CN115885850B - Tissue culture medium for regenerating old mango and method for regenerating mature embryo of old mango - Google Patents
Tissue culture medium for regenerating old mango and method for regenerating mature embryo of old mango Download PDFInfo
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- 238000000034 method Methods 0.000 title claims description 8
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of plant cultivation, and particularly relates to a dedifferentiated medium for regenerating old mango and a tissue culture method for regenerating mature embryos of the old mango. The invention provides a dedifferentiation culture medium, wherein dicamba and thidiazuron are added into the dedifferentiation culture medium to improve embryology of old mango callus, improve differentiation rate of the callus, and shorten time for regenerating mature embryo of old mango, so that a high regeneration frequency old mango tissue culture regeneration system is established. The results of the examples show that: the dedifferentiation culture medium can improve the callus differentiation rate, obtain robust old mango plant seedlings and provide a large number of seedlings for artificial cultivation of the old mango.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a tissue culture medium for regenerating old mango and a tissue culture method for regenerating mature embryos of the old mango.
Background
The old mango (Elymussibiricus) is also called barley grass, siberian elymus, which is a perennial herb of the genus elymus of the family Gramineae. Old mango is widely distributed in northeast, north China, northwest, qinghai-Tibet plateau and other areas of China, and is a dominant species and colonisation species in meadow grasslands and meadow communities. The old mango has strong tillering power, good cold resistance and drought resistance, high yield and good palatability, and is widely cultivated as excellent pasture. At present, the domestic old mango seed is deficient in variety, and the high grain falling property causes that the seed yield of the old mango is difficult to meet the production requirement. At present, an efficient genetic transformation platform of the old mango 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 of establishing a genetic transformation system, the nearer tissue culture regeneration system of the old mango takes young spikes as explants, but the young spikes are difficult to obtain as the explants, and the development conditions are inconsistent under different planting conditions and different sampling times, so that the experimental repeatability is poor, the selection of a mature seed embryo as the explants is convenient, and the experimental repeatability is good, but the tissue culture of Shi Laomang wheat callus by taking the mature seed embryo as the explants has low differentiation rate (Du Pengfei and the like, and the establishment of the genetic transformation system of the old mango mediated by a gene gun [ J ]. Plant theory, 2021,56 (01): 62-70).
Disclosure of Invention
In view of the technical problems, the invention aims to provide a dedifferentiated medium for tissue culture regeneration of old mango, and the tissue culture medium provided by the invention is used for tissue culture regeneration seedling culture of the old mango, so that the callus differentiation rate is high.
In order to solve the problems, the invention provides the following technical scheme:
The invention provides a dedifferentiated medium for regenerating old mango, which is based on a basic medium and further comprises the following components in concentration: 3 to 5mg/L of dicamba and 0.2 to 0.3mg/L of thidiazuron.
Preferably, the dedifferentiation medium further comprises the following components in the following concentration on the basis of the basal medium: maltose 28-35 g/L.
Preferably, the dedifferentiation medium is based on an 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.
Preferably, the composition also comprises 1100-1200 mg/L of proline and 270-330 mg/L of casein.
The invention provides a method for regenerating and tissue-culturing mature embryo of old mango, which comprises the following steps:
inoculating the aseptic explant of the old mango seed to the dedifferentiation culture medium according to any one of the technical schemes for dedifferentiation culture to obtain the old mango callus with differentiated regeneration buds; the length of the regeneration bud is 0.2-0.8 cm;
transferring the callus of the old mango bud differentiated into a rooting medium for rooting culture to obtain the old mango seedling;
and (3) domesticating and culturing the regenerated seedlings of the old mango to obtain seedlings of the old mango plants.
Preferably, the explant comprises mature embryo of aged mango seed.
Preferably, the preparation method of the mature embryo comprises the following steps: and (5) transversely cutting endosperm of the old mango seeds to obtain mature embryo of the old mango seeds.
