CN115885850A - Tissue culture medium for regeneration of old mango wheat and tissue culture method for regeneration of mature embryo of old mango wheat - Google Patents
Tissue culture medium for regeneration of old mango wheat and tissue culture method for regeneration of mature embryo of old mango wheat Download PDFInfo
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Abstract
The invention belongs to the technical field of plant cultivation, and particularly relates to a dedifferentiation culture medium for old mango regeneration and a mature embryo regeneration tissue culture method for old mango wheat. The invention provides a dedifferentiation culture medium, wherein dicamba and thidiazuron are added into the dedifferentiation culture medium, so that the embryogenesis of the old miscanthus tissue is improved, the differentiation rate of the callus is improved, and the regeneration tissue culture time of the mature embryo of the old miscanthus is shortened, thereby establishing a high-regeneration-frequency old miscanthus tissue culture regeneration system. The results of the examples show that: the dedifferentiation culture medium can improve the differentiation rate of the callus, obtain robust awn wheat plant seedlings and provide a large number of seedlings for artificial cultivation of awn wheat.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a tissue culture medium for regeneration of old mango wheat and a tissue culture method for regeneration of mature embryos of old mango wheat.
Background
Old mango (Elymussibiricas), also known as Dahurian wheatgrass, siberian shavings, is a perennial herb of the genus Togae of the family Gramineae. The Amberryptium pratense is widely distributed in northeast, northwest and Qinghai-Tibet plateaus of China, and is a dominant species and a group-building species in meadow steppes and meadow communities. The old mango wheat is widely cultivated as excellent pasture due to strong tillering capacity, good cold resistance and drought resistance, high yield and good palatability. At present, the variety of the old awn wheat in China is deficient, and the high grain-dropping property causes that the seed yield of the old awn wheat is difficult to meet the production requirement. At present, an efficient genetic transformation platform of Miscanthus tragopus is urgently needed to be established, and a foundation is laid for developing gene editing and molecular design breeding of key genes.
The establishment of a mature tissue culture regeneration system is the key for establishing a genetic transformation system, the more recent old mango tissue culture regeneration system takes young ears as explants, but the young ears as the explants are difficult to obtain materials, and the development conditions are inconsistent under different planting conditions and different material-obtaining times, so that the experimental repeatability is poor, while the selection of the mature seed embryo as the explant is convenient to obtain materials and good in experimental repeatability, but the old mango callus differentiation rate is low when the mature seed embryo is taken as the explant for tissue culture at present (Du Pengfei and the like, the establishment of a gene gun-mediated old mango genetic transformation system [ J ]. Plant science and report, 2021,56 (01): 62-70).
Disclosure of Invention
In view of the technical problems, the invention aims to provide a dedifferentiation culture medium for tissue culture and regeneration of the old mango wheat, and the tissue culture medium provided by the invention is applied to tissue culture and regeneration seedling of the old mango wheat, so that the callus differentiation rate is high.
In order to solve the above problems, the present invention provides the following technical solutions:
the invention provides a dedifferentiation culture medium for regeneration of old awn wheat, which further comprises the following components in concentration on the basis of a basic culture medium: 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron.
Preferably, the dedifferentiation medium further comprises the following components in concentration on the basis of a basal medium: maltose 28-35 g/L.
Preferably, the dedifferentiation medium takes an N6 medium as a basic medium, and further comprises the following components in concentration: 28-35 g/L of maltose, 3-5 mg/L of dicamba, 0.2-0.3 mg/L of thidiazuron and 6.5-7.5 g/L of agar.
Preferably, the food also comprises proline 1100-1200 mg/L and casein 270-330 mg/L.
The invention provides a regeneration tissue culture method for mature embryos of old mango wheat, which comprises the following steps:
inoculating the sterile aweto explant to a dedifferentiation culture medium in any one of the technical schemes for dedifferentiation culture to obtain aweto callus differentiated to generate regeneration buds; the length of the regeneration bud is 0.2-0.8 cm;
transferring the callus of the old awn wheat from which the regeneration bud is differentiated to a rooting culture medium for rooting culture to obtain the regenerated seedling of the old awn wheat;
and carrying out acclimatization culture on the regenerated old mango wheat seedlings to obtain old mango wheat plant seedlings.
Preferably, the explant comprises mature embryo of Miscanthus somnifera seed.
Preferably, the preparation method of the mature embryo comprises the following steps: and transversely cutting off endosperm of the old mango wheat seeds to obtain mature embryos of the old mango wheat seeds.
