CN110066848B - Method for researching cytological influence of exogenous ABA treatment on tomato pollen maturation - Google Patents
Method for researching cytological influence of exogenous ABA treatment on tomato pollen maturation Download PDFInfo
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Abstract
The invention discloses a method for exploring cytological influence of exogenous ABA treatment on tomato pollen maturation, which relates to the technical field of agriculture and comprises the following steps: s1, sample preparation: the tomato macadamia powder 208 inbred line is an experimental sample; s2, preparation of reagents: abscisic acid (ABA): a,0 μ M concentration of ABA; b, 10. mu.M concentration of ABA; c, 100. mu.M concentration of ABA; d, ABA at a concentration of 1000 μ M; dihydroguaiaretic acid (NDGA): e, NDGA at a concentration of 0 μ M; f,10 μ M concentration of NDGA; g, NDGA at a concentration of 100. mu.M; h, NDGA at a concentration of 1000. mu.M. The research results of the method for treating tomato anthers by using ABA and NDGA solutions with different concentrations respectively to explore effective medicament concentrations for inducing pollen abortion provide certain theoretical basis for further clarifying a molecular mechanism of ABA regulation and control of plant stamen development, disclosing a regulation and control mechanism of male sterility and simultaneously providing application of ABA-related male sterility in breeding work.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to a method for researching cytological influence of exogenous ABA treatment on tomato pollen maturation.
Background
The maturation of pollen has a decisive influence on the reproduction of plants, in particular on the yield and quality of crops. Pollen maturation is regulated by a very complex hormone network, wherein the mechanism of abscisic acid regulation of pollen maturation is not clear.
Tomatoes are hermaphroditic plants, which are usually propagated by the combination of male and female gametes. The normal development of stamens has a decisive influence on the reproduction of tomatoes, the preservation of germplasm resources and particularly the yield. However, the discovery, research and utilization of male sterility effectively promote the application and popularization of various crop hybrids, and the male sterility has important development potential and application value in crop breeding. Tomato male sterility is divided into pollen abortion type, stamen degeneration type and functional sterility type, although the current tomato male sterility genes are positioned to each chromosome, further fine positioning is needed, and the factors influencing the tomato male sterility are various, such as hormone, environment, substance metabolism, energy metabolism and the like.
In order to research the effect of abscisic acid in the pollen maturation process and better understand the influence of abscisic acid on the yield and quality of crops, the invention provides a research method of the cytological influence of exogenous ABA treatment on tomato pollen maturation.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for researching the cytological influence of exogenous ABA treatment on tomato pollen maturation, and solves the problems that the regulation and control mechanism of abscisic acid on pollen maturation is not clear at present and the quality and yield of agricultural products cannot be accurately controlled through abscisic acid.
(II) technical scheme
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: a method for exploring the cytological influence of exogenous ABA treatment on tomato pollen maturation comprises the following steps:
s1, sample preparation: the tomato macadamia powder 208 inbred line is an experimental sample;
s2, preparation of reagents:
abscisic acid (ABA): a, 0. mu.M concentration of ABA; b, 10. mu.M concentration of ABA; c, 100. mu.M concentration of ABA; d, ABA at a concentration of 1000 μ M;
dihydroguaiaretic acid (NDGA): e, NDGA at a concentration of 0 μ M; f,10 μ M concentration of NDGA; g, NDGA at a concentration of 100. mu.M; h, NDGA at a concentration of 1000. mu.M; the reagents are respectively put into standby vessels, so that the subsequent use is convenient;
s3, detecting the percentage of normal pollen after the reagent is used
(1) Selecting buds in microspore period, taking liquid medicine by using an injector, slightly breaking sepals by using a needle head, and injecting the liquid medicine into anthers;
(2) immediately putting the flower buds into FAA fixing liquid, vacuumizing for 20-30Min, and fixing for 24h and then carrying out paraffin section preparation;
(3) after the sample fixed by the FAA stationary liquid is subjected to gradient dehydration by tert-butyl alcohol, the sample is embedded in paraffin, the sample is sliced on a rotary microtome, then the slice is placed on a clean glass slide, a deionized water floating sheet is added, and then the sample is placed in an oven for baking;
(4) after a sample is baked, dewaxing is carried out in dimethylbenzene, and then the sample is dyed in Hai's hematoxylin dyeing solution after being rehydrated by gradient ethanol;
(5) and then dehydrated by gradient ethanol, redyeed by 1% fast green, observed by Canada gum sealing piece after being transparent by dimethylbenzene, and counted by a microscope to obtain the percentage of normal pollen.