Preferably, the rooting culture medium is based on a barley rooting culture medium and further comprises 28-32 g/L maltose and 3-4 g/L plant gel.
Preferably, the temperature period of the dedifferentiation culture is 15.5-16.5 hours at 24-26 ℃ per day, and 7.5-8.5 hours at 20-22 ℃; the dedifferentiation culture is dark culture, and the dedifferentiation culture time is 33-45 d.
Preferably, 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.
The invention has the beneficial effects that: the invention provides a dedifferentiated medium for regenerating old mango, which is based on a basic medium and further comprises the following components in concentration: 3 to 5mg/L of dicamba and 0.2 to 0.3mg/L of thidiazuron. In the dedifferentiation medium provided by the invention, dicamba and thidiazuron are added into the dedifferentiation medium to improve the differentiation rate of the callus. The results of the examples show that: the dedifferentiation 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 that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a cross-cut of the mature embryo from the mature embryo and scutellum portion of comparative example 2 old mango seed;
FIG. 2 is a differentiation and rooting chart of the non-browned calli of example 1;
FIG. 3 is a differentiated regenerated seedling of old mango of example 1;
FIG. 4 is a regenerated seedling of old mango of example 1;
FIG. 5 is a domestication culture diagram of the regenerated seedlings of old mango of example 1;
FIG. 6 shows the cross-cut endosperm-cutting portion of mature embryo and scutellum of the old mango seed of example 1.
Detailed Description
The invention provides a dedifferentiated medium for regenerating old mango, which is based on a basic medium and further comprises the following components in concentration: 3 to 5mg/L of dicamba and 0.2 to 0.3mg/L of thidiazuron.
The dedifferentiation medium according to the invention is preferably based on a basal medium and comprises the following components in the following concentrations: 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron; preferably also comprises the following concentrations of components: 28-35 g/L of maltose, 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron; it is further preferred that the composition further comprises the following concentrations: 28-35 g/L maltose, 3-5 mg/L dicamba, 0.2-0.3 mg/L thidiazuron and 6.5-7.5 g/L agar, more preferably also 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 invention, the dedifferentiation medium more preferably contains 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron or 28-35 mg/L of maltose, 3-5 mg/L of dicamba and 0.2-0.3 mg/L of thidiazuron or 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 or 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 on the basis of the basic medium. The dedifferentiation medium of the present invention comprises dicamba at a concentration of 3 to 5mg/L, preferably 3.5 to 4.5mg/L, more preferably 4mg/L. The dedifferentiated medium provided by the invention comprises thidiazuron at a concentration of 0.2-0.3 mg/L, preferably 0.22-0.27 mg/L, more preferably 0.25mg/L. The dedifferentiated medium according to the present invention preferably comprises maltose at a concentration of 28 to 35g/L, more preferably 29 to 33g/L, still more preferably 30g/L. The dedifferentiation medium provided by the invention comprises agar with a concentration of 6.5-7.5 g/L, more preferably 7g/L. The dicamba and thidiazuron in the dedifferentiation medium can improve embryogenic property of old mango callus and improve differentiation rate of callus.
In the present invention, the basal medium is preferably an N6 medium; the basal medium is further preferably a modified N6 medium, and the modified N6 medium is preferably added with 1100-1200 mg/L of proline and 270-330 mg/L of casein on the basis of the N6 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 disodium ethylenediamine tetraacetate, 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 of proline and 270-330 mg/L of casein.
The modified N6 medium provided by the invention preferably comprises casein with a concentration of 270-330 mg/L, more preferably 285-315 mg/L, and even more preferably 300mg/L.
The modified N6 culture medium provided by the invention preferably comprises proline with the concentration of 1100-1200 mg/L, more preferably 1150mg/L.