Preferably, the rooting medium is based on a barley rooting medium, and further comprises 28-32 g/L of maltose and 3-4 g/L of plant gel.
Preferably, the temperature cycle of the dedifferentiation culture is that the dedifferentiation culture is carried out for 15.5 to 16.5 hours at the temperature of 24 to 26 ℃ every day, and then the dedifferentiation culture is carried out for 7.5 to 8.5 hours at the temperature of 20 to 22 ℃; the dedifferentiation culture is dark culture, and the time of the dedifferentiation culture is 33-45 days.
Preferably, the temperature of the domestication culture is 23-27 ℃, the domestication culture is carried out under the light and dark alternate condition, the light culture time is 15-17 h, and the dark culture time is 7-9 h.
The invention has the beneficial effects that: the invention provides a dedifferentiation culture medium for regeneration of old mango wheat, which further comprises the following components in concentration on the basis of a basic culture medium: 3 to 5mg/L of dicamba and 0.2 to 0.3mg/L of thidiazuron. In the dedifferentiation culture medium provided by the invention, dicamba and thidiazuron are added in the dedifferentiation culture medium to improve the differentiation rate of the callus. The results of the examples show that: the dedifferentiation culture medium can improve the differentiation efficiency of the callus.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.
FIG. 1 is a cross-cut mature embryo of a mature barley seed of comparative example 2 with endosperm-retaining mature embryo and scutellum portions removed;
FIG. 2 is the differentiation and rooting profiles of non-browned callus of example 1;
FIG. 3 shows the Miscanthus perenniana differentiated regenerated plantlets of example 1;
FIG. 4 shows the regenerated seedlings of Miscanthus stramineus of example 1;
FIG. 5 is a diagram showing the acclimated culture of the regenerated seedlings of Miscanthus stramineus of example 1;
FIG. 6 shows the cross-section of the mature embryo and scutellum of the old mango wheat seed in example 1 with the endosperm remaining.
Detailed Description
The invention provides a dedifferentiation culture medium for regeneration of old awn wheat, which further comprises the following components in concentration on the basis of a basic culture medium: 3 to 5mg/L of dicamba and 0.2 to 0.3mg/L of thidiazuron.
The dedifferentiation medium of the invention preferably comprises the following components in concentration on the basis of a basic medium: 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron; preferably further comprising the following components in the following concentrations: 28-35 g/L of maltose, 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron; further preferred are compositions also comprising the following concentrations: 28-35 g/L of maltose, 3-5 mg/L of dicamba, 0.2-0.3 mg/L of thidiazuron and 6.5-7.5 g/L of agar, and more preferably further comprises the following components in concentration: 28-35 g/L of maltose, 3-5 mg/L of dicamba, 0.2-0.3 mg/L of thidiazuron, 1100-1200 mg/L of proline, 270-330 mg/L of casein and 6.5-7.5 g/L of agar.
In the present invention, the dedifferentiation medium more preferably contains, in addition to the basic medium, only 3 to 5mg/L of dicamba and 0.2 to 0.3mg/L of thidiazuron, or further contains only 28 to 35g/L of maltose, 3 to 5mg/L of dicamba, 0.2 to 0.3mg/L of thidiazuron and 6.5 to 7.5g/L of agar, or further contains only 28 to 35g/L of maltose, 3 to 5mg/L of dicamba, 0.2 to 0.3mg/L of thidiazuron, 1100 to 1200mg/L of proline, 270 to 330mg/L of casein and 6.5 to 7.5g/L of agar. The dedifferentiation medium of the present invention contains dicamba at a concentration of 3 to 5mg/L, preferably 3.5 to 4.5mg/L, and more preferably 4mg/L. The dedifferentiation medium provided by the invention comprises thidiazuron with the concentration of 0.2-0.3 mg/L, preferably 0.22-0.27 mg/L, and more preferably 0.25mg/L. The dedifferentiation medium provided by the present invention preferably contains maltose at a concentration of 28 to 35g/L, more preferably 29 to 33g/L, and still more preferably 30g/L. The dedifferentiation medium provided by the invention comprises agar with the concentration of 6.5-7.5 g/L, and more preferably 7g/L. The dicamba and the thidiazuron in the dedifferentiation culture medium can improve the embryogenesis of the callus of the Miscanthus stramonium and improve the differentiation rate of the callus.
