CN113973715B - Method for improving sporogenous rate of microspores of black-bone vegetables - Google Patents
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Abstract
The invention relates to the technical field of plant tissue culture, and provides a method for improving the germ rate of Wucai microspores, wherein 0.05-0.2 mg/L brassinolide BR is sprayed on the flower buds of Wucai plants 24 hours before the flower buds of Wucai are picked; meanwhile, in the microspore culture stage after the picking of the black-flowered cabbage buds, 100-300 mg/L polyvinylpyrrolidone PVP is added into a microspore culture medium. The invention has the advantages that: the invention develops a method for improving the embryogenic rate of the microspores of the brassica oleracea by spraying brassinolide on the brassica oleracea plants and adding a nonionic polymer compound PVP.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for improving the embryogenic rate of spicebush microspores.
Background
Wucai (Brassica campestris L. Ssp. Chinensis) is a variety of Brassica Brassica subspecies of Brassicaceae, and has abundant nutrition and high economic value, and is mainly eaten by leaves. At present, the black-boned vegetables are planted in most regions of China, wherein the south has a plurality of planting regions, particularly the fields of Anhui, shanghai, jiangsu and the like, and the planting area is large. The Wucai from Anhui province is one of famous specialty of Anhui province, and has the advantages of wide distribution, high yield, good flavor, long supply period and the like.
The microspore stage is a transient and important stage in the development process of male gametophytes in the life history of higher plants, and the microspores are mononuclear cells released from tetrads after meiosis, namely immature pollen which develops to a certain stage. The culture of the free microspore is based on the totipotency principle of plant cells, and the microspore of the plant is subjected to tissue culture under specific conditions, so that a regeneration plant is obtained. The isolated microspore culture technology is a haploid breeding technology, and is a process of separating microspores in a certain development period from anthers and developing the microspores into complete fertile plants through certain treatment under the aseptic condition. In the process of culturing the sporophore of the lindera aggregata, the condition of low rate of embryo emergence of the lindera aggregata caused by genotype is common, and the condition is imminent to be changed.
At present, in the process of culturing brassica microspores, different additives are mainly added into an NLN culture medium to optimize a microspore culture system, wherein the addition of active carbon, silver nitrate and the like is common, but the culture effect on the brassica microspores is not obvious. Different phytohormones such as IAA and 6-BA are added into a culture medium to influence the culture of the brassica microspores to different degrees, but a system or a method with obvious effect is difficult to find to improve the embryo emergence rate of the brassica microspores.
Disclosure of Invention
The invention aims to provide a method for improving the germ extraction rate of the spicebush spores with a remarkable effect.
The invention adopts the following technical scheme to solve the technical problems:
a method for improving the embryo-growing rate of the Wucai microspore comprises the steps of spraying 0.05-0.2 mg/L brassinolide BR on the flower buds of the Wucai plants 24 hours before the flower buds of the Wucai are picked; meanwhile, in the microspore culture stage after the picking of the black-flowered cabbage buds, 100-300 mg/L polyvinylpyrrolidone PVP is added into a microspore culture medium.
The method comprises the following specific steps as one of the preferable modes of the invention:
(1) Spraying 0.05-0.2 mg/L brassinolide BR on flower buds of the Brassica oleracea plants in a bolting and flowering period, and picking corresponding flower bud samples after spraying treatment for 24 hours for subsequent microspore culture;
(2) Pre-cooling the picked flower bud sample at 4 ℃ for 1d;
(3) After precooling, selecting a bud sample at the mononuclear border stage, sterilizing the bud sample by using 70% alcohol for 30s and 0.1% HgCI for 6min in sequence, and finally washing the bud sample by using sterile water for 3 times, wherein each time is 5min; then, the B5 medium was added to the glass and the buds were ground with a glass rod to release the microspores; then, filtering the microspore solution in the glass by using a steel wire mesh screen into a new sterilized glass, filtering the sterilized glass into a centrifuge tube by using a cell mesh screen for the second time, and centrifuging the sterilized glass; finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again to obtain a precipitate, namely the purified microspore required by the target;
(4) Re-suspending the purified microspores obtained in the step (3) by using an NLN culture medium, and adding polyvinylpyrrolidone PVP with the concentration of 100-300 mg/L into the purified microspores;
(5) Carrying out heat shock treatment on the culture obtained in the step (4) at the temperature of 33 ℃ for 24 hours, and then transferring to dark culture at the temperature of 25 ℃; after the macroscopic embryoid bodies appeared, they were cultured with shaking on a shaker at 25 ℃.