The sources of the respective components in the above-mentioned medium are not particularly limited unless otherwise specified, and conventional commercially available products may be used. The maltose is applied as a carbon source in a dedifferentiation medium, a regeneration medium and a rooting medium, and can reduce the browning of the callus.
The invention also provides a method for regenerating and tissue-culturing mature embryo of old mango, which comprises the following steps:
inoculating the aseptic explant of the old mango seed to the dedifferentiation culture medium in the technical scheme for dedifferentiation culture to obtain the old mango seed callus with differentiated regeneration buds;
transferring the callus of the old mango bud differentiated to a rooting culture medium for rooting culture to obtain the old mango seedling; the length of the regeneration bud is 0.2-0.8 cm;
and (3) domesticating and culturing the regenerated seedlings of the old mango to obtain seedlings of the old mango plants.
The invention inoculates aseptic explant of old mango to dedifferentiation culture medium for dedifferentiation culture to obtain the old mango callus with differentiated regeneration bud.
In the present invention, the aged miscanthus seed explant preferably comprises an aged miscanthus seed mature embryo. The preparation method of the mature embryo of the invention preferably comprises the following steps: the mature embryo of the old mango seed is obtained after the endosperm is transected and cut off, more preferably the endosperm of the seed is transected and cut off by the old mango seed, and the mature embryo and the old mango seed mature embryo of the scutellum part are reserved.
The old mango seeds are preferably full seeds without mildew and are preserved under the low-temperature condition. The laomao seed of the present invention is preferably treated at a temperature of 4 ℃ for one week prior to endosperm removal. The invention uses mature embryo of old mango which transects endosperm as explant to carry out regeneration tissue culture, because the mature embryo of old mango has the capability of differentiating embryogenic callus, and meanwhile, the embryo cutting treatment solves the problem of germination of the mature embryo in a culture medium.
The present invention preferably further comprises sterilizing the explant prior to inoculating said sterile explant of laomang barley. The sterilization mode is not particularly limited, and preferably, after the sterilization mode is cleaned, the old mango explant is sterilized for 55-65 s by alcohol with the volume concentration of 70% -75%, sterilized by water for 3-4 times, sterilized by sodium hypochlorite solution with the mass concentration of 30% for 19-21 min, and sterilized by sterilized water for 5-6 times; more preferably, the method comprises sterilizing with 70% ethanol for 60s, washing with sterile water for 3 times, sterilizing with 30% sodium hypochlorite solution for 20min, and washing with sterile water for 5 times.
The invention is not particularly limited to sterile water, and is preferably obtained after sterilization of distilled water; the alcohol and sodium hypochlorite solutions of the invention are preferably used in amounts sufficient to submerge the explants. The invention preferably uses a drop of tween-20 added dropwise to sodium hypochlorite solution, and then shakes gently for later use.
After the aseptic explant of the old mango is obtained, the invention inoculates the mature embryo of the old mango and the scutellum part in a dedifferentiation culture medium for dedifferentiation culture. In the inoculation of the invention, the scutellum of mature embryo of old mango is preferably directly contacted with the dedifferentiation medium downwards.
The dedifferentiation culture is preferably dark culture, and the dark culture is favorable for the mature embryo of the old mango to produce the callus.
The temperature period of the dedifferentiation culture is preferably 15.5 to 16.5 hours at 24 to 26 ℃ per day, and 7.5 to 8.5 hours at 20 to 22 ℃. The temperature period of the dedifferentiation culture according to the present invention is preferably 15.5 to 16.5 hours per day at 24 to 26 ℃, more preferably 16 hours per day at 25 ℃; the temperature cycle of the dedifferentiation culture according to 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 according to the present invention is preferably repeated daily until the dedifferentiation culture is ended when the callus differentiated from the regenerated shoot is obtained.
The time of the dedifferentiation culture according to the present invention is preferably 33 to 45d, more preferably 35 to 42d, and still more preferably 40d.
The dedifferentiation media according to the present invention are already discussed above and are not described here again.