In the present invention, the basal medium is preferably N6 medium; the basic culture medium is further preferably an improved N6 culture medium, and the improved N6 culture medium is preferably prepared by adding proline 1100-1200 mg/L and casein 270-330 mg/L to the N6 culture medium.
The improved N6 culture medium preferably comprises 2.8g/L of potassium nitrate, 0.463g/L of ammonium sulfate, 0.4g/L of monopotassium phosphate, 0.185g/L of magnesium sulfate heptahydrate, 0.165g/L of calcium chloride dihydrate, 37.3mg/L of ethylene diamine tetraacetic acid disodium, 27.8mg/L of ferrous sulfate heptahydrate, 4.4mg/L of manganese sulfate monohydrate, 1.5mg/L of zinc sulfate heptahydrate, 1.6mg/L of boric acid, 0.8mg/L of potassium iodide, 0.01mg/L of sodium molybdate dihydrate, 0.01mg/L of cobalt chloride hexahydrate, 1.25mg/L of copper sulfate pentahydrate, 10mg/L of thiamine hydrochloride, 1mg/L of pyridoxine hydrochloride, 1mg/L of nicotinic acid, 2mg/L of glycine, 1100-1200 mg/L and 270-330 mg/L of casein.
The improved N6 culture medium provided by the invention preferably comprises casein with the concentration of 270-330 mg/L, more preferably 285-315 mg/L, and even more preferably 300mg/L.
The improved N6 culture medium provided by the invention preferably comprises proline with the concentration of 1100-1200 mg/L, and more preferably 1150mg/L.
In the present invention, the source of each component in the above-mentioned medium is not particularly limited, and a conventional commercially available product may be used unless otherwise specified. The maltose is applied as a carbon source in a dedifferentiation culture medium, a regeneration culture medium and a rooting culture medium, and the maltose can reduce the browning of callus.
The invention also provides a mature embryo regeneration tissue culture method of the old mango wheat, which comprises the following steps:
inoculating the sterile aweto explant to the dedifferentiation culture medium in the technical scheme for dedifferentiation culture to obtain an aweto callus with differentiated regeneration buds;
transferring the callus of the old aweto from which the regeneration buds are differentiated to a rooting culture medium for rooting culture to obtain the regenerated seedling of the old aweto; the length of the regeneration bud is 0.2-0.8 cm;
and carrying out acclimatization culture on the regenerated old mango wheat seedlings to obtain old mango wheat plant seedlings.
The invention inoculates the awn sterile explant in a dedifferentiation culture medium for dedifferentiation culture to obtain awn callus differentiated to regenerate buds.
In the present invention, the old mango wheat explant preferably comprises the mature embryo of the old mango wheat seed. The preparation method of the mature embryo of the invention is preferably as follows: and transversely cutting off endosperm of the old mango seeds to obtain mature embryos of the old mango seeds, more preferably transversely cutting off endosperm of the old mango seeds, and keeping mature embryos and mature embryo of the old mango seeds of the scutellum part.
The Miscanthus floridulus seeds are preferably full seeds which are not mildewed and are stored at low temperature. The Amur wheat seeds of the present invention are preferably treated at a temperature of 4 ℃ for one week before cutting off the endosperm. The invention takes the mature embryo of old mango wheat with transected endosperm as an explant to carry out regenerative tissue culture, because the mature embryo of old mango wheat has the capacity of differentiating embryonic callus, and the embryo cutting treatment solves the problem that the mature embryo germinates in a culture medium.
Before inoculating the sterile Miscanthus stramineus explant, the invention preferably further comprises sterilizing the explant. The sterilization mode is not specially limited, preferably, after cleaning, the aweto explant is sterilized by alcohol with the volume concentration of 70-75% for 55-65 s, and is washed by sterile water for 3-4 times, and then is sterilized by sodium hypochlorite solution with the mass concentration of 30% for 19-21 min, and is washed by sterile water for 5-6 times; more preferably, the mixture is sterilized by alcohol with the volume concentration of 70 percent for 60s, washed by sterile water for 3 times, sterilized by sodium hypochlorite solution with the mass concentration of 30 percent for 20min and washed by sterile water for 5 times.
The present invention is not particularly limited to sterile water, and is preferably obtained by sterilizing distilled water; the alcohol and sodium hypochlorite solution are preferably used in an amount to submerge the explant. The invention preferably drops a drop of tween-20 in the sodium hypochlorite solution and gently shakes for standby.