In a preferred embodiment of the present invention, in the step (1), the spraying concentration of the brassinolide is specifically 0.1mg/L.
In a preferred embodiment of the present invention, in the step (3), the wire mesh screen is 300 mesh and the cell mesh screen is 40um.
In a preferred embodiment of the present invention, in the step (3), the centrifugal rotation speeds of the two centrifugal treatments are 1000r/min.
In the step (4), the purified microspore is diluted by NLN culture medium to adjust the density; when the density of the microspore reaches 1 multiplied by 10 5 ～5×10 5 At one/mL, the cells were dispensed into petri dishes and polyvinylpyrrolidone was added to each dish at the corresponding concentration.
In a preferred embodiment of the present invention, the polyvinylpyrrolidone is added in the step (4) at a concentration of 150mg/L.
In a preferred embodiment of the present invention, in the step (5), after the macroscopic embryoid body appears, the culture is placed on a shaking table for shaking culture at 25 ℃ and the shaking rotation speed is 50r/min.
As one of the preferable modes of the invention, the B5 culture medium and the NLN culture medium are conventional basal culture media in the field, and the components are not described again.
The principle is as follows:
(1) During microspore culture, harmful substances (such as phenols, abscisic acid and the like) can be generated due to proliferation and growth of cells, so that embryogenesis is inhibited. Polyvinylpyrrolidone (PVP), a synthetic water-soluble polymer, has the general properties of water-soluble polymers, namely colloid protection, film-forming properties, adhesion, hygroscopicity, solubilization or coacervation. By adding polyvinylpyrrolidone into the culture medium, harmful substances such as phenols, abscisic acid and the like can be precipitated and separated out, so that the embryo yield of microspore culture is effectively improved.
(2) The brassinolide is a plant hormone with extremely high physiological activity, and can participate in various regulation processes such as inhibition of plant organ senescence, promotion of photosynthesis and seed germination, participation in stress resistance and the like; among the known brassinolide substances, brassinolide has the highest physiological activity and has huge application potential in agricultural production. In the prior art, there are few cases that the hormone is added into NLN culture medium to promote microspore embryo emergence, but the method is not obvious in the microspore embryo emergence effect of the brassica oleracea through experiments. The method changes the application period of the brassinolide, and directly acts on the flower buds of the samples before picking, so that the embryo emergence rate of subsequent microspore culture is greatly improved.
Compared with the prior art, the invention has the advantages that: the invention researches and develops a method for improving the rate of embryos of spicebush microspores by spraying brassinolide on a spicebush plant and adding a nonionic macromolecular compound PVP.
Drawings
FIG. 1 is a diagram showing the embryoid bodies of microspores of Brassica oleracea in example 3 after shaking culture for 19 days.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The method for improving the embryogenic rate of the sporozoites of the brassica oleracea comprises the following specific steps of:
(1) In the bolting and flowering period, 0.05mg/L brassinolide BR is sprayed on the flower buds of the Brassica oleracea plants, and corresponding flower bud samples are picked after spraying treatment for 24 hours and are used for subsequent microspore culture.
(2) And (3) pre-cooling the picked bud sample for 1d at the temperature of 4 ℃.
(3) After precooling, selecting a bud sample at the mononuclear border stage, sterilizing the bud sample by using 70% alcohol for 30s and 0.1% HgCI for 6min in sequence, and finally washing the bud sample by using sterile water for 3 times, wherein each time is 5min; then, the B5 medium was added to the glass and the buds were ground with a glass rod to release the microspores; then, filtering the microspore solution in the glass by using a 300-mesh steel wire mesh screen into a new sterilized glass, secondarily filtering the sterilized glass by using a 40-um cell mesh screen into a centrifugal tube, and centrifuging at the rotating speed of 1000 r/min; and finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again at the centrifugal speed of 1000r/min to finally obtain the precipitate, namely the purified microspore required by the target.
(4) Resuspending and diluting the purified microspore obtained in the step (3) by using NLN culture medium to adjust the density of the microspore; when the density of the microspore reaches 1 multiplied by 10 5 ～5×10 5 At one/mL, polyvinylpyrrolidone PVP was added at a concentration of 100 mg/L.
(5) Carrying out heat shock treatment on the culture obtained in the step (4) at the temperature of 33 ℃ for 24 hours, and then transferring to dark culture at the temperature of 25 ℃; after the appearance of macroscopic embryoid bodies, they were placed on a shaker at 25 ℃ for shaking culture (rotation speed 50 r/min).
Example 2
The method for improving the embryogenic rate of the sporozoites of the brassica oleracea comprises the following specific steps of:
(1) In the bolting and flowering period, 0.2mg/L brassinolide BR is sprayed on the flower buds of the lindera glauca plants, and corresponding flower bud samples are picked after spraying treatment for 24 hours and used for subsequent microspore culture.
(2) And (3) pre-cooling the picked bud sample for 1d at the temperature of 4 ℃.
(3) After precooling, selecting a bud sample at the mononuclear border stage, sterilizing the bud sample by using 70% alcohol for 30s and 0.1% HgCI for 6min in sequence, and finally washing the bud sample by using sterile water for 3 times, wherein each time is 5min; then, the B5 medium was added to the glass and the buds were ground with a glass rod to release the microspores; then, filtering the microspore solution in the glass by using a 300-mesh steel wire mesh screen into a new sterilized glass, secondarily filtering the microspore solution into a centrifuge tube by using a 40-um-mesh cell mesh screen, and centrifuging the centrifuge tube at the rotating speed of 1000 r/min; and finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again at the centrifugal speed of 1000r/min to finally obtain the precipitate, namely the purified microspore required by the target.
(4) Resuspending and diluting the purified microspore obtained in step (3) with NLN culture medium to adjust the density of the microspore; when the density of the microspore reaches 1 multiplied by 10 5 ～5×10 5 At one/mL, polyvinylpyrrolidone PVP was added at a concentration of 300 mg/L.
(5) Carrying out heat shock treatment on the culture obtained in the step (4) at the temperature of 33 ℃ for 24 hours, and then transferring to dark culture at the temperature of 25 ℃; after the appearance of macroscopic embryoid bodies, the cells were subjected to shaking culture (rotation speed 50 r/min) at 25 ℃ on a shaker.
Example 3
The method for improving the embryogenic rate of the sporozoites of the brassica oleracea comprises the following specific steps of:
(1) In the bolting and flowering period, 0.1mg/L brassinolide BR is sprayed on flower buds of the Brassica oleracea plants, and corresponding flower bud samples are picked after spraying treatment for 24 hours and are used for subsequent microspore culture.
(2) And (3) pre-cooling the picked flower bud sample at 4 ℃ for 1d.
(3) After precooling, selecting a bud sample at the mononuclear border stage, sterilizing the bud sample by using 70% alcohol for 30s and 0.1% HgCI for 6min in sequence, and finally washing the bud sample by using sterile water for 3 times, wherein each time is 5min; then, the B5 medium was added to the glass and the buds were ground with a glass rod to release the microspores; then, filtering the microspore solution in the glass by using a 300-mesh steel wire mesh screen into a new sterilized glass, secondarily filtering the sterilized glass by using a 40-um cell mesh screen into a centrifugal tube, and centrifuging at the rotating speed of 1000 r/min; and finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again at the centrifugal speed of 1000r/min to finally obtain the precipitate, namely the purified microspore required by the target.
(4) Resuspending and diluting the purified microspore obtained in the step (3) by using NLN culture medium to adjust the density of the microspore; when the density of the microspore reaches 1 multiplied by 10 5 ～5×10 5 At one/mL, polyvinylpyrrolidone PVP was added at a concentration of 150mg/L.
(5) Carrying out heat shock treatment on the culture obtained in the step (4) at the temperature of 33 ℃ for 24 hours, and then transferring to dark culture at the temperature of 25 ℃; after the macroscopic embryoid appears, the leaf mustard is placed on a shaking table for shaking culture at 25 ℃ (the rotating speed is 50 r/min), and the microspore embryo state of the leaf mustard after being cultured for 19 days is shown in figure 1.
Example 4
The embodiment is used for verifying the influence of different concentrations of BR on the germ production rate of the black-bone dish under different concentrations of PVP treatment:
1. the experimental method comprises the following steps:
1. 40 parts of Huainan yellow cabbage are used as materials and are numbered from 1 to 40 respectively; and carrying out conventional cultivation on 40 parts of materials, and managing until bolting and flowering.
2. Averaging 40 parts of the material into 4 groups of 10 plants each; each group was treated as follows:
group 1: as a control, no treatment was done;
group 2: spraying 0.05mg/L brassinolide BR on flower buds of the Brassica oleracea plants;
group 3: spraying 0.1mg/L brassinolide BR on flower buds of the Brassica oleracea plants;
group 4: 0.2mg/L brassinolide BR is sprayed on flower buds of the lindera aggregata plants.
After 24h of spraying treatment, corresponding flower bud samples are picked for subsequent microspore culture.
3. And (3) pre-cooling the picked bud samples for 1d in an environment of 4 ℃.
4. After precooling, selecting a bud sample in a mononuclear border period, sequentially sterilizing the bud sample by using 70% alcohol for 30s (fully shaking) and 0.1% HgCI for 6min, and finally washing the bud sample by using sterile water for 3 times, wherein each time lasts for 5min; then, the B5 medium was added to the glass and the buds were ground with a glass rod to release the microspores; then, filtering the microspore solution in the glass by using a 300-mesh steel wire mesh screen into a new sterilized glass, secondarily filtering the sterilized glass by using a 40-um cell mesh screen into a centrifugal tube, and centrifuging at the rotating speed of 1000 r/min; and finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again at the centrifugal speed of 1000r/min to finally obtain the precipitate, namely the purified microspore required by the target.
5. Resuspending and diluting each purified microspore obtained in step (3) with NLN medium to adjust the density; when the density of the microspore reaches 1 multiplied by 10 5 ～5×10 5 At individual/mL, each spore suspension was aliquoted into four petri dishes and treated as follows:
processing one: polyvinylpyrrolidone was not added to the separately packed petri dishes a for comparison;
and (5) processing: adding polyvinylpyrrolidone PVP (polyvinylpyrrolidone) with the concentration of 100mg/L into the separately packaged culture dish B;
and (3) treatment III: adding polyvinylpyrrolidone PVP with the concentration of 150mg/L into the separately packaged culture dish C;
and (4) treatment: polyvinylpyrrolidone PVP with a concentration of 300mg/L was added to the separately packed petri dishes D.
6. Placing each culture dish at the temperature of 33 ℃ for heat shock treatment for 24 hours, and then transferring to the dark culture at the temperature of 25 ℃; after the embryoid body which can be seen by naked eyes appears, placing the embryoid body on a shaking bed for shaking culture at 25 ℃ (the rotating speed is 50 r/min); and counting the whole embryo emergence condition of the brassica oleracea microspores after 25 days of culture.
