Method for in-vitro induction of tetraploid grapes

Abstract

The invention discloses a method for in-vitro induction of tetraploid grapes, and belongs to the technical field of grape tetraploid induction, and the method comprises the following steps: S1, taking tender diploid grape tissue culture seedlings, removing terminal buds, cutting into stem segments, and pre-culturing for 3-5 days; s2, performing colchicine induction on the pre-cultured stem segments, and inoculating the stem segments into a first culture medium for first culture after the induction is finished; s3, after the first culture is completed, each tissue culture seedling is cut into stem segments, the stem segments are transferred into a second culture medium for second culture, and finally the grape tetraploid tissue culture seedlings are obtained. The invention provides an induction method of tetraploid grapes, which provides a practical basis for polyploid breeding work of grapes. By means of ploidy breeding, the defects that diploid grapes are small in fruit size, large in seed number and poor in resistance are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of grape tetraploid induction, and more particularly to a method for in vitro induction of tetraploid grapes. Background Art

[0002] Grape (Vitis vinifera L.), as a deciduous vine fruit tree of the genus Vitis in the family Vitaceae, has extremely high nutritional value and economic benefits globally. Grape fruits can not only be used for wine-making, juicing, making raisins and fresh consumption, but also are widely planted around the world. Especially in China, grape cultivation has a history of thousands of years and is one of the important fruit tree species in China.

[0003] Fruit tree polyploids have advantages such as thick branches, thick leaves, large fruits, strong growth, high yield, strong adaptability and strong stress resistance. These advantages can be maintained stably without segregation and can be propagated asexually. Therefore, the cultivation of polyploids has attracted the attention of fruit tree breeders. Since the chromosome number of polyploid plants increases, the morphological characteristics of the plant body will also change, and they are stronger in terms of resistance than the original body, and have great value in breeding research work and biological evolution. Polyploids are more adaptable to new environments than diploids, and have characteristics such as gigantism, high nutrient content and low fertility.

[0004] The development of fresh table grapes in China is rapid. Currently, the mainly cultivated grape varieties are diploids, and a small number are triploids and tetraploids. Among them, tetraploid grapes show prominent advantages such as dark green leaves, thick stems, short internodes, large fruit grains, few seeds, high assimilate content and strong resistance. Therefore, through ploidy breeding, the fruit grain size and resistance of diploid grapes can be significantly improved. There are many ways of polyploid breeding. Among them, sexual hybridization breeding is a traditional way to obtain polyploids, but this method has a relatively long cycle, while using colchicine to induce grape tetraploids in vitro has various prominent advantages such as short cycle and quick effect, and has increasingly become the first choice of breeders.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] In view of the above technical problems, the present invention can obtain tetraploid grape tissue culture seedlings with large fruit grains, few seeds and strong resistance by inducing polyploidy in diploid grapes.

[0007] The present invention is implemented as follows:

[0008] The present invention provides a method for in vitro induction of tetraploid grapes, comprising the following steps:

[0009] S1. Take young diploid grape tissue culture seedlings, perform decapitation treatment, cut them into stem segments, and carry out pretreatment for 3 - 5 days;

[0010] S2. Induce the pre-cultured stem segments with colchicine. After the induction is completed, inoculate them in the first culture medium for the first culture;

[0011] S3. After the first culture is completed, cut each tissue culture seedling into stem segments and transfer them to the second culture medium for the second culture to finally obtain tetraploid tissue culture seedlings of grapes.

[0012] S4. Identify the tetraploid plants by flow cytometry. The specific method is as follows: Take 0.2 g of young leaves, lyse the cell nuclei with the CyStain UVPrecise P kit, detect after DAPI staining for 2 minutes, and screen the tetraploid tissue culture seedlings with the peak fluorescence intensity located on the right side of the diploid and more concentrated distribution.

[0013] In some preferred embodiments, the diploid grape is Jintian Rose grape.

[0014] In some preferred embodiments, the colchicine is a colchicine solution of 2000 mg / L - 6000 mg / L or a colchicine solid medium with a concentration of 200 mg / L - 400 mg / L.

[0015] In some preferred embodiments, the composition of the first culture medium is a medium of 1 / 2MS + 5.0 g / L agar + 20 - 30 g / L sucrose.

[0016] In some preferred embodiments, the second culture medium is a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose + 0.1 - 0.5 mg / L IBA.

[0017] In some preferred embodiments, when containing the colchicine solution, the fixation time is 24 - 72 h.

[0018] In some preferred embodiments, the treatment conditions are: temperature 24 - 26°C, under light-shielded conditions, and the rotation speed of the constant-temperature shaker is 180 rpm.

[0019] In some preferred embodiments, the treatment time of the colchicine solid medium is 20 - 40 d.

[0020] In some preferred embodiments, the culture conditions for the treatment are: temperature 23 - 25°C, 2000 lx light for 16 h, and dark conditions for 8 h.