Preferably, multiple sets of samples are taken at step 1 in step S3, and are sampled at days 2, 4, 6, and 8 after treatment, respectively.
Preferably, the rotary microtome slices the sample to a slice thickness of 8-10 μ M.
Preferably, the observation and statistics of pollen percentage are carried out under an OLYMPUS BX41 optical microscope, and the photographs are photographed by OLYMPUS C-7070Wide Zoom and processed and edited by Adobe Photoshop CS5 software.
Preferably, the temperature for drying the sample is 43 ℃, and a drying process needs to be carried out for 24 hours.
Preferably, when the percentage of normal pollen is counted in step 5 of step S3, after hematoxylin and fast green staining, and observing pollen under a microscope, some pollen with empty, flat, transparent and abnormal malformation is abnormal pollen and some pollen with full, regular and opaque pollen is normal pollen can be clearly observed, and thus counting the percentage of normal pollen can preliminarily judge the quality of pollen, and the higher the percentage of normal pollen is, the higher the quality of pollen is.
(III) advantageous effects
The invention has the beneficial effects that: ABA treatment found that excessive ABA also causes abnormal pollen maturation, and similar results were shown when the anthers were treated with different concentrations of ABA solution, and the 10. mu.M, 100. mu.M and 1000. mu.M ABA solutions used herein all showed large amounts of misshapen pollen grains at day 2 after treatment, and thereafter the effect was increased with the increase of treatment time, but the overall trend remained similar. This shows that, in the ripening process of tomato pollen, it is not better that ABA concentration is higher, and when the ABA concentration exceeds a certain threshold, the growth of tomato pollen is inhibited, whereas ABA solution of 10 μ M in this text far exceeds the threshold required for pollen growth, and the search for the ABA threshold required for pollen growth is needed for further research in the later period, and pollen abortion can be effectively caused by treating anther with ABA of concentration exceeding the threshold.
The research results of the method for treating tomato anthers by using ABA and NDGA solutions with different concentrations respectively to explore effective medicament concentrations for inducing pollen abortion provide certain theoretical basis for further clarifying a molecular mechanism of ABA regulation and control of plant stamen development, disclosing a regulation and control mechanism of male sterility and simultaneously providing application of ABA-related male sterility in breeding work.
Drawings
FIG. 1 is a schematic diagram of the developmental stage of tomato floral organs;
FIG. 2 is a schematic representation of microscopic observation of NDGA treated tomato anthers;
FIG. 3 is a schematic representation of microscopic observation of ABA treated tomato anthers;
FIG. 4 is a graph showing the percentage of normal pollen under different treatment conditions.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 to 4, the present invention provides a technical solution: a method for exploring the cytological influence of exogenous ABA treatment on tomato pollen maturation comprises the following steps:
s1, sample preparation: the tomato macadamia powder 208 inbred line is an experimental sample;
s2, preparation of reagents:
abscisic acid (ABA): a,0 μ M concentration of ABA; b, 10. mu.M concentration of ABA; c, 100. mu.M concentration of ABA; d, ABA at a concentration of 1000 μ M;
dihydroguaiaretic acid (NDGA): e, NDGA at a concentration of 0 μ M; f, NDGA at a concentration of 10 μ M; g, NDGA at a concentration of 100. mu.M; h, NDGA at a concentration of 1000. mu.M; the reagents are respectively put into standby vessels, so that the subsequent use is convenient;
s3, detecting the percentage of normal pollen after the reagent is used
(1) Selecting buds in microspore period, taking liquid medicine by using an injector, slightly breaking sepals by using a needle head, and injecting the liquid medicine into anthers;
taking multiple groups of samples, and sampling on days 2, 4, 6 and 8 after treatment;
(2) immediately putting the flower buds into FAA fixing liquid, vacuumizing for 20-30Min, and fixing for 24h and then carrying out paraffin section preparation;