After the sterile explant of the old awn wheat is obtained, the mature embryo and scutellum part of the old awn wheat are inoculated in a dedifferentiation culture medium for dedifferentiation culture. In the inoculation process, the mature embryo scutellum of the old mango wheat is preferably downwards and directly contacted with a dedifferentiation culture medium.
The dedifferentiation culture is preferably dark culture, and the dark culture is favorable for generating callus by mature embryos of old mango wheat.
The temperature cycle of the dedifferentiation culture is preferably 15.5 to 16.5 hours of culture at 24 to 26 ℃ per day and 7.5 to 8.5 hours of culture at 20 to 22 ℃. The temperature cycle of the dedifferentiation culture is preferably 15.5 to 16.5 hours of culture at 24 to 26 ℃ per day, and more preferably 16 hours of culture at 25 ℃ per day; the temperature cycle of the dedifferentiation culture of the present invention is preferably 7.5 to 8.5 hours per day at 20 to 22 ℃, and more preferably 8 hours per day at 21 ℃.
The temperature cycle of the dedifferentiation culture of the invention is preferably repeated every day until callus with differentiated regeneration buds is obtained and the dedifferentiation culture is finished.
The time for the dedifferentiation culture according to the present invention is preferably 33 to 45 days, more preferably 35 to 42 days, and still more preferably 40 days.
The dedifferentiation medium according to the invention has already been discussed above and will not be described in detail here.
In the prior art, in a mature embryo tissue culture regeneration system of old mango wheat, MS culture medium is taken as a basic culture medium in a dedifferentiation culture medium, 2,4-D is selectively added, and the differentiation rate of callus is low. According to the invention, dicamba and thidiazuron are added into the dedifferentiation culture medium, so that the differentiation efficiency of the callus is improved.
After the callus of the Miscanthus strawberries with the differentiated regeneration buds is obtained, the callus of the Miscanthus strawberries with the differentiated regeneration buds is transferred to a rooting culture medium for rooting culture, and the Miscanthus strawberries regeneration seedlings are obtained. The length of the regenerated bud is 0.2-0.8 cm, and more preferably 0.3-0.7 cm.
In the invention, the rooting culture medium is based on a barley rooting culture medium and also comprises maltose 28 ～ 32g/L, plant gel 3 ～ 4g/L。
The barley rooting medium preferably comprises 16.5g/L of ammonium nitrate, 3.7g/L of magnesium sulfate heptahydrate, 3.32g/L of calcium chloride, 19g/L of potassium nitrate, 1.7g/L of potassium dihydrogen phosphate, 1.25mg/L of copper sulfate pentahydrate, 22.3mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.25mg/L of sodium molybdate dihydrate, 0.025mg/L of cobalt chloride hexahydrate, 0.1g/L of inositol, 0.4mg/L of thiamine hydrochloride and 0.75g/L of glutamine; more preferably, the ammonium nitrate solution only contains 16.5g/L of ammonium nitrate, 3.7g/L of magnesium sulfate heptahydrate, 3.32g/L of calcium chloride, 19g/L of potassium nitrate, 1.7g/L of monopotassium phosphate, 1.25mg/L of copper sulfate pentahydrate, 22.3mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.25mg/L of sodium molybdate dihydrate, 0.025mg/L of cobalt chloride hexahydrate, 0.1g/L of inositol, 0.4mg/L of thiamine hydrochloride and 0.75g/L of glutamine.
The rooting medium preferably comprises 16.5g/L of ammonium nitrate, 3.7g/L of magnesium sulfate heptahydrate, 3.32g/L of calcium chloride, 19g/L of potassium nitrate, 1.7g/L of potassium dihydrogen phosphate, 1.25mg/L of copper sulfate pentahydrate, 22.3mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.25mg/L of sodium molybdate dihydrate, 0.025mg/L of cobalt chloride hexahydrate, 0.1g/L of inositol, 0.4mg/L of thiamine hydrochloride, 0.75g/L of glutamine, 28-32 g/L of maltose and 3-4 g/L of plant gel; more preferably, the plant gel only contains 16.5g/L of ammonium nitrate, 3.7g/L of magnesium sulfate heptahydrate, 3.32g/L of calcium chloride, 19g/L of potassium nitrate, 1.7g/L of monopotassium phosphate, 1.25mg/L of copper sulfate pentahydrate, 22.3mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.25mg/L of sodium molybdate dihydrate, 0.025mg/L of cobalt chloride hexahydrate, 0.1g/L of inositol, 0.4mg/L of thiamine hydrochloride, 0.75g/L of glutamine, 28-32 g/L of maltose and 3-4 g/L of plant gel. The rooting medium comprises maltose with the concentration of 28-32 g/L, and more preferably 30g/L. The rooting medium comprises plant gel with the concentration of 3-4 g/L, and more preferably 3.5g/L. The plant gel has the function of solidifying the culture medium, and simultaneously, the hardness of the culture medium is lower than that of agar so as to be beneficial to the rooting of the callus.
In the present invention, the rooting culture is preferably performed under light and dark alternate conditions, and the light culture temperature of the rooting culture of the present invention is preferably 24 to 26 ℃, more preferably 25 ℃. The dark culture temperature for rooting culture in the invention is preferably 20-22 ℃, and more preferably 21 ℃. The light-dark alternating time is preferably 15-17 h of light culture and 7-9 h of dark culture, and more preferably 16h of light culture and 8h of dark culture; the intensity of the illumination is preferably 800 to 1200lux, more preferably 900 to 1100lux, and still more preferably 1000lux. In the present invention, the time for the rooting culture is preferably 21 to 28 days, more preferably 23 to 28 days, still more preferably 26 to 28 days, and still more preferably 28 days.
And (4) obtaining the regeneration seedling of the old mango wheat after rooting culture, and more preferably obtaining the robust regeneration seedling of the old mango wheat. The invention preferably performs acclimatization culture on the regenerated seedlings of the old mango wheat to obtain the old mango wheat plant seedlings. The acclimatization culture method is characterized in that the preferred old mango regenerated seedlings are adapted to a greenhouse at the temperature of 23-27 ℃ for one day, then the root culture medium of the old mango regenerated seedlings is flushed by running water, the roots of the old mango regenerated seedlings are soaked in sterile water for culture, and the roots are transferred to a substrate for acclimatization culture after absorbing the residual solid culture medium. The matrix of the present invention is preferably a matrix having a volume ratio of 3:1 vermiculite and nutrient soil composite matrix.
In the present invention, the temperature of the acclimatization culture is preferably 23 to 27 ℃, more preferably 22 to 26 ℃, and still more preferably 25 ℃.
In the invention, the domestication culture is preferably carried out under light-dark alternating conditions, the light-dark alternating time is preferably 15-17 h, the dark culture time is 7-9 h, and more preferably 16h and 8h; the light intensity for the light culture is preferably 2800 to 3200lux, more preferably 2900 to 3100lux, and still more preferably 3000lux. The time for the acclimatization culture of the present invention is preferably 7 to 8 days, and more preferably 7 days.
The acclimatization culture method is used for preferably selecting the old mango wheat plant seedlings with developed root systems.
The optimal concentration ranges of all components of the dedifferentiation medium and the rooting medium provided by the invention are controlled between 98% and 102% of the optimal concentration value, the component with the content of less than 200mg/L can be prepared into 100 times or 1000 times of mother liquor for dilution and then used, and the components which are easily decomposed at high temperature, such as copper sulfate, maltose, glutamine and dicamba, need to be filtered and sterilized and then added after being sterilized at high temperature and high pressure. The prepared culture medium needs to be placed until the condensed water is completely volatilized, and the storage life of the prepared culture medium at room temperature is two weeks.
The invention discloses a method for inducing callus formation by transversely cutting mature embryos, cutting endosperms off, placing the mature embryos in a dedifferentiation culture medium containing dicamba and thidiazuron, and simultaneously forming regeneration buds at the later stage of callus induction. Transferring the callus tissues differentiated to the regeneration buds to a rooting culture medium to form robust regeneration seedlings. In the rooting culture medium of the dedifferentiation culture medium provided by the invention, dicamba and thidiazuron are added, so that the embryogenesis of the callus of Miscanthus floridum is improved, and the differentiation rate of the callus is improved; the setting of the primary dedifferentiation culture medium, the rooting culture medium, the culture temperature, the culture illumination intensity and the light-dark alternate conditions provided by the invention is favorable for quickly obtaining the strong old mango wheat plant seedlings.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1 culture Medium
The components of the N6 culture medium are as follows: 2.8g/L potassium nitrate, 0.463g/L ammonium sulfate, 0.4g/L potassium dihydrogen phosphate, 0.185g/L magnesium sulfate heptahydrate, 0.165g/L calcium chloride dihydrate, 37.3mg/L disodium ethylene diamine tetraacetate, 27.8mg/L ferrous sulfate heptahydrate, 4.4mg/L manganese sulfate monohydrate, 1.5mg/L zinc sulfate heptahydrate, 1.6mg/L boric acid, 0.8mg/L potassium iodide, 0.01mg/L sodium molybdate dihydrate, 0.01mg/L cobalt chloride hexahydrate, 1.25mg/L copper sulfate pentahydrate, 10mg/L thiamine hydrochloride, 1mg/L pyridoxine hydrochloride, 1mg/L nicotinic acid and 2mg/L glycine.
