 1/2 the MS culture medium is obtained by halving the concentration of each solute in the MS culture medium.
Claims (8)
1. The preparation method of the coix seed regeneration plant comprises the following steps:
1) culturing coix sprout on callus induction culture medium to obtain embryonic callus; the callus induction culture medium consists of an MS culture medium, 1mg/L NAA and 1 mg/L2,4-D, and the pH value is 5.8-6.0; the culture conditions were: the illumination intensity is 3000-3500lx, the temperature is 25-28 ℃, and the light period is 16 h/8 h of illumination/darkness;
2) culturing the embryonic callus on a differentiation culture medium to obtain a regeneration bud; the differentiation culture medium consists of an MS culture medium, 250mg/L of cefuroxime, 2mg/L of 6-BA and 2mg/L of kinetin, and the pH value is 5.8-6.0; the culture conditions were: the illumination intensity is 3000-3500lx, the temperature is 25-28 ℃, and the light period is illumination 12 h/darkness 12 h;
3) culturing the regeneration bud on a rooting culture medium to obtain a coix seed regeneration plant; the rooting culture medium consists of 1/2MS culture medium and 0.5mg/L IBA, and the pH value is 5.8-6.0; the 1/2MS culture medium is obtained by halving the concentration of each solute in the MS culture medium; the culture conditions were: the illumination intensity is 3000-3500lx, the temperature is 25-28 ℃, and the light period is 16 h/8 h.
2. The method of claim 1, wherein: the coix seed sprout is obtained by germinating coix seed.
3. The method of claim 1, wherein: the length of the coix sprout is 0.5 cm.
4. A method according to any one of claims 1-3, characterized in that: the method further comprises subculturing the embryogenic callus.
5. The method of claim 4, wherein: the subculture is carried out on the callus induction medium or subculture medium; the subculture medium is obtained by adding copper sulfate or silver nitrate into the callus induction culture medium.
6. The method of claim 5, wherein: the addition amount of copper sulfate or silver nitrate in the subculture medium is 0.85mg/L of the culture medium.
7. The induction method of the coix seed embryonic callus comprises the following steps: culturing said coix sprout on said callus induction medium of claim 1 to obtain coix embryogenic callus.
8. Use of the method of any one of claims 1-7 for genetic transformation of coix.
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| CN110199877A (en) * | 2019-07-04 | 2019-09-06 | 安徽省农业科学院棉花研究所 | A method of the stable homozygous coix lacryma-jobi resource of phenotype is obtained by microspores culture |
| CN110218738A (en) * | 2019-07-04 | 2019-09-10 | 安徽省农业科学院棉花研究所 | A kind of method of antiweed coix lacryma-jobi resource acquisition |
| CN110199878A (en) * | 2019-07-04 | 2019-09-06 | 安徽省农业科学院棉花研究所 | A kind of abductive approach of the embryo callus of coix lacryma-jobi |
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