CN116849128A - Method for culturing anther of Kazakii autotetraploid plant - Google Patents
Method for culturing anther of Kazakii autotetraploid plant Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for culturing anther of a soviet-gasaki homotetraploid plant, which comprises the operations of bud collection at a proper development stage, bud pretreatment before anther culture, anther preculture, embryoid induction, embryoid germination, seedling strengthening, seedling hardening, transplanting and the like. Compared with the existing reported technology for culturing the anther of the diploid eggplant, 3 diploid haploids are obtained from anther culture of Su Qijia autotetraploid plants, and each diploid haploid plant is derived from different independent embryoids, so that the homozygosity of the genotype of the tetraploid plant can be judged according to the difference between different diploid haploid plants or the separation condition of inbred offspring of the diploid haploid plants, the difficulty in the process of identifying the homozygosity of the genotype of the autotetraploid plant by inbreeding on the tetraploid level is overcome, and the success of culturing the anther of the diploid Su Qijia variety is realized.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and particularly relates to a method for culturing anther of a soviet-gasaki homotetraploid plant and application thereof.
Background
Eggplant (Solarium melongena L) is cultivated in various provinces in China in Asian tropical zone of original production. Herbs or sub-shrubs can be used for vegetable and fruit, and root, stem, leaf and seed can be used as medicines. In nature, diploid is the main factor, haploid and polyploid plants are mainly obtained by anther culture or diploid doubling treatment. The apoploid eggplant plant obtained by anther culture has abnormal meiosis process compared with a diploid plant, tetraploid lines with good marketability and high yield are difficult to screen, and the conventional commercial tetraploid lines or hybrid lines obtained by anther culture are not reported at present because the test method for whether the genotypes of the apoploid eggplant plants are homozygous or not is more difficult than that of the diploid plant.
Although, at the beginning of the 70 s, riana et al succeeded in growing seedlings by callus routes through diploid eggplant anther culture, and thereafter, whether eggplant anther or pollen (microspores) culture has been reported to be successful, the technical method for culturing the tetraploid plant anther and the application thereof in practice have not been reported.
Disclosure of Invention
The invention aims to: aiming at the problems that the plant is difficult to be induced by the influence of the genotype on the part of the variety of the existing diploid anther culture, or the induced polyploid plants such as tetraploid and the like have low propagation coefficient, poor economic character, difficult stability of ploidy, difficult acquisition of homozygous diploid inbred line and the like, the invention provides the method for culturing the anther of the autotetraploid plant of the eggplant, which can indirectly identify the genotype homozygosity of the autotetraploid plant on the one hand, and reduce the ploidy of the plant through anther culture on the other hand, so as to obtain the homozygous diploid eggplant and the inbred line, thereby improving the efficiency of the haploid breeding technology of the eggplant.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the method for culturing the anther of the Kazakii autotetraploid plant comprises the following steps:
(1) Collecting flower buds on tetraploid eggplant plants, putting the flower buds into a plastic self-sealing pocket for packaging, immediately putting into a heat preservation box filled with ice cubes, and putting the sealing pocket and the flower buds in the sealing pocket into a refrigerator at 4 ℃ for low-temperature treatment for 2-5 d;
(2) Taking out the flower buds subjected to low-temperature treatment in the step (1), stripping off sepal splinters, and carrying the sepal splinters into a sterile room for surface sterilization;
(3) Taking the flower buds in the step (2), peeling out complete anthers under the aseptic condition, inoculating the complete anthers into a preparation culture medium of a culture dish, sealing, placing the culture medium in a 35 ℃ incubator for culturing for 1-3d under the dark condition, and then taking the culture medium out and placing the culture medium in a 25 ℃ incubator for culturing for 15-30d under the light-dark alternating condition;
(4) Transferring anthers in the preparation culture medium in the step (3) into an embryoid induction culture medium, and culturing for 10-30d under the same light-dark alternation condition until embryoids appear; transferring embryoid and anther into germination culture medium, culturing under the same condition, transferring into strong seedling culture medium after embryoid develops complete root and bud, and transplanting after plant height reaches above 2 cm;
(5) Gently clamping the rooting and seedling strengthening in the step (4) by using forceps, washing off the culture medium stained on the root, soaking in carbendazim solution for 10-30min, taking out, dipping the root system in IBA or NAA solution, then planting in unused seedling culture matrix, watering thoroughly without water accumulation, covering the seedling tray by using a plastic film, placing the seedling tray in the above light-dark alternating incubator, gradually removing the film for 7-15d, and keeping the leaves of the seedling from wilting and growing gradually.