[0021] In some preferred embodiments, the morphological characteristics of the tetraploid plants in step S4 include: the stem diameter increases by 20% - 30%, the leaf area increases by 40% - 50%, and the flow cytometry detection shows that the chromosome multiple doubles.

[0022] The present invention has the following beneficial effects:

[0023] In the present invention, the diploid grape variety Jintian Rose (Muscat Hamburg and Red Globe) is transformed into a tetraploid grape through induction technology. During the induction process, the diploid grape Jintian Rose is treated with a colchicine solution at a concentration of 6000 mg / L for 48 hours. The results show that this method has a significant induction effect, the plants grow rapidly and are less poisoned, and the induction success rate reaches 17.78%. In addition, in the method of treating with a solid colchicine medium, treating with a solid colchicine medium at a concentration of 400 mg / L for 30 days can obtain the highest induction rate, reaching 8.89%. These results indicate that the present invention provides an effective method for inducing polyploid grapes, which helps to improve the genetic diversity and production performance of tetraploid grape varieties. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a comparison diagram of a diploid grape plant and a tetraploid grape plant;

[0026] Figure 2 It is a comparison diagram of the leaves of diploid and tetraploid grapes;

[0027] Figure 3 It is a comparison diagram of the rooting of diploid and tetraploid grapes;

[0028] Figure 4 It is an identification diagram of diploid grapes and tetraploid grapes by flow cytometry. Detailed Embodiments

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0030] The following specifically describes a method for in vitro inducing tetraploid grapes provided by the present invention.

[0031] A method for in vitro inducing tetraploid grapes includes the following steps:

[0032] S1. Take young diploid grape tissue culture seedlings, perform decapitation treatment, cut them into stem segments, and perform a pretreatment for 3 - 5 days;

[0033] S2. Induce the pre-cultured stem segments with colchicine. After the induction ends, inoculate them in the first culture medium for the first culture;

[0034] S3. After the first culture is completed, cut each tissue culture seedling into stem segments, transfer them to the second culture medium for the second culture, and finally obtain tetraploid grape tissue culture seedlings.

[0035] Colchicine, as a commonly used inducer, is used to induce plants to produce tetraploids. Its main mechanism is to bind to tubulin during cell division, inhibit the formation of the spindle, and cause the chromosomes of the cell to double. The autopolyploids induced by colchicine are often accompanied by changes in aspects such as plant morphology, anatomy, physiology, and cultivation characteristics. Therefore, in this application, colchicine is used to treat diploid grapes, and the obtained tetraploid grapes have characteristics such as large fruit grains and strong resistance.

[0036] In some preferred embodiments, the diploid grape is Jintian Rose grape.

[0037] Among them, Jintian Rose grape is bred by crossing diploid grape Muscat Hamburg and Red Globe. Both Muscat Hamburg and Red Globe are good parental materials. Muscat Hamburg has a strong rose fragrance and good quality, and Red Globe has advantages such as large fruit grains, hard pulp, and high disease resistance. The Jintian Rose obtained by their hybridization can combine these characteristics and has higher fruit quality and better adaptability.

[0038] In some preferred embodiments, the stem segment is a single-bud stem segment.

[0039] Using single-bud stem segments to prepare tetraploid grapes, because the preparation method of single-bud stem segments is relatively simple, a large number of samples can be processed quickly, reducing the preparation cost.

[0040] In some preferred embodiments, the colchicine-containing solution is a colchicine solution with a concentration of 2000 mg / L - 6000 mg / L or a colchicine solid medium with a concentration of 200 mg / L - 400 mg / L.

[0041] In some preferred embodiments, the composition of the first culture medium is a medium of 1 / 2MS + 5.0 g / L agar + 20 - 30 g / L sucrose.

[0042] In some preferred embodiments, the second culture medium is a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose + 0.1 - 0.5 mg / L IBA.

[0043] In some preferred embodiments, when using the colchicine solution, the treatment time is 24 - 72 h.

[0044] In some preferred embodiments, the conditions for the treatment are as follows: temperature 24 - 26°C, under light - avoiding conditions, and the rotation speed of the constant - temperature shaker is 180 rpm.

[0045] In some preferred embodiments, the treatment time of the colchicine solid medium is 20 - 40 d.

[0046] In some preferred embodiments, the culture conditions for the treatment are as follows: temperature 23 - 25°C, 16 - hour illumination with 2000 lx light, and 8 - hour dark conditions.

[0047] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0048] Example 1

[0049] A method for in vitro induction of tetraploid grapes provided in this example is as follows: Using Jintian Rose, which is bred by crossing Muscat Hamburg grapes and Red Globe grapes, as the explant, inoculating it into a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose, culturing it for 30 d under the conditions of temperature 25°C and 16 - hour illumination with 2000 lx tissue - culture - specific LED light, then removing the apical bud and cutting it into single - bud stem segments, and then pre - culturing it on the medium for 4 d.