(3) after the sample fixed by the FAA stationary liquid is subjected to gradient dehydration by tert-butyl alcohol, the sample is embedded in paraffin, the sample is sliced on a rotary microtome, then the slice is placed on a clean glass slide, a deionized water floating sheet is added, and then the sample is placed in an oven for baking;
a rotary microtome slices the sample, and the slice thickness is 8-10 mu M;
(4) after a sample is baked, dewaxing is carried out in dimethylbenzene, and then the sample is dyed in Hai's hematoxylin dyeing solution after being rehydrated by gradient ethanol;
the sample drying temperature is 43 ℃, a 24-hour drying process is required, after hematoxylin and green fixing dyeing, pollen is observed under a microscope, some pollen is empty, flat, transparent and abnormal, some pollen is full, regular and light-tight, and therefore the quality of the pollen can be preliminarily judged by counting the percentage of normal pollen, and the higher the percentage of normal pollen is, the higher the quality of the pollen is;
(5) and dehydrating by gradient ethanol, counterstaining by 1% fast green, observing by Canadian gum seal after being transparent by dimethylbenzene, and counting the percentage of normal pollen by using a microscope.
When the pollen percentage is observed and counted, the pollen percentage is observed under an OLYMPUS BX41 optical microscope, and is photographed by an OLYMPUS C-7070Wide ZooM, and the photograph is processed and edited by Adobe Photoshop CS5 software.
Experimental analysis:
in FIG. 1, A is the microstructure of anthers at various developmental stages; b is a flower bud real object picture of 9 developmental stages. Wherein I is the sporogenous cell period, and the bud length is 4 mm; II is microsporocyte period, bud length 6 mm; III is in tetrad period, the bud is 8mm long; IV, in the microspore period, the bud is 10mm long; v is the primary pollen grain period, and the bud is 15mm long; VI, the sepals are loosened to expose the petals, and the pollen is mature; VII, loosening petals and maturing pollen; VIII is 1 day before full bloom; IX flourishing. The treatment with the agent was carried out at the IV (arrow) stage. Sporogenous cell Sp; microsporocyte MI; a tetrad Tds; microspore MSp; pollen grains PG; the lower right angle scale in A is 50. mu.M.
In FIG. 2A 1-A4 are controls, B1-B4 are 10 μ M NDGA solution treated tomato anthers, C1-C4 are 100 μ M NDGA solution treated tomato anthers, and D1-D4 are 1000 μ M NDGA solution treated tomato anthers; A1-D1, A2-D2, A3-D3, and A4-D4, respectively, 2 days after treatment, 4 days after treatment, 6 days after treatment, and 8 days after treatment; the scale in A1 is 200. mu.M, and the other scales are 50. mu.M. Malformed pollen grains APG; and (4) normal pollen PG.
Previous studies found that pollen abnormalities in transgenic tomatoes with suppressed genes for key genes for ABA biosynthesis started after the tetrad stage (Dai et al, 2018), and therefore we treated the anthers with 0 μ M, 10 μ M, 100 μ M and 1000 μ M NDGA solutions at stage iv, when pollen development was in the microspore cell stage, and then sampled and observed on days 2, 4, 6 and 8 after treatment. By using microtomy, we observed that the control group developed well after 2, 4, 6, and 8 days of treatment, microspore cells developed further and completely with the maturation of anthers, the shape gradually developed into regular circles, and the staining was also uniform and thick (fig. 2, a1-a 4); more than 75% pollen grains have matured already at day 8 (FIG. 2, A4 and FIG. 4, A), however, treatment with NDGA solution of different concentrations causes pollen maturation abnormalities to a different extent, and the 10. mu.M NDGA solution treated anthers found a small amount of malformed pollen at day 2, while still a large amount of malformed pollen appeared at day 4, and the number of malformed pollen grains gradually increased at day 6 until the rate of malformed pollen grains reached 90% at day 8 after treatment (FIG. 2, B1-B4 and FIG. 4, A), treatment with 100. mu.M NDGA solution, significant malformed pollen appeared at day 4, and the normal pollen rate was only around 12% (FIG. 4, A), and thereafter the normal pollen rate remained around 10% with the increase of treatment time, but the degree of pollen malformation increased (FIG. 2, C1-C4 and FIG. 4, A), on the other hand, the 1000. mu.M NDGA solution showed significant abnormality on day 2 after the treatment (FIG. 2, D1), the normal pollen rate decreased to about 10% (FIG. 4, A), and the normal pollen rate was maintained at about 10% as the treatment time was extended (FIG. 2, D1-D4 and FIG. 4, A).