Further, in the step (1), collecting buds on the tetraploid plant of the eggplant, wherein the morphological characteristics are that the thickness of the stem end is larger than that of the petal end, the petal cylinder is basically flush with the calyx cylinder, the calyx split is slightly separated, at the moment, pollen is in a microspore stage, and the buds which are strong in development, free of diseases and insects and tightly wrapped in the petals are selected.
Further, in the step (2), a two-step method is adopted for sterilization, firstly, the finished buds are put into a sterile triangular flask, 70% -75% of alcohol is poured into the sterilized triangular flask, the buds are deeply soaked, and the sterilized buds are manually shaken or oscillated for 1-5min, and then the alcohol is poured out; adding 1-2 drops of Tween-80 into the triangular flask, pouring 6.5% sodium hypochlorite solution, shaking or oscillating for 5-15min, pouring sodium hypochlorite solution, washing with sterile water for 3-5 times, sucking the liquid on the bud surface with sterilized absorbent paper or filter paper, and sealing the bottle mouth with sealing film.
Further, in the step (3), the preparation medium is MS solid medium added with 0.1-2mg/L of auxin and mitogen.
Further, in the step (4), 0.1-1mg/L of auxin (IAA/NAA/IBA) and cytokinin (BA/KT) are added to the embryoid body induction medium which is NLN or B5 solid medium. .
Further, in the step (4), 0.1-1mg/L of auxin, mitogen and gibberellin are added to the germination medium which is NLN or B5 solid medium, 1 fresh medium is replaced every 20 days or so, 2 fresh medium is replaced at most, and all anthers which do not produce embryoids are discarded; the strong seedling culture medium is a B5 solid culture medium, and 0.1-0.2mg/L of auxin is added or not added according to the growth state of the seedling. After the embryoid develops complete roots and buds, the embryoid is transferred into a strong seedling culture medium until 3 normal leaves or more are developed (no water stain phenomenon), the plant height reaches more than 2cm, and the seedlings are acclimatized and transplanted in time. 2,4-D is not used in the whole culture process, which is beneficial to the germination of embryoid into seedlings.
Further, in the step (5), the carbendazim solution is obtained by diluting carbendazim wettable powder with the active ingredient content of 50wt% with water according to the mass ratio of 1:500-800; immersing the root into carbendazim solution for 10-20 min. Effectively preventing pathogen infection from occurring in the seedling hardening process and reducing the survival rate of the seedlings.
Further, in the step (5), the concentration of the IBA or NAA solution is 200-1000 ppm, and the soaking time of the seedling roots in the solution is 1 s-20 min; the seedling substrate is a common seedling substrate for vegetable sowing or vermiculite, the thickness of the seedling substrate is not less than 8cm, and the seedling substrate is thoroughly poured but does not accumulate water; the root is inserted into the matrix and the leaf is exposed out of the matrix, and the plant row spacing is not less than 5cm.
Further, in the steps (3) to (5), the light-dark alternate culture conditions are as follows: the temperature is 25 ℃, the photoperiod is 12-16 h, the dark period is 8-12 h, and the illumination intensity is 1000-1500 lx.
Further, in the step (5), the plastic film is not opened within 1 week after being sealed, the film can be opened after 1 week, the wilting dead plants can be pulled out, the film is covered again after watering or spraying water, gaps are gradually reserved for ventilation and moisture dispersion, the film is completely removed after 15 days, and a small amount of clean water is sprayed to the leaf surfaces when the leaves wilt.
Further, tender leaves (without affecting the continuous growth of seedlings) at the lower part of the survival plant are taken, the DNA content of the tender leaves is measured by a flow cytometry method, and diploid seed seedlings are used as a control.
The DNA content in the cells is determined by a flow cytometry method. The fluorescence intensity X-Mean (abscissa) of the sample peak is proportional to the cell DNA content, so the ploidy is judged from the proportional relationship between the X-Mean of each sample. For example, by adjusting the diploid peak of the control to the 100-axis position, haploids will appear at 50 and the peak at 200-axis position is tetraploid. The ordinate of the graph represents the cell number, and the peak heights reflect the difference in cell ratios.
The beneficial effects are that:
by applying the method of the invention, flower buds collected from Su Qijia tetraploid plants obtained in 3 months of 2022 are subjected to anther culture in 3 months of 2023, 3 regenerated plants obtained in 6 months of 2023 are transplanted and survived, and all the regenerated plants are diploid. The anther culture of the diploid hybrid 'Su Qijia' variety is easy to obtain embryoid bodies, but is difficult to seedling, and the technique can indirectly realize the success of the anther culture of the diploid Su Qijia variety before 1 tetraploid plant (donor plant in the test) which is transplanted to survive is obtained in 2022.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 leaf DNA content of diploid control.