[0050] Taking the pretreated single - bud stem segments of tetraploid grapes, after removing the leaves, drying the remaining moisture on the stem segments with a sterilized filter paper, putting them into a solution containing 6000 mg / L colchicine, placing them in a 26°C light - avoiding environment, soaking them with a constant - temperature shaker at 180 rpm for 48 h. After soaking, rinsing the stem segments 5 times with sterile water to wash away the residual colchicine agent on the stem segments, then drying the remaining moisture with a sterilized filter paper, inoculating the treated stem segments into a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose, continuously culturing them under the conditions of temperature 25°C and 16 - hour illumination with 2000 lx tissue - culture - specific LED light. After culturing for 40 d until the stem segments grow, cutting each plant into single - bud stem segments, transferring them into a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose + 0.2 mg / L IBA for separate culture, sub - culturing twice, and performing chromosome ploidy identification when the leaves grow to 6 - 8 leaves.

[0051] Example 2

[0052] Other steps and parameters of this example are the same as those of Example 1, and the only difference is that:

[0053] The concentration of the colchicine solution into which the pretreated single - bud stem segments are put is 4000 mg / L.

[0054] Example 3

[0055] This example is the same as Example 1 in other steps and parameters, except that:

[0056] The concentration of the colchicine solution into which the pre-treated single-bud stem segments are placed is 2000 mg / L.

[0057] Example 4

[0058] This example is the same as Example 1 in other steps and parameters, except that:

[0059] The treatment time for placing the pre-treated single-bud stem segments into the colchicine solution is 24 h.

[0060] Example 5

[0061] This example is the same as Example 1 in other steps and parameters, except that:

[0062] The treatment time for placing the pre-treated single-bud stem segments into the colchicine solution is 72 h.

[0063] Example 6

[0064] An induction method for in vitro tetraploid grapes provided in this example is as follows: Using Jintian Rose, which is bred from the hybridization of grape Muscat Hamburg and Red Globe, as the explant, inoculating it into a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose, culturing it for 30 d under the conditions of a temperature of 25 °C and a special LED light for tissue culture of 2000 lx for 16 h, then removing the apical bud and cutting it into single-bud stem segments, and then pre-culturing it on the medium for 4 d.

[0065] Taking the pre-treated diploid grape single-bud stem segments, placing them into a solid medium containing 400 mg / L colchicine, performing a 30-d medicament treatment under the conditions of a temperature of 25 °C and a special LED light for tissue culture of 2000 lx for 16 h, inoculating the treated stem segments into a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose, continuously culturing them under the conditions of a temperature of 25 °C and a special LED light for tissue culture of 2000 lx for 16 h, then culturing until 40 d, cutting each plant into single-bud stem segments, transferring them into a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose + 0.2 mg / L IBA for separate culture, subculturing twice, and performing chromosome ploidy identification when the leaves grow to 6 - 8 leaves.

[0066] Example 7

[0067] This example is the same as Example 4 in other steps and parameters, except that:

[0068] Placing the pre-treated single-bud stem segments into a solid medium with a colchicine concentration of 300 mg / L.

[0069] Example 8

[0070] This example is the same as Example 4 in other steps and parameters, except that:

[0071] The pretreated single-bud stem segments are placed in a solid medium with a colchicine concentration of 200 mg / L.

[0072] Example 9

[0073] This example is the same as Example 4 in other steps and parameters, except that:

[0074] The time for placing the pretreated single-bud stem segments in the colchicine solid medium is 20 days.

[0075] Example 10

[0076] This example is the same as Example 4 in other steps and parameters, except that:

[0077] The time for placing the pretreated single-bud stem segments in the colchicine solid medium is 40 days.

[0078] Comparative Example 1

[0079] This comparative example is the same as Example 1 in other steps and parameters, except that:

[0080] The pre-culture time of the single-bud stem segments in the comparative example is 0 days.

[0081] Comparative Example 2

[0082] This comparative example is the same as Example 1 in other steps and parameters, except that:

[0083] The pre-culture time of the single-bud stem segments in the comparative example is 8 days.

[0084] Comparative Example 3

[0085] This comparative example is the same as Example 1 in other steps and parameters, except that:

[0086] The stem segments used for treatment in the comparative example are double-bud stem segments.

[0087] Comparative Example 4

[0088] This comparative example is the same as Example 2 in other steps and parameters, except that:

[0089] The stem segments used for treatment in the comparative example are double-bud stem segments.

[0090] Comparative Example 5

[0091] This comparative example is the same as Example 3 in other steps and parameters, except that:

[0092] The stem segments used for treatment in the comparative example were double-bud stem segments.

[0093] Test Example 1

[0094] The tetraploid grape plants obtained by preparing in Example 1 were compared with the plants of diploid grapes.

[0095] The tetraploid grape seedlings and ordinary diploid grape seedlings obtained by preparing in Example 1 were subjected to ploidy identification. The flow cytometer used in the test was the CyFlow Space flow cytometer of the Sysmex Partec brand. After the tissue culture seedlings treated with the colchicine solution were cultured for 100 days and when the seedlings had 6 - 8 leaves, about 0.2 g of young leaves of the plants were taken and placed in a culture dish. 500 μl of the nuclