Gibberellin synergizes vernalization to induce flowering in strawberry

Abstract

The application discloses a gibberellin synergistic vernalization strawberry flowering experiment method, and can effectively verify the influence of gibberellin synergistic vernalization on the growth and development of a dormant period strawberry plant and the influence on the gene expression of the flowering of the strawberry plant, the gibberellin synergistic vernalization promotes the expression of flowering genes FT, SOC1, LYF and CO, and simultaneously inhibits the expression of flowering inhibition genes SVP and TFL1, so that the strawberry plant can flower in advance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant culture, and particularly relates to a gibberellin synergistic vernalization strawberry flowering experiment method. BACKGROUND

[0002] As a popular fruit, strawberries are loved for their delicious taste and versatile uses. However, the growth and flowering of strawberries are influenced by seasons and environments, which poses certain challenges to agricultural production. Strawberry plants usually start flowering in spring, but early flowering is crucial for commercial production and yield increase. To address this challenge, plant biologists and agricultural researchers have been seeking innovative methods to promote flowering in strawberry plants at the right time.

[0003] Gibberellin is a plant growth hormone that plays a key role in plant growth and flowering. Gibberellin can promote stem length and flower bud formation, both of which are important components of strawberry flowering. In some cases, by externally applying gibberellin, the level of gibberellin in strawberry plants can be increased to stimulate flowering. Vernalization is one of the adaptive strategies of plants to cold environments. In natural environments, strawberry plants need to experience a cold winter to enter the flowering stage in spring. Vernalization involves the perception and signaling of low temperature by plants, which leads to the activation of flowering-related genes.

[0004] TFL1, as a flowering inhibitor, inhibits flower bud formation and flowering. During vernalization, the expression level of TFL1 decreases, reducing the inhibition of flower bud differentiation and helping to promote the flowering of strawberries. SVP, similar to TFL1, is also a flowering inhibitor that inhibits flower bud differentiation and prolongs the growth period of plants. During vernalization, the expression level of SVP decreases, thereby reducing the inhibition of flower bud differentiation and promoting the flowering of strawberries. SOC1 is an important flowering integrator that promotes flower bud differentiation and flowering. During vernalization, the expression level of SOC1 increases, thereby increasing the likelihood of flowering. LYF is a key transcription factor that is crucial for the regulation of flower bud differentiation and flowering. During vernalization, the expression of LYF usually increases, helping to promote the flowering of strawberries. FT is a signal molecule that is produced in leaves and transmitted to the stem tip, promoting flower bud differentiation and flowering. During vernalization, the expression of FT usually increases, which is one of the key signals for flowering. CO is also an important flowering regulator that plays a role in the photoperiod pathway and is related to the expression of FT. During vernalization, the regulation of the photoperiod pathway helps to increase the expression of CO, thereby promoting the production of FT and flowering. These factors interact in a complex manner and collectively participate in the regulation of flower bud differentiation and flowering in strawberry plants.

[0005] Therefore, a strawberry cultivation method capable of gibberellin synergistic vernalization is needed to realize controllable flowering time of strawberries. SUMMARY

[0006] The present application aims to solve the above technical problems, and provides a gibberellin synergistic vernalization strawberry flowering experiment method, which can effectively verify the influence of gibberellin synergistic vernalization on the growth and development of dormant period strawberry plants and the influence on the gene expression of strawberry plant flowering.

[0007] The present application solves the above technical problems by adopting the technical scheme of a gibberellin synergistic vernalization strawberry flowering experiment method, comprising the following steps:

[0008] Step one: select a virus-free strawberry stolon plant, plant the strawberry stolon stem seedlings in a hole tray filled with a mixture of peat and vermiculite, divide them into several treatment groups, spray gibberellin, paclobutrazol or water on the leaves of strawberry plants in different treatment groups, and transfer the plants to a vernalization condition plant growth box for flower induction culture;

[0009] Step two: place the plants after flower induction in a greenhouse with natural light and temperature conditions, use the plants treated by flower induction as a blank control group, collect the plant leaves, stems, stem tips and flower samples, and record the plant leaf length, leaf width, plant height and flowering conditions;

[0010] Step three: after collecting the leaf, stem, stem tip and flower samples, wrap them with tin paper and freeze them in liquid nitrogen, then place the samples in an ultra-low temperature refrigerator, grind the strawberry tissue into powder in liquid nitrogen, and transfer it to a centrifuge tube;