FIG. 2 flower bud morphology suitable for culture.
FIG. 3 process of regenerating plants by anther culture with autotetraploid eggplant plant ploidy;
a: the tetraploid plant generated by anther culture of the 2022 diploid eggplant Su Qijia is also a donor plant of the tetraploid flower bud in the test, and can bloom and fruit normally;
b: FIG. 3-A plant leaf DNA content, tetraploid;
c: the inoculated 1 anther produces embryoid bodies, and the embryoid-derived anther rate is 5.6% (1/18);
d: the embryogenic anthers and embryoids in FIG. 3-C were transferred into embryoid-forming medium;
e: 3 embryoid bodies in FIG. 3-D develop into plants suitable for transplanting after transfer into glass flasks;
f: plant leaf DNA content, diploid in FIG. 3-E.
FIG. 4 shows the results of plant ploidy identification generated by anther culture of the autotetraploid eggplant;
a and a': seedling 1 in FIG. 3-E is transplanted into a surviving plant and its leaf DNA content, diploid;
b and B': transplanting the seedling 2 in FIG. 3-E into a surviving plant, and its leaf DNA content, diploid;
c and C': seedling 3 in FIG. 3-E was transplanted into a surviving plant and its leaf DNA content, diploid.
Detailed Description
The invention will be better understood from the following examples.
The test was performed in the genetic breeding laboratory of the national academy of science and technology, 1 month 2023 to 8 month 2023. The eggplant tetraploid plant is derived from a tetraploid plant obtained by culturing a diploid hybrid 'Su Qijia' anther in 2022 and 3 months. The 50% wp carbendazim powder is produced by Shanghai Yue Ling chemical industry Co., ltd, the content of active ingredient is 50wt%, each liter of tap water contains 1.25 g powder, and the suspension is prepared by diluting 800 times; indole-3-butyric acid (IBA, molecular weight 203.24) or Naphthalene Acetic Acid (NAA), produced by Shanghai chemical reagent company of the chinese medical group, the powder was first dissolved with a small amount of 100% alcohol and then diluted to 1000ppm with ultra-pure water.
(1) Collecting flower buds on tetraploid plants: flower buds on autotetraploid plants (figures 3-A, B) were collected on sunny days, with pollen in microspore development stage (flower bud morphology see figure 2). The picked flower buds are put into a plastic bag for sealing or tightly binding, and are immediately put into a heat preservation box (preventing the water of the flower buds from being lost) and brought back to a laboratory for standby.
(2) Pretreatment of flower buds: the flower buds before surface disinfection are pretreated for 2-5 days at the low temperature of 4 ℃, and sepal fragments are stripped, so that the disinfection effect is improved.
(3) Anther surface sterilization and culture: sterilizing by a two-step method, sucking the liquid on the surface of the sterilized flower buds by using sterilized absorbent paper or filter paper, peeling off complete anther under a sterile condition, inoculating to a prepared culture medium in a culture dish with the diameter of 5cm, sealing, placing into a 35 ℃ incubator for culturing for 1-3d under a dark condition, then taking out, placing into a condition of alternating light and dark at 25 ℃ for 12h/12h and illumination intensity of 1500lx, and continuously culturing for 7-20d; transferring anthers into embryoid induction medium for culturing for 10-30d until embryoid appears (FIG. 3-C); embryoid bodies and anthers were then transferred to germination medium (FIG. 3-D), culture was continued under the same conditions, with 1 fresh medium change every 20D or so, up to 2 changes, and all anthers that did not produce embryoid bodies were discarded. After the embryoid develops complete roots and buds, transferring the embryoid into a strong seedling culture medium in a triangular flask or a can, and timely hardening and transplanting after more than 3 leaves grow and the leaves are in a water stain-free state and the plant height reaches more than 2cm (figure 3-E). The preparation culture medium is MS solid culture medium added with NAA and 6-BA in 0.1-2 mg/L; the embryoid induction culture medium is NLN or B5 solid culture medium, and 0.1-1mg/L of auxin and mitogen are added; the embryoid germination medium is NLN or B5 solid medium, 0.1-1mg/L of auxin, mitogen and gibberellin are added, the seedling strengthening medium is B5 solid medium, and 0.1mg/L of auxin is added or not according to the growth state of seedlings.
(4) Hardening and transplanting: the rooting and seedling strengthening method is characterized in that rooting and seedling strengthening suitable for transplanting are gently clamped from a culture flask by forceps, a culture medium stained on the root is carefully washed away under the condition of not damaging the root, the rooting and seedling strengthening is soaked in a carbendazim solution of 800 times for 10-30min, the root system is dipped with 1000ppm IBA or NAA after being taken out, then the rooting and seedling strengthening is planted into an unused seedling raising matrix, watering is carried out without water accumulation, a seedling tray is covered by a plastic bag or a plastic film, the seedling tray is placed into an illumination culture box at the temperature of 25 ℃ above, the film is gradually removed for 7-15d, the leaf of the seedling is kept from wilting, the plant survives, and the leaf grows up gradually.
(5) Plant ploidy detection: the ploidy of the plants was identified by flow cytometry when the regenerated plantlets grew to 3-4 true leaves (FIG. 3-F, FIG. 4). The flow cytometry method for ploidy identification was to collect fresh young leaves, the leaf numbers were in one-to-one correspondence with the plant numbers, the diploid control provided by the test was the seedling of diploid plant 87-4 (FIG. 1), and the leaf size required for each test was approximately the thumbnail cap size. The amount of leaf to be tested should be more than 4 times this amount in preparation for repeated experiments.
The testing mechanism comprises: beijing Jin Di future Biotechnology Inc.
Detection instrument: partec CyFlow Space.
The kit comprises: partec CyStain UV Precise P.
The result judging method comprises the following steps: the fluorescence intensity X-Mean (abscissa) of the sample peak is proportional to the cell DNA content, so the ploidy is judged from the proportional relationship between the X-Mean of each sample. For example, by adjusting the diploid peak of the control to the 100-axis position (FIG. 1), haploids will appear at 50 and the peak at 200-axis position is tetraploid. The ordinate of the graph represents the cell number, and the peak height reflects the difference in cell ratio.
The bud pretreated by the method is inoculated for 3 months and 5 days, embryoid appears in 3 months 23, embryoid grows into seedlings for 4 months and 19 days, transplanting survival is carried out for 6 months and 16 days, and ploidy is tested, wherein only more than 3 months are needed.
Compared with the existing reported diploid eggplant anther culture technology, the method has the advantages that the autotetraploid anther culture obtains a plurality of diploid haploids, and as each diploid haploid plant is derived from different independent embryoids, the homozygosity of the tetraploid plant genotype can be judged according to the difference between different diploid haploid plants or the separation condition of inbred offspring thereof, and the difficulty in the selfing identification of the homozygosity of the tetraploid plant genotype on the tetraploid level is overcome; and secondly, the obtained doubled haploid plant is subjected to anther culture again to obtain doubled haploid plant, and the doubled haploid anther is cultured to obtain doubled haploid inbred line (DH line), so that the method is the most reliable method for ensuring that the homozygous diploid eggplant inbred line is obtained from the heterozygous plant. The method is a way for obtaining doubled haploid inbred line indirectly for genotype heterozygosis and only tetraploid diploid eggplant varieties which are obtained by anther culture without obtaining diploid and haploid regeneration plants. Experiments 1 tetraploid plant generated by anther culture of diploid eggplant variety Su Qijia is subjected to anther culture, 3 plants (1 embryo-out anther from 3 flower buds) are induced to be transplanted to survive from 16 inoculated anthers, and all the plants are diploid plants through flow cytometry identification, as shown in fig. 3 and fig. 4.