2. The experimental results are as follows:
the results are shown in Table 1.
TABLE 1 influence of different concentrations of BR on the germination rate of Umbelliferae after PVP treatment at different concentrations
As can be seen from Table 1, compared with the absolute control group which is not treated, the method provided by the invention can effectively improve the embryogenic rate of the microspore of the brassica campestris no matter spraying brassinolide BR on the brassica campestris plants or adding polyvinylpyrrolidone PVP into the culture medium; especially under the combination of '0.1 mg/L BR +150mg/L PVP', the germination rate of the brassica rapa microspore reaches 4.78 embryos/bud.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (5)
1. A method for improving the germ extraction rate of the microspores of the black-bone vegetables is characterized by comprising the following specific steps:
(1) Spraying 0.1mg/L brassinolide BR on flower buds of Brassica oleracea plants in a bolting and flowering period, and picking corresponding flower bud samples after spraying for 24h for subsequent microspore culture;
(2) Pre-cooling the picked flower bud sample at 4 ℃ for 1d;
(3) After precooling, selecting a bud sample at the mononuclear border stage, sterilizing the bud sample by using 70% alcohol for 30s and 0.1% HgCI for 6min in sequence, and finally washing the bud sample by using sterile water for 3 times, wherein each time is 5min; then, the B5 medium was added to the glass and the buds were ground with a glass rod to release the microspores; then, filtering the microspore solution in the glass by using a steel wire mesh screen into a new sterilized glass, filtering the sterilized glass into a centrifuge tube by using a cell mesh screen for the second time, and centrifuging the sterilized glass; finally, resuspending the centrifugal precipitate by using a B5 culture medium, and centrifuging again to obtain a precipitate, namely the purified microspore required by the target;
(4) Resuspending the purified microspores obtained in step (3) in NLN medium, and adding polyvinylpyrrolidone PVP with concentration of 150 mg/L;
(5) Carrying out heat shock treatment on the culture obtained in the step (4) at the temperature of 33 ℃ for 24 hours, and then transferring to dark culture at the temperature of 25 ℃; after the macroscopic embryoid bodies appeared, they were cultured with shaking on a shaker at 25 ℃.
2. The method for increasing the embryogenic rate of sporozoites of Wucai dish as claimed in claim 1, wherein in step (3), the steel wire mesh screen is 300 mesh and the cell mesh screen is 40um.
3. The method for increasing the embryogenic rate of sporozoite of Wucai dish as claimed in claim 1, wherein in step (3), the centrifugation speed of the two centrifugation treatments is 1000r/min.
4. The method for improving the embryogenic rate of the microspores of the lindera aggregate according to claim 1, wherein in the step (4), the purified microspores are diluted by NLN medium to adjust the density; when the density of the microspore reaches 1 multiplied by 10 5 ～5×10 5 At one/mL, the cells were dispensed into petri dishes and polyvinylpyrrolidone was added to each dish at the corresponding concentration.
5. The method for improving the embryogenic rate of microspores of lindera aggregate according to claim 1, wherein in the step (5), after the macroscopic embryoid body appears, the culture is placed on a shaking table for shaking culture at 25 ℃ and the shaking rotation speed is 50r/min.
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| CN114982632A (en) * | 2022-04-21 | 2022-09-02 | 安徽农业大学 | Method for reducing browning rate of regeneration plant of black-bone vegetable microspore |
| CN114916441B (en) * | 2022-05-24 | 2023-03-31 | 安徽农业大学 | Method for improving germ production rate of free microspores of lindera aggregate by using melatonin |
| CN115316280A (en) * | 2022-09-16 | 2022-11-11 | 安徽农业大学 | Method for improving leaf-shaped embryo formation rate and one-step seedling rate of black cabbage |
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