[0011] Step four: add preheated CTAB to the centrifuge tube, then add mercaptoethanol in the fume hood, shake to mix thoroughly, place the sample mixed with the medicine in a 65℃ water bath, then add phenol-chloroform-isoamyl alcohol in the fume hood, then invert, ice bath, and then centrifuge at 4℃;

[0012] Step five: add phenol-chloroform-isoamyl alcohol to a new centrifuge tube in the fume hood, then take an equal volume of supernatant of the sample after step four centrifugation and add it to the new centrifuge tube, invert, ice bath, and then centrifuge at 4℃;

[0013] Step six: add ethanol to the new centrifuge tube, add the supernatant obtained in step five to the ethanol, shake evenly, then precipitate in the refrigerator, and centrifuge the precipitated solution at 4℃ in the centrifuge;

[0014] Step seven: remove the supernatant of the solution obtained by centrifugation, add pre-cooled anhydrous ethanol on ice, fully dissolve and mix by flicking, and continue to centrifuge in a centrifuge cooled to 4 DEG C;

[0015] Step eight: remove the supernatant of the solution after centrifugation until there is no excess supernatant, place on a workbench under ventilation, add DEPC water to fully dissolve the precipitate, and use a microspectrophotometer to determine the purity and concentration of total RNA.

[0016] Step nine: according to the instructions of the reverse transcription kit, mix 1 μg of total RNA with PrimeScript RT Enzyme Mix I for reverse transcription, dilute the obtained cDNA by three times, and then perform real-time quantitative PCR cycles.

[0017] Preferably, in step one, before spraying gibberellin, paclobutrazol or water, the strawberry stolon shoots are cultured for two weeks under 12 hours of light and 30 DEG C environment to prevent flower induction.

[0018] Preferably, in step one, the gibberellin solution is 10 mg / L -1 or 20 mg / L -1 , and the paclobutrazol solution is 10 mg / L -1 .

[0019] Preferably, in step one, the vernalization conditions are 8 hours of light, 12 DEG C, and 16 hours of darkness, 8 DEG C.

[0020] Preferably, in step four, 588 μL of CTAB with a concentration of 2% is added after preheating, and 12 μL of mercaptoethanol with a concentration of 0.4% is added in a fume hood.

[0021] Preferably, in step four, 600 μL of phenol-chloroform-isoamyl alcohol is added in a fume hood, and in step five, 500 μL of phenol-chloroform-isoamyl alcohol is added in a fume hood, and the ratio of phenol-chloroform-isoamyl alcohol in steps four and five is 25:24:1.

[0022] Preferably, in step six, 400 μL of ethanol is added in a new centrifuge tube, and 400 μL of supernatant obtained in step five is added in the ethanol.

[0023] Preferably, the real-time quantitative PCR cycles in step nine include the following: denaturation at 95 DEG C for 1 minute, then 40 cycles of 95 DEG C for 20 seconds, 58 DEG C for 20 seconds and 72 DEG C for 20 seconds.

[0024] The present application has the beneficial effects of:

[0025] 1. The present application can effectively verify the influence of gibberellin on the growth and development of strawberry plants during the dormant period, and the influence on the gene expression of strawberry plant flowering. 2. The present application can effectively verify the influence of vernalization on the growth and development of strawberry plants during the dormant period, and the influence on the gene expression of strawberry plant flowering.

[0026] 2、GA3 synergistic vernalization promotes the expression of FT, SOC1, LYF and CO, and inhibits the expression of SVP and TFL1, so that the strawberry plants can flower earlier. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a statistical diagram of the influence of different conditions on the leaf area of strawberry plants.

[0028] Figure 2 is a statistical diagram of the influence of different conditions on the leaf area of strawberry plants.

[0029] Figure 3 is a statistical diagram of the influence of different conditions on the leaf area of strawberry plants.

[0030] Figure 4 is a statistical diagram of the influence of different conditions on the leaf area of strawberry plants.

[0031] Figure 5 is a statistical diagram of the influence of different conditions on the leaf area of strawberry plants.

[0032] Figure 6 is a statistical diagram of the influence of different conditions on the leaf area of strawberry plants. DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with the drawings and embodiments.