The results show that the tomato anthers treated by different concentrations of the ABA biosynthesis inhibitor NDGA influence the pollen maturation to different degrees, and the higher the concentration of NDGA, the more serious the influence of the longer the treatment time on the pollen maturation.
In FIG. 3A 1-A4 are controls, B1-B4 are 10 μ M NDGA solution treated tomato anthers, C1-C4 are 100 μ M NDGA solution treated tomato anthers, and D1-D4 are 1000 μ M NDGA solution treated tomato anthers; A1-D1, A2-D2, A3-D3, and A4-D4, respectively, 2 days after treatment, 4 days after treatment, 6 days after treatment, and 8 days after treatment; the scale in A1 is 200. mu.M, and the other scales are 50. mu.M. Malformed pollen grains APG; and (4) normal pollen PG.
For ABA treatment, the control group gradually increased the percentage of normal pollen as development proceeded normally, reached 80% by day 8 (FIG. 3, A1-A4 and FIG. 4, B), the anther was treated with 10. mu.M ABA solution, a small amount of abnormal pollen appeared by day 2 (FIG. 3, B1), the percentage of normal pollen gradually decreased by treatment time, the percentage of normal pollen remained only around 10% by day 8 after treatment (FIG. 3, B1-B4 and FIG. 4, B), the ABA solution treatments of 100. mu.M and 1000. mu.M showed similar trends, the small amount of abnormal pollen appeared by day 2 after treatment, and the large amount of abnormal pollen appeared by day 4 after treatment, and the normal percentage had decreased to around 10%, and thereafter remained around 10% by treatment time (FIG. 3, C1-C4, D1-D4 and FIG. 4, B).
The data in fig. 4 can be seen as: excessive ABA also causes abnormal pollen maturation, and similar results were shown for different concentrations of ABA solution treated anthers.
In summary, the following results can be obtained: in the part of the treatment of the ABA biosynthesis inhibitor NDGA, the tomato anthers are treated with NDGA in different concentrations to affect the pollen maturation to different degrees, and the higher the concentration of NDGA, the more serious the effect of the longer treatment time on the pollen maturation (the earlier and more abnormal pollen appears). This suggests that ABA may play a crucial role in the maturation of tomato pollen and that treatment of tomato pollen with NDGA solutions at concentrations above 10 μ M is effective in causing sterility of tomato pollen.