In the prior art, in the tissue culture regeneration system of mature embryo of old mango, the dedifferentiation culture medium is based on an MS culture medium, 2,4-D is selected and 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 old mango callus differentiated with the regeneration bud is obtained, the invention transfers the old mango callus differentiated with the regeneration bud into a rooting culture medium for rooting culture, so as to obtain the old mango regeneration seedling. The length of the regeneration bud according to the present invention is 0.2 to 0.8cm, more preferably 0.3 to 0.7cm.
In the invention, the rooting medium is based on barley rooting medium, and further comprises 28 ～ g/L maltose and 3 ～ g/L plant gel.
The barley rooting culture 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 monopotassium phosphate, 1.25mg/L of cupric 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, it contains only 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 cupric 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 culture 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 monopotassium phosphate, 1.25mg/L of cupric 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, 28-32 g/L of maltose and 3-4 g/L of plant gel; more preferably, the plant gel contains only 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 cupric 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, 28 to 32g/L of maltose and 3 to 4g/L of plant gel. The rooting culture medium comprises maltose with the concentration of 28-32 g/L, and more preferably 30g/L. The rooting culture 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 a solidification culture medium, and the hardness of the culture medium is lower than that of agar, so that the callus is favorable for rooting.
In the present invention, the rooting culture is preferably performed under alternate light and dark conditions, and the light culture temperature of the rooting culture is preferably 24-26 ℃, more preferably 25 ℃. The dark culture temperature of rooting culture is preferably 20-22 ℃, more preferably 21 ℃. The time of light and dark alternation is preferably 15-17 hours of dark culture for 7-9 hours of light culture, and more preferably 16 hours of dark culture for 8 hours of light culture; the intensity of the illumination is preferably 800 to 1200lux, more preferably 900 to 1100lux, and even more preferably 1000lux. In the present invention, the rooting culture time is preferably 21 to 28d, more preferably 23 to 28d, still more preferably 26 to 28d, and still more preferably 28d.
And obtaining regenerated seedlings of the old mango, more preferably, robust regenerated seedlings of the old mango after rooting culture. The invention preferably carries out domestication culture on the regenerated seedlings of the old mango to obtain the seedlings of the old mango plants. The domestication culture method is characterized in that the domestication culture method is that the regenerated seedlings of the old mango is preferably adapted to one day in a greenhouse with the temperature of 23-27 ℃, then root culture mediums are flushed out of the regenerated seedlings of the old mango by using running water, the roots of the regenerated seedlings of the old mango are soaked in sterile water for culture, and the roots are transferred into a substrate for domestication culture after absorbing residual solid culture mediums. The matrix according to the invention preferably has a volume ratio of 3:1 and nutrient soil.
In the present invention, the temperature of the domestication culture is preferably 23 to 27 ℃, more preferably 22 to 26 ℃, and even more preferably 25 ℃.
In the invention, the domestication culture is preferably performed under a light-dark alternation condition, wherein the light-dark alternation time is preferably 15-17 hours of light culture time, 7-9 hours of dark culture time, and more preferably 16 hours of light culture and 8 hours of dark culture; the illumination intensity of the illumination culture is preferably 2800 to 3200lux, more preferably 2900 to 3100lux, and still more preferably 3000lux. The time for the domestication culture according to the present invention is preferably 7 to 8 days, more preferably 7 days.
The domestication culture of the invention is preferably performed on the old mango seedlings with developed root systems.
The optimal concentration ranges of all components of the dedifferentiation medium and the rooting medium are controlled between 98% -102% of the optimal concentration values, the components with the content less than 200mg/L can be firstly prepared into mother liquor with the content of 100 times or 1000 times for dilution and then used, and components which are easy to decompose at high temperature, such as copper sulfate, maltose, glutamine and dicamba, need to be filtered and sterilized and are added after high-temperature and high-pressure sterilization. After the culture medium is prepared, the culture medium can be used after the condensed water is completely volatilized, and the storage life of the prepared culture medium at room temperature is two weeks.