[0034] As shown in Figure 1-6 , the present application is a method for promoting flowering of strawberry plants by synergistic vernalization of GA3, comprising the following steps:

[0035] Step 1: Select a virus-free strawberry plant with 5-6 leaves and plant it in a 10 cm wide, 15 cm deep hole plate filled with a mixture of peat and vermiculite, and divide it into several treatment groups under 12 hours of light and 30°C for two weeks to prevent flower induction; then, spray GA3, paclobutrazol or water on the leaves of the strawberry plants in different treatment groups, the GA3 solution is 10 mg L -1 or 20 mg L -1 , the paclobutrazol solution is 10 mg L -1 , and the plants are transferred to a vernalization plant growth chamber for 3 weeks of flower induction culture, the vernalization conditions are 8 hours of light, 12°C, and 16 hours of darkness, 8°C;

[0036] Step two: the plants are placed in a greenhouse with natural light and temperature conditions, the light time is 10-11h, the temperature is 20±2℃, and the plants treated by flower induction are used as blank control group, the plants are cultured under 12h light and 30℃ for two weeks, then placed in a greenhouse with natural light and temperature conditions, the leaf samples of the blank control group and the treated group are collected at 0, 3, 6, 9, 12, 24 and 48 hours after the beginning of the flower induction experiment, and the leaf, stem, stem tip and flower samples of the plants are collected every seven days (if there is no sample, it is not taken), and the leaf length, leaf width, plant height and flowering of the plants are recorded every seven days;

[0037] Step three: after the leaf, stem, stem tip and flower samples are collected, they are wrapped with tin paper and frozen in liquid nitrogen, then the samples are placed in a-80℃ ultra-low temperature refrigerator, the strawberry tissue is ground into powder in liquid nitrogen and transferred into a 2ml centrifuge tube;

[0038] Step four: 588μL of CTAB with a concentration of 2% is added to the 2ml centrifuge tube, then 12μL of mercaptoethanol with a concentration of 0.4% is added in the fume hood, and the mixture is shaken to mix thoroughly, the sample mixed with the medicine is preheated in a 65℃ water bath for 10min, and it is inverted and shaken every 2min during the period, 600μL of phenol-chloroform-isoamyl alcohol with a ratio of 25:24:1 is added in the fume hood, then after being inverted, it is ice-bathed for 10min, and the temperature of the centrifuge is reduced to 4℃, and then it is centrifuged at 11000rpm / min for 10min;

[0039] Step five: 500μL of phenol-chloroform-isoamyl alcohol with a ratio of 25:24:1 is added to a new 1.5ml centrifuge tube in the fume hood, then an equal volume of supernatant of the sample centrifuged in step four is added to the new centrifuge tube, it is inverted and ice-bathed for 10min, and then the temperature of the centrifuge is reduced to 4℃, and it is centrifuged at 11000rpm / min for 10min;

[0040] Step six: 400μL of ethanol is added to a new 1.5ml centrifuge tube, 400μL of supernatant obtained in step five is added to the ethanol, and after being shaken evenly, it is precipitated in a 4℃ refrigerator for 60-90min, and then the precipitated solution is centrifuged in a centrifuge with a temperature reduced to 4℃, at 11000rpm / min for 15min;

[0041] Step seven: the supernatant of the solution obtained by centrifugation is removed, 600μL of pre-cooled anhydrous ethanol is added on ice, and it is fully dissolved and mixed by tapping, and then it is centrifuged in a centrifuge with a temperature reduced to 4℃, at 11000rpm / min for 10min;

[0042] Step eight: remove the supernatant until no extra supernatant, place on the bench for 2 min, add 20 μL DEPC water to dissolve the precipitate, use micro spectrophotometer to determine the purity and concentration of total RNA;

[0043] Step nine: according to the instructions of reverse transcription kit, take 1 μg total RNA mixed with PrimeScript RT Enzyme Mix I for reverse transcription, then dilute the obtained cDNA three times, and then perform real-time quantitative PCR cycle, use TBGreen TM Premix Ex Taq TM Real-time quantitative PCR was performed on a quantitative PCR instrument, and the real-time quantitative cycle program was as follows: denaturation at 95℃ for 1 min, then 95℃ for 20 s, 58℃ for 20 s and 72℃ for 20 s for 40 cycles, and the relative expression level was normalized by the untreated 0 h strawberry 26 s rRNA as an internal reference gene to standardize the original data.

[0044] The experimental results of this example are as follows:

[0045] (1) Effect of gibberellin synergistic vernalization on growth and development of dormant strawberry plants

[0046] As shown in Figure 1 , Figure 2 , vernalization treatment and gibberellin inhibitor had a significant effect on leaf area, and vernalization significantly increased leaf area, while gibberellin inhibitor significantly reduced leaf area. The difference between vernalization treatment + 10 mg / L gibberellin and vernalization treatment + 20 mg / L gibberellin was not significant. In summary, vernalization can significantly increase leaf area, different concentrations of gibberellin have no significant effect on leaf area, but gibberellin inhibitor can significantly reduce leaf area.

[0047] Plant height: as shown in Figure 3 , Figure 4 , vernalization can significantly increase the height of strawberry plants, and gibberellin synergistic vernalization treatment can also increase the height of strawberry plants, and 20 mg / L gibberellin treatment has the best effect on increasing the height of strawberry plants. Gibberellin inhibitor treatment can alleviate the effect of vernalization on the height of strawberry plants, so that the height of the treated group is not significantly different from the control group, which indicates that gibberellin inhibitor can eliminate the effect of vernalization. In summary, vernalization + 20 mg / L gibberellin treatment has the best effect on increasing plant height, and gibberellin inhibitor can eliminate the effect of vernalization on plant height.

[0048] In summary, the most suitable conditions for promoting the dormancy of strawberry plants are vernalization + 20 mg / L gibberellin treatment. Vernalization at 12°C for 21 days and 20 mg / L gibberellin treatment can significantly increase leaf area and plant height, and can significantly increase leaf area (52.60%) and plant height (50.60%) compared with the blank control group.

[0049] (2) Vernalization combined with gibberellin treatment can accelerate flower bud differentiation of strawberry plants

[0050] As shown in Figure 5 , different treatments were performed on dormant strawberry plants, and it was found that the vernalization + gibberellin treatment group could significantly increase the flowering rate compared with the control group. Figure 5 The vernalization + 20 gibberellin treatment group began to show buds at 28 days after treatment, while the control group began to show buds at 49 days. Compared with the control group, the vernalization + 20 gibberellin treatment group could accelerate flower bud differentiation by 21 days. Among different vernalization treatment groups, the flowering rate of strawberry plants was vernalization + gibberellin 20 mg / L treatment group > vernalization + gibberellin 10 mg / L treatment group > vernalization treatment group > vernalization + gibberellin inhibitor group, and the flowering rate of the vernalization + gibberellin 20 mg / L treatment group was as high as 95.00% at 84 days.

[0051] In summary, for promoting flower bud differentiation of strawberry plants, vernalization at 12°C for 21 days combined with 20 mg / L gibberellin spraying on leaves is the best, which can accelerate the flowering by 21 days compared with the control group, thereby realizing the early maturation of fruits.

[0052] (3) Effect of gibberellin combined with vernalization on gene expression of flowering of strawberry plants

[0053] As shown in Figure 6As shown in the figure, it can be found that the vernalization + gibberellin inhibitor group has higher expression of flowering inhibition gene TFL1 at each time point. With the increase of cultivation time, the low expression of TFL1 gene in the vernalization group re-started to express at the fourth week and reached a peak at the sixth week, but the expression of TFL1 in the 10, 20 ppm GA3 groups remained low. The expression of flowering inhibitor SVP is similar to TFL1, but its expression is down-regulated in each group with the decrease of light time and temperature, and the vernalization combined with gibberellin treatment has lower expression than the simple vernalization treatment. The expression of flowering integrator FT is opposite to TFL1. The treatment of gibberellin inhibitor completely silences FT, and the change of temperature and light has no effect on the expression of FT, while the 20 ppm gibberellin group has higher FT expression. The key flowering genes SOC1, LYF and CO show certain commonality. In 1-10 weeks, the expression of these three genes is vernalization + gibberellin 20 mg / L treatment group > vernalization + gibberellin 10 mg / L treatment group > vernalization treatment group > vernalization + gibberellin inhibitor group. The treatment of gibberellin up-regulates the expression of genes, and within a certain range, the higher the concentration of gibberellin, the higher the expression of genes, while the treatment of gibberellin inhibitor offsets the effect of vernalization treatment, so that the paclobutrazol group and the CK group have no significant difference.

[0054] In summary, gibberellin combined with vernalization promotes the expression of flowering genes FT, SOC1, LYF and CO, and inhibits the expression of flowering inhibition genes SVP and TFL1, which indicates that this synergistic effect explains the gene principle of early flowering at the gene level.

[0055] It should be understood that the specific embodiments described herein merely serve to explain the present application and are not intended to limit the present application.