The invention provides a thought and a method for culturing the anther of the homotetraploid plant of the Suzaki plant, and the method and the way for realizing the technical scheme are numerous, the above description is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and the modifications are also regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. A method for culturing anther of a soyabean autotetraploid plant, which is characterized by comprising the following steps:
(1) Collecting flower buds on tetraploid eggplant plants, putting the flower buds into a plastic self-sealing pocket for packaging, immediately putting into a heat preservation box filled with ice cubes, and putting the sealing pocket and the flower buds in the sealing pocket into a refrigerator at 4 ℃ for low-temperature treatment for 2-5 d;
(2) Taking out the flower buds subjected to low-temperature treatment in the step (1), stripping off sepal splinters, and carrying the sepal splinters into a sterile room for surface sterilization;
(3) Taking the flower buds in the step (2), peeling out complete anthers under the aseptic condition, inoculating the complete anthers into a preparation culture medium of a culture dish, sealing, placing the culture medium in a 35 ℃ incubator for culturing for 1-3d under the dark condition, and then taking the culture medium out and placing the culture medium in a 25 ℃ incubator for culturing for 15-30d under the light-dark alternating condition;
(4) Transferring anthers in the preparation culture medium in the step (3) into an embryoid induction culture medium, and culturing for 10-30d under the same light-dark alternation condition until embryoids appear; transferring embryoid and anther into germination culture medium, culturing under the same condition, transferring into strong seedling culture medium after embryoid develops complete root and bud, and transplanting after plant height reaches above 2 cm;
(5) Gently clamping the rooting and seedling strengthening in the step (4) by using forceps, washing off the culture medium stained on the root, soaking in carbendazim solution for 10-30min, taking out, dipping the root system in IBA or NAA solution, then planting in unused seedling culture matrix, watering thoroughly without water accumulation, covering the seedling tray by using a plastic film, placing the seedling tray in the above light-dark alternating incubator, gradually removing the film for 7-15d, and keeping the leaves of the seedling from wilting and growing gradually.
2. The method for anther culture of a Su Qijia autotetraploid plant of claim 1, wherein in step (1), the buds of the eggplant tetraploid plant are collected, and the morphology is characterized in that the stem end thickness is larger than that of the buds of which the petal cylinder is basically flush with the calyx cylinder and the calyx split is slightly separated, and the pollen is in a microspore stage, and the buds with strong development, no disease and tightly packed petals are selected.
3. The method for culturing the anther of the Su Qijia autotetraploid plant according to claim 1, wherein in the step (2), a two-step method is adopted for sterilization, the finished buds are put into a sterile triangular flask, 70% -75% of alcohol is poured into the sterile triangular flask, the buds are deeply soaked, and the solution is manually shaken or oscillated for 1-5min, and then the alcohol is poured out; adding 1-2 drops of Tween-80 into the triangular flask, pouring 6.5% sodium hypochlorite solution, shaking or oscillating for 5-15min, pouring sodium hypochlorite solution, washing with sterile water for 3-5 times, sucking the liquid on the bud surface with sterilized absorbent paper or filter paper, and sealing the bottle mouth with sealing film.
4. The method for anther culture of a Su Qijia autotetraploid plant of claim 1, wherein in step (3), said preliminary medium is MS solid medium supplemented with 0.1-2mg/L of auxin and mitogen.
5. The method for culturing an anther of a Su Qijia autotetraploid plant of claim 1, wherein in step (4), said embryoid body induction medium is a solid medium of NLN or B5 supplemented with 0.1-1mg/L of auxin, cytokinin.
6. The method for anther culture of Su Qijia autotetraploid plants of claim 1, wherein in step (4), 0.1-1mg/L auxin, mitogen and gibberellin are added to the germination medium as NLN or B5 solid medium; the strong seedling culture medium is a B5 solid culture medium, and 0.1-0.2mg/L of auxin is added or not added according to the growth state of the seedling.
7. The Su Qijia autotetraploid plant anther culture method of claim 1, wherein in the step (5), the carbendazim solution is obtained by diluting carbendazim wettable powder with the active ingredient content of 50wt% with water according to the mass ratio of 1:500-800; immersing the root into carbendazim solution for 10-20 min.
8. The method for anther culture of Su Qijia autotetraploid plants according to claim 1, wherein in the step (5), the concentration of the IBA or NAA solution is 200-1000 ppm, and the soaking time of the root of the seedling in the solution is 1 s-20 min; the seedling substrate is a common seedling substrate for vegetable sowing or vermiculite, the thickness of the seedling substrate is not less than 8cm, and the seedling substrate is thoroughly poured but does not accumulate water; the root is inserted into the matrix and the leaf is exposed out of the matrix, and the plant row spacing is not less than 5cm.
9. The method for anther culture of Su Qijia autotetraploid plants of claim 1, wherein in steps (3) to (5), the alternate light and dark culture conditions are as follows: the temperature is 25 ℃, the photoperiod is 12-16 h, the dark period is 8-12 h, and the illumination intensity is 1000-1500 lx.
10. The method for anther culture of Su Qijia autotetraploid plants according to claim 1, wherein in step (5), the plastic film is sealed without opening the film within 1 week, the film can be opened after 1 week, the wilting dead plants are removed, the film is covered again after watering or spraying water, and the slit is gradually left for ventilation and moisture dispersion, the film is completely removed after 15 days, and a small amount of clear water is sprayed to the leaf surfaces when the wilting of leaves occurs.
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