The dedifferentiation culture medium comprises the following components: taking N6 culture medium as basic culture medium, proline 1150mg/L, casein 300mg/L, maltose 30g/L, dicamba 4mg/L, thidiazuron 0.25mg/L and agar 7g/L.
The barley rooting culture medium comprises the following components: 16.5g/L of ammonium nitrate, 3.7g/L of magnesium sulfate heptahydrate, 3.32g/L of calcium chloride, 19g/L of potassium nitrate, 1.7g/L of monopotassium phosphate, 1.25mg/L of copper sulfate pentahydrate, 22.3mg/L of manganese sulfate monohydrate, 8.6mg/L of zinc sulfate heptahydrate, 6.2mg/L of boric acid, 0.83mg/L of potassium iodide, 0.25mg/L of sodium molybdate dihydrate, 0.025mg/L of cobalt chloride hexahydrate, 0.1g/L of inositol, 0.4mg/L of thiamine hydrochloride and 0.75g/L of glutamine.
The rooting medium comprises the following components: the barley rooting medium, the maltose 30g/L and the plant gel are 3.5g/L.
The regeneration tissue culture method of the mature embryo of the old mango comprises the following steps:
(1) Obtaining seeds: in 9 months at 2021, the ripe Amur wheat seed No. 1 in North forest is harvested in Zhongxian county in Qinghai province, the Amur wheat seed is stored in a low-temperature dry mode, the thousand seed weight is 3.75g after the palea is removed, and the seed is treated for 7 days at the temperature of 4 ℃ before the beginning of culture.
(2) Obtaining an explant: soaking 200 Miscanthus stra seeds treated in the step (1) at the temperature of 4 ℃ in 70% ethanol for 1min, washing with sterile water for 3 times, adding sodium hypochlorite with the mass concentration of 30%, soaking for 20min, and washing with sterile water for 5 times. Placing the sterilized old mango wheat seeds on sterile dry filter paper for 20min to absorb water, transversely cutting off endosperm under a body type lens, and keeping mature embryo and scutellum parts of the old mango wheat seeds, wherein the mature embryo and scutellum parts of the old mango wheat seeds are shown in figure 6.
(3) And (3) dedifferentiation culture: and (3) in a dedifferentiation culture medium, directly contacting the mature embryo of the old aweto seed obtained in the step (2) downwards with the culture medium to induce the formation of callus, wherein 15 parallel experiments are arranged, 8 explants are cultured in each parallel experiment, the obtained mixture is placed in a tissue culture box to perform dedifferentiation culture under the dark condition, the temperature cycle of the dedifferentiation culture is 24-26 ℃ for 16h, then the culture is performed at 20-22 ℃ for 8h, and the temperature cycle is repeated every day until the culture is finished, so that the old aweto callus with differentiated regeneration buds is obtained. Callus formation was observed on all explants after 21d dedifferentiation dark culture, and browned calli accounted for 50 ± 9% of the total number of calli after 28d dedifferentiation medium culture. After culturing in dedifferentiation medium for 35d to 42d, 58. + -. 18% of calli in the non-browned calli were seen to have regenerated shoots, and differentiation of the non-browned calli is shown in FIG. 2.
(4) Rooting culture: transferring the aweto callus with the length of 0.2-0.8 cm obtained by differentiating the regenerated bud in the step (3) to a rooting culture medium for rooting culture.
Rooting culture is carried out in a tissue culture box, and the circulation period of the tissue culture box is set as follows: under the illumination condition, the temperature is 24-26 ℃, the illumination culture time is 16h, and the illumination intensity is 1000lux; the temperature is 20-22 ℃ under the dark condition, and the dark culture time is 8h. After 14-21 d of culture, all the calli of the differentiated regeneration buds can develop developed root systems, which is shown in figure 2, and robust regeneration seedlings can be seen in figure 3 after three to four weeks of culture.