The invention transversely cuts mature embryo, cuts endosperm, and places the mature embryo in dedifferentiated medium containing dicamba and thidiazuron to induce callus formation, and at the same time, a regeneration bud can be formed in the later stage of callus induction. Transferring the callus differentiated from the regeneration buds to a rooting culture medium to form robust regeneration seedlings. In the dedifferentiation medium rooting medium provided by the invention, dicamba and thidiazuron are added to improve embryogenic property of old mango callus and increase differentiation rate of callus; the setting of the primary dedifferentiation culture medium, the rooting culture medium, the culture temperature, the culture illumination intensity and the light-dark alternation condition provided by the invention is favorable for quickly obtaining robust old mango seedlings.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1 Medium
The N6 culture medium comprises the following components: 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 disodium ethylenediamine tetraacetate, 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 and 2mg/L of glycine.
The dedifferentiation medium comprises the following components: n6 medium is used as basic medium, 1150mg/L of proline, 300mg/L of casein, 30g/L of maltose, 4mg/L of dicamba, 0.25mg/L of thidiazuron and 7g/L of agar.
The barley rooting 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 cupric 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 culture medium comprises the following components: barley rooting medium, 30g/L maltose and 3.5g/L plant gel.
The 2-step method for regenerating and tissue-culturing mature embryo of old mango comprises the following steps:
(1) Seed acquisition: and harvesting full and mildew-free old mango seeds of North forest No. 1 in Huangzhong county of Qinghai province in 2021, drying and preserving the old mango seeds at a low temperature, wherein the thousand seed weight of the seeds after the palea is removed is 3.75g, and treating the seeds for 7d at the temperature of 4 ℃ before starting culture.
(2) Obtaining an explant: soaking 200 grains of the aged mango seeds treated in the step (1) at the temperature of 4 ℃ for 1min with 70% ethanol, washing 3 times with sterile water, adding sodium hypochlorite with the mass concentration of 30% for soaking 20min, and washing 5 times with sterile water. Placing sterilized old mango seeds on sterile dry filter paper for 20min to suck seed water, transversely cutting endosperm under a split mirror, and reserving mature embryo and scutellum parts of the old mango seeds, wherein the mature embryo and scutellum parts of the old mango seeds are shown in fig. 6.
(3) Dedifferentiation culture: in the dedifferentiation culture medium, mature embryo of the old mango seed obtained in the step (2) is directly contacted with the culture medium downwards to induce the formation of callus, 15 parallel experiments are provided, 8 explants are cultured in each parallel experiment, the culture is carried out in a tissue culture box under the dark condition for dedifferentiation culture, the dedifferentiation culture temperature period is 24-26 ℃ for 16h, the culture is carried out at 20-22 ℃ for 8h, and the temperature period is repeated every day until the culture is finished, so that the old mango callus with differentiated regeneration buds is obtained. Callus formation was observed in all explants after 21d of dedifferentiated dark culture, and after 28d of dedifferentiated medium culture, the browned callus accounted for 50±9% of the total callus number. It was found that 58.+ -.18% of the calli in the non-brown calli showed regeneration buds after 35d to 42d culture in the dedifferentiating medium, and differentiation of the non-brown calli was shown in FIG. 2.
(4) Rooting culture: transferring the old mango callus with the length of 0.2-0.8 cm obtained from the step (3) to a rooting medium for rooting culture.
Rooting 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 16 hours, and the illumination intensity is 1000lux; the temperature is 20-22 ℃ under the dark condition, and the time of dark culture is 8 hours. After 14-21 d culture, the calli of all differentiated regenerated buds can develop developed root systems as shown in figure 2, and the robust regenerated seedlings can be cultured for three to four weeks as shown in figure 3.