And the ABA treatment discovers that excessive ABA can cause abnormal pollen maturation, and similar results are shown when ABA solutions with different concentrations are used for treating anthers. As used herein, 10. mu.M, 100. mu.M and 1000. mu.M ABA solutions all exhibited large amounts of misshapen pollen grains at day 2 after treatment, after which the effect was exacerbated as treatment time was extended, but generally remained similar. This shows that, in the ripening process of tomato pollen, it is not better that ABA concentration is higher, and when the ABA concentration exceeds a certain threshold, the growth of tomato pollen is inhibited, whereas ABA solution of 10 μ M in this text far exceeds the threshold required for pollen growth, and the search for the ABA threshold required for pollen growth is needed for further research in the later period, and pollen abortion can be effectively caused by treating anther with ABA of concentration exceeding the threshold.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for exploring the cytological influence of exogenous ABA treatment on tomato pollen maturation is characterized by comprising the following steps of:
s1, sample preparation: the tomato macadamia powder 208 inbred line is an experimental sample;
s2, preparation of reagents:
abscisic acid (ABA): a,0 μ M concentration of ABA; b, 10. mu.M concentration of ABA; c, 100. mu.M concentration of ABA; d, ABA at a concentration of 1000 μ M;
dihydroguaiaretic acid (NDGA): e, NDGA at a concentration of 0 μ M; f,10 μ M concentration of NDGA; g, NDGA at a concentration of 100. mu.M; h, NDGA at a concentration of 1000. mu.M; the reagents are respectively placed into standby vessels, so that the subsequent use is convenient;
s3, detecting the percentage of normal pollen after the reagent is used
(1) Selecting buds in microspore period, taking liquid medicine by using an injector, slightly breaking sepals by using a needle head, and injecting the liquid medicine into anthers;
(2) immediately putting the flower buds into FAA fixing liquid, vacuumizing for 20-30Min, and fixing for 24h and then carrying out paraffin section preparation;
(3) after the sample fixed by the FAA stationary liquid is subjected to gradient dehydration by tert-butyl alcohol, the sample is embedded in paraffin, the sample is sliced on a rotary microtome, then the slice is placed on a clean glass slide, a deionized water floating sheet is added, and then the sample is placed in an oven for baking;
(4) after a sample is baked, dewaxing is carried out in dimethylbenzene, and then the sample is dyed in Hai's hematoxylin dyeing solution after being rehydrated by gradient ethanol;
(5) and then dehydrated by gradient ethanol, redyeed by 1% fast green, observed by Canada gum sealing piece after being transparent by dimethylbenzene, and counted by a microscope to obtain the percentage of normal pollen.
2. The method for exploring the cytological effect of exogenous ABA treatment on tomato pollen maturation according to claim 1, wherein the method comprises the following steps: multiple sets of samples were taken at step 1 in step S3 and were sampled at days 2, 4, 6, and 8 after treatment, respectively.
3. The method for exploring the cytological effect of exogenous ABA treatment on tomato pollen maturation according to claim 1, wherein the method comprises the following steps: and the rotary wheel microtome slices the sample, and the slice thickness is 8-10 mu M.
4. The method for exploring the cytological effect of exogenous ABA treatment on tomato pollen maturation according to claim 1, wherein the method comprises the following steps: when the pollen percentage is observed and counted, the pollen percentage is observed under an OLYMPUS BX41 optical microscope, and is photographed by an OLYMPUS C-7070Wide ZooM, and the photograph is processed and edited by Adobe Photoshop CS5 software.
5. The method for exploring the cytological effect of exogenous ABA treatment on tomato pollen maturation according to claim 1, wherein the method comprises the following steps: the temperature for drying the sample is 43 ℃, and a drying process needs to be carried out for 24 hours.
6. The method for exploring the cytological effect of exogenous ABA treatment on tomato pollen maturation according to claim 1, wherein the method comprises the following steps: when the percentage of normal pollen is counted in the step 5 in the step S3, after hematoxylin and green fixing staining, the pollen is observed under a microscope, some pollen is very clearly observed to be empty, flat, transparent and abnormal, and some pollen is full, regular and opaque to be normal, so that the quality of the pollen can be preliminarily judged by counting the percentage of normal pollen, and the higher the percentage of normal pollen is, the higher the quality of the pollen is.
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| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN108827964A (en) * | 2018-07-05 | 2018-11-16 | 南京林业大学 | A kind of method of quick determining green bamboo Pistil And Stamen developmental process |
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|---|---|---|---|---|
| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN108827964A (en) * | 2018-07-05 | 2018-11-16 | 南京林业大学 | A kind of method of quick determining green bamboo Pistil And Stamen developmental process |
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| K.N. Chandra Sekhar等.Role of ABA in stamen and pistil development in the normal and solanifolia mutant of tomato (Lycopersicon esculentum).《Sex Plant Reprod》.1991,第4卷 * |
| 周云等.外源ABA对拟南芥花粉生长的影响.《河南大学学报(自然科学版)》.2010,第40卷(第3期), * |
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