(5) Domestication of regenerated seedlings: and (4) carrying out acclimation culture on the regenerated seedlings of the old mango wheat obtained in the step (6) in a greenhouse. Removing a sealing film of a culture bottle for the regenerated old mango wheat seedlings to allow the regenerated old mango wheat seedlings to adapt to a greenhouse for 1d, flushing agar by running water, slightly separating roots by using small tweezers, allowing the regenerated old mango wheat seedlings to be shown in figure 4, soaking roots of the regenerated seedlings in sterile water for culture to allow the roots to absorb residual solid culture medium, transplanting the roots into a vermiculite and nutrient soil (German Wo Duo nutrient soil) composite matrix with the volume ratio of 3:1 for acclimation culture, and allowing the regenerated old mango wheat seedlings in the acclimation culture process to be shown in figure 5. The temperature of the acclimatization culture is 23-27 ℃, the illumination culture time is 16h, the dark culture time is 8h, the illumination intensity is 3000lux, the acclimatization culture time is 1 week, and the old mango wheat plant seedling is obtained after the acclimatization culture.
Comparative example 1: composition change of dedifferentiation medium, transected endosperm of explants
1. Dedifferentiation medium
The dedifferentiation culture medium comprises the following components: potassium nitrate 2.8g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate heptahydrate 0.185g/L, calcium chloride dihydrate 0.165g/L, disodium ethylenediaminetetraacetate 37.3mg/L, ferrous sulfate heptahydrate 27.8mg/L, manganese sulfate monohydrate 4.4mg/L, zinc sulfate heptahydrate 1.5mg/L, boric acid 1.6mg/L, potassium iodide 0.8mg/L, sodium molybdate dihydrate 0.01mg/L, cobalt chloride hexahydrate 0.01mg/L, copper sulfate pentahydrate 1.25mg/L, thiamine hydrochloride 10mg/L, pyridoxine hydrochloride 1mg/L, nicotinic acid 1mg/L, glycine 2mg/L, proline 1150mg/L, casein 300mg/L, maltose 30g/L, dicamba 4mg/L, butyryl hydrazine 25mg/L and agar 7g/L.
2. The mature embryo of the old mango wheat is directly developed into a seedling from an embryo radicle without a dedifferentiation and redifferentiation stage:
(1) Obtaining seeds: the same as in example 1.
(2) Obtaining an explant: soaking 200 aweto seeds treated in the step (1) at the temperature of 4 ℃ in 70% ethanol for 1min, washing with sterile water for 3 times, adding sodium hypochlorite with the mass concentration of 30%, soaking for 20min, and washing with sterile water for 5 times. Placing the sterilized old mango wheat seeds on sterile dry filter paper for 20min to absorb water, transversely cutting off endosperm under a body type lens, and keeping mature embryo and scutellum parts of the old mango wheat seeds, wherein the mature embryo and scutellum parts of the old mango wheat seeds are shown in figure 6.
(3) And (3) dedifferentiation culture: the dedifferentiation culture medium used for dedifferentiation culture induces the formation of callus by directly contacting the mature embryo of the old mango wheat seed obtained in the step (2) downwards with the culture medium, and the number of the mature embryos is 12 in parallel, and each explant is cultured in parallel by 5.
Placing the mixture in a tissue culture box to perform dedifferentiation culture under the dark condition, wherein the temperature cycle of the dedifferentiation culture is 24-26 ℃ for 16h, then culturing the mixture at 20-22 ℃ for 8h, and repeating the temperature cycle every day until the culture is finished. After the mature wheat embryos are subjected to dedifferentiation and dark culture for 21d, all mature wheat embryos germinate and root, no callus is formed, the mature wheat embryos are cultured for about 42-49 d, and the mature wheat embryos are not subjected to dedifferentiation and redifferentiation stages, and the germ radicles germinate into the mature wheat seedlings.
Comparative example 2: the mature embryo is longitudinally cut and then transversely cut, and the endosperm is cut
(1) Obtaining seeds: the same as in example 1.
(2) Obtaining an explant: soaking 200 seeds in 70% ethanol for 1min, washing with sterile water for 3 times, adding sodium hypochlorite with the mass concentration of 30%, soaking for 20min, and washing with sterile water for 5 times. Placing the seeds on sterile dry filter paper for 20min, removing water, longitudinally cutting mature embryo under body type microscope, transversely cutting off endosperm, and keeping mature embryo and scutellum part, wherein the obtained mature embryo is shown in figure 1.