(5) Domestication of regenerated seedlings: and (3) carrying out domestication culture on the aged miscanthus sinensis regenerated seedlings obtained in the step (6) in a greenhouse. Removing a sealing film of a culture bottle for the regenerated seedlings of the old mango, enabling the regenerated seedlings of the old mango to adapt to 1d in a greenhouse, flushing agar with flowing water, slightly separating roots by using small tweezers, enabling the regenerated seedlings of the old mango to see a graph 4, immersing the roots of the regenerated seedlings in sterile water for culture, enabling the roots to absorb residual solid culture medium, transplanting the roots into a vermiculite and nutrient soil (Devoduo nutrient soil) composite matrix with a volume ratio of 3:1 for domestication culture, and enabling the regenerated seedlings of the old mango in the domestication culture process to see a graph 5. The temperature of the domestication culture is 23-27 ℃, the illumination culture time is 16 hours, the dark culture time is 8 hours, the illumination intensity is 3000lux, the domestication culture time is 1 week, and the aged mango seedlings are obtained after the domestication culture.
Comparative example 1: cross-cutting endosperm of explants with altered dedifferentiation medium composition
1. Dedifferentiation medium
The dedifferentiation medium comprises the following components: 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 disodium ethylenediamine tetraacetate, 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, 1150mg/L of proline, 300mg/L of casein, 30g/L of maltose, 4mg/L of dicamba, 25mg/L of hydrazine and 7g/L of agar.
2. Mature embryo of old mango is directly developed into seedling from embryo radicle without dedifferentiation and redifferentiation stage:
(1) Seed acquisition: as in example 1.
(2) Obtaining an explant: soaking 200 grains of the aged mango seeds treated in the step (1) at the temperature of 4 ℃ for 1min with 70% ethanol, washing 3 times with sterile water, adding sodium hypochlorite with the mass concentration of 30% for soaking 20min, and washing 5 times with sterile water. Placing sterilized old mango seeds on sterile dry filter paper for 20min to suck seed water, transversely cutting endosperm under a split mirror, and reserving mature embryo and scutellum parts of the old mango seeds, wherein the mature embryo and scutellum parts of the old mango seeds are shown in fig. 6.
(3) Dedifferentiation culture: and (3) the dedifferentiation culture medium is applied, mature embryo of the old mango seed obtained in the step (2) is directly contacted with the culture medium downwards to induce the formation of callus, and 12 parallel culture plates are arranged, and 5 explants are cultured in each parallel culture plate.
Placing the culture medium in a tissue culture box to perform dedifferentiation culture under dark condition, wherein the dedifferentiation culture temperature period is 24-26 ℃ and is cultured for 16h, then the culture medium is cultured for 8h at 20-22 ℃, and the temperature period is repeated every day until the culture is finished. After 21d of dedifferentiation and darkness culture, all mature embryos of the old mango are germinated and rooted, no callus is formed, the mature embryos are cultured for about 42-49 d, and the mature embryos are not subjected to dedifferentiation and redifferentiation stages, and germinate from embryo radicle into the old mango seedlings.
Comparative example 2: mature embryo is slit and transected and endosperm is excised
(1) Seed acquisition: as in example 1.
(2) Obtaining an explant: soaking 200 seeds in 70% ethanol for 1min, washing with sterile water for 3 times, adding 30% sodium hypochlorite, soaking for 20min, and washing with sterile water for 5 times. Placing the seeds on sterile dry filter paper for 20min to absorb water, longitudinally cutting the mature embryo under a split mirror, transversely cutting endosperm, and retaining the mature embryo and scutellum part to obtain the mature embryo shown in figure 1.