Claims

1. A strawberry flowering experimental method using gibberellins in conjunction with vernalization, characterized by: The following steps are involved: Step 1: Select virus-free strawberry runners and plant them in plug trays filled with peat-vermiculite mixture to divide them into several treatment groups. Spray the leaves of strawberry plants in different treatment groups with gibberellin, paclobutrazol or water. The gibberellin solution is 10 mg / L -1 or 20 mg / L -1 Paclobutrazol solution is 10 mg L -1 and transferring the plants into a plant growth chamber under vernalization conditions for flower induction culture; Step 2: Place the plants that have completed floral induction in a greenhouse with natural light and temperature conditions for cultivation. Use the plants that have undergone floral induction as a blank control group. Collect samples of the leaves, stems, stem tips, and flowers of the plants and record the leaf length, leaf width, plant height, and flowering status of the plants. Step 3: Collect samples of leaves, stems, stem tips, and flowers, wrap them in tin foil, and freeze them in liquid nitrogen. After freezing, place the samples in an ultra-low temperature freezer. Grind the strawberry tissue into powder in liquid nitrogen and transfer it to a centrifuge tube. Step 4: Add preheated CTAB to the centrifuge tube, then add mercaptoethanol in a fume hood and shake to mix thoroughly. Preheat the sample mixed with the drug in a 65°C water bath. Add phenol-chloroform-isoamyl alcohol in a fume hood, then invert the tube, place on ice, and then cool the centrifuge to 4°C before centrifugation. Step 5: In a fume hood, add phenol-chloroform-isoamyl alcohol to a new centrifuge tube. Then, take an equal volume of the supernatant from the sample centrifuged in step 4 and add it to the new centrifuge tube. Invert the tube and place it on ice. Then, cool the centrifuge to 4°C and centrifuge. Step 6: Add ethanol to a new centrifuge tube, add the supernatant obtained in step 5 to the ethanol, shake evenly and precipitate in the refrigerator, and centrifuge the precipitated solution in a centrifuge at 4°C; Step 7: Remove the supernatant from the solution obtained by centrifugation, add pre-cooled anhydrous ethanol on ice, flick to fully dissolve and mix, and continue centrifugation in a centrifuge cooled to 4°C; Step 8: Remove the supernatant from the centrifuged solution until there is no excess supernatant, let it stand on the workbench with ventilation, add DEPC water to fully dissolve the precipitate, and use a micro-spectrophotometer to measure the purity and concentration of the total RNA; Step 9: According to the instructions of the reverse transcription kit, 1 μg of total RNA was mixed with PrimeScript RT Enzyme Mix I for reverse transcription. The obtained cDNA was diluted three times and then subjected to real-time quantitative PCR cycles. The relative expression levels of genes SVP, SOC1, TFL1, CO, FT, and LYF were determined by real-time quantitative PCR cycles.

2. The strawberry flowering experimental method of gibberellin-assisted vernalization according to claim 1, characterized in that: In step 1, before spraying gibberellin, paclobutrazol or water, the strawberry runner seedlings were cultured under 12 hours of light and 30°C for two weeks to prevent flower induction.

3. The strawberry flowering experimental method of gibberellin-assisted vernalization according to claim 1, characterized in that: The vernalization conditions in step 1 were 8 hours of light, 12°C and 16 hours of darkness, 8°C.

4. The strawberry flowering experimental method of gibberellin-assisted vernalization according to claim 1, characterized in that: In step 4, 588 μL of preheated 2% CTAB was added, and 12 μL of 0.4% mercaptoethanol was added in a fume hood.

5. The strawberry flowering experimental method of gibberellin-assisted vernalization according to claim 1, characterized in that: In step 4, 600 μL of phenol-chloroform-isoamyl alcohol was added to the fume hood, and in step 5, 500 μL of phenol-chloroform-isoamyl alcohol was added to the fume hood. The ratio of phenol-chloroform-isoamyl alcohol in steps 4 and 5 was 25:24:

1.

6. The strawberry flowering experimental method of gibberellin-assisted vernalization according to claim 1, characterized in that: In step 6, 400 μL of ethanol was added to a new centrifuge tube, and 400 μL of the supernatant obtained in step 5 was added to the ethanol.

7. The strawberry flowering experimental method of gibberellin-assisted vernalization according to claim 1, characterized in that: The real-time quantitative PCR cycle in step nine includes the following: denaturation at 95°C for 1 minute, followed by 40 cycles of 95°C for 20 seconds, 58°C for 20 seconds, and 72°C for 20 seconds.

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