(3) Dedifferentiation culture: and (3) applying the dedifferentiation culture medium in the comparative example 1 to dedifferentiate and culturing 5 explants in parallel, wherein the mature embryo of the Amur aweto seed obtained in the step (2) is directly contacted with the culture medium downwards to induce the formation of callus. Placing the mixture in a tissue culture box to perform dedifferentiation culture under the dark condition, wherein the temperature cycle of the dedifferentiation culture is 24-26 ℃ for 16h, then culturing the mixture at 20-22 ℃ for 8h, and repeating the temperature cycle every day until the culture is finished. Callus formation was observed on all explants after 21d dedifferentiation and dark culture.
(4) Differentiation culture: transferring the callus obtained in the step (3) to a regeneration medium (the regeneration medium is the same as the example 1) for differentiation culture, wherein the differentiation culture is carried out in a tissue culture box, and the cycle period of the tissue culture box is set as follows: under the illumination condition, the temperature is 24-26 ℃, the illumination culture time is 16h, and the illumination intensity is 1000lux; the temperature is 20-22 ℃ under the dark condition, and the dark culture time is 8h. No callus differentiation was seen after 42 days of culture.
The callus browning rates and the callus regeneration efficiencies of examples 1 and comparative examples 1 to 2 were calculated according to the following formulas:
differentiation efficiency of callus = number of differentiated germinated calli/number of non-browned calli
TABLE 1 callus browning rates and callus regeneration efficiencies of example 1 and comparative examples 1 to 4
| Example 1 | Comparative example 1 | Comparative example 2 | |
| |
100％ | 0％ | 100％ |
| Browning rate of callus | 50±9％ | 0％ | 0％ |
| Differentiation rate of callus | 58±18％ | 0％ | 0％ |
In conclusion, the tissue culture method and the tissue culture medium can improve the regeneration efficiency of the callus and can obtain the robust seedlings of the old mango wheat plants.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.
Claims (10)
1. The dedifferentiation culture medium for regeneration of old awnlow is characterized by further comprising the following components in concentration on the basis of a basic culture medium: 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron.
2. The dedifferentiation medium according to claim 1, characterized in that it also comprises the following components in the following concentrations: 28-35 g/L of maltose.
3. The dedifferentiation medium according to claim 1 or 2, characterized in that it is a basal medium in N6 medium, further comprising the following components in the following concentrations: 28-35 g/L of maltose, 3-5 mg/L of dicamba, 0.2-0.3 mg/L of thidiazuron and 6.5-7.5 g/L of agar.
4. The dedifferentiation medium according to claim 3, further comprising proline 1100 to 1200mg/L and casein 270 to 330mg/L.
5. A mature embryo regeneration tissue culture method of Miscanthus floridulus is characterized by comprising the following steps:
inoculating a sterile aweto explant to the dedifferentiation culture medium of any one of claims 1 to 4 for dedifferentiation culture to obtain aweto callus differentiated to generate regenerated buds; the length of the regeneration bud is 0.2-0.8 cm;
transferring the callus of the old awn wheat from which the regeneration bud is differentiated to a rooting culture medium for rooting culture to obtain the regenerated seedling of the old awn wheat;
and carrying out acclimatization culture on the regenerated old mango wheat seedlings to obtain old mango wheat plant seedlings.
6. The tissue culture method of claim 5, wherein the explant comprises mature embryo of Amymus formosanus seed.
7. The tissue culture method of claim 6, wherein the mature embryo is prepared by the following steps: and transversely cutting off endosperm of the old mango wheat seeds to obtain mature embryos of the old mango wheat seeds.
8. The tissue culture method of claim 5, wherein the rooting medium is based on a barley rooting medium, and further comprises 28-32 g/L of maltose and 3-4 g/L of plant gel.
9. The tissue culture method according to claim 5, wherein the temperature cycle of the dedifferentiation culture is 15.5-16.5 h at 24-26 ℃ per day, and then 7.5-8.5 h at 20-22 ℃; the dedifferentiation culture is dark culture, and the time of the dedifferentiation culture is 33-45 days.
10. The tissue culture method according to claim 5, wherein the temperature of the domestication culture is 23-27 ℃, the domestication culture is carried out under the condition of light and dark alternation, the light culture time is 15-17 h, and the dark culture time is 7-9 h.
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