(3) Dedifferentiation culture: dedifferentiation culture Using the dedifferentiation medium of comparative example 1, mature embryo of old mango seed obtained in step (2) was directly contacted with the medium downward to induce formation of callus, and 18 parallel were set up, 5 explants were cultivated in each parallel. Placing the culture medium in a tissue culture box to perform dedifferentiation culture under dark condition, wherein the dedifferentiation culture temperature period is 24-26 ℃ and is cultured for 16h, then the culture medium is cultured for 8h at 20-22 ℃, and the temperature period is repeated every day until the culture is finished. Callus formation was observed for all explants after 21d of dedifferentiated dark culture.
(4) And (3) differentiation culture: transferring the callus obtained in the step (3) to a dedifferentiation medium (the dedifferentiation medium is the same as in example 1) for differentiation culture, wherein the differentiation culture is performed in a tissue culture incubator, and the cycle period of the tissue culture incubator is set as follows: under the illumination condition, the temperature is 24-26 ℃, the illumination culture time is 16 hours, and the illumination intensity is 1000lux; the temperature is 20-22 ℃ under the dark condition, and the time of dark culture is 8 hours. Callus differentiation was not seen after 42d of culture.
The browning rate of the calli and the regeneration efficiency of the calli of example 1 and comparative examples 1 to 2 were calculated as follows:
Differentiation efficiency of callus = number of callus differentiated to bud/number of callus without browning
TABLE 1 callus browning rate and callus regeneration efficiency of example 1 and comparative examples 1 to 2
| Example 1 | Comparative example 1 | Comparative example 2 | |
| Callus induction rate | 100％ | 0％ | 100％ |
| Callus browning rate | 50±9％ | 0％ | 0％ |
| Callus differentiation rate | 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 strong old mango seedlings.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (5)
1. The method for regenerating and tissue-culturing mature embryo of old mango is characterized by comprising the following steps:
Inoculating the aseptic explant of the old mango to a dedifferentiation culture medium for dedifferentiation culture to obtain old mango callus with differentiated regeneration buds; the length of the regeneration bud is 0.2-0.8 cm;
the explant is mature embryo of old mango seeds; the mature embryo of the old mango seed is obtained by transversely cutting endosperm from the old mango seed;
the dedifferentiation medium is based on an N6 medium, and also contains only the following components in the following concentrations: 3-5 mg/L of dicamba, 0.2-0.3 mg/L of thidiazuron, 28-35 g/L of maltose, 1100-1200 mg/L of proline, 270-330 mg/L of casein and 6.5-7.5 g/L of agar;
transferring the callus of the old mango bud differentiated into a rooting medium for rooting culture to obtain the old mango seedling;
and (3) domesticating and culturing the regenerated seedlings of the old mango to obtain seedlings of the old mango plants.
2. The tissue culture method according to claim 1, wherein the rooting medium is based on barley rooting medium, and further comprises 28-32 g/L maltose and 3-4 g/L plant gel; the barley rooting 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 cupric 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.
3. The tissue culture method according to claim 1, wherein the dedifferentiation culture temperature period is 15.5 to 16.5 hours at 24 to 26 ℃ per day and 7.5 to 8.5 hours at 20 to 22 ℃; the dedifferentiation culture is a dark culture, and the dedifferentiation culture time is 33-45 d.
4. The tissue culture method according to claim 1, wherein the temperature of the domestication culture is 23-27 ℃, the domestication culture is performed under alternate light and dark conditions, the light culture time is 15-17 h, and the dark culture time is 7-9 h.
5. The tissue culture method according to claim 1, wherein the dedifferentiation medium is based on an N6 medium and further comprises only the following components in the following concentrations: dicamba 4mg/L, thidiazuron 0.25mg/L, maltose 30g/L, proline 1150mg/L, casein 300mg/L and agar 7g/L.
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| US20070039069A1 (en) * | 2004-03-22 | 2007-02-15 | Rogers James A | Nucleic acid molecules associated with oil in plants |
| CN100491535C (en) * | 2005-06-27 | 2009-05-27 | 四川大学 | Chuancao-II Laomangmai wheat pest-resisting gene transferring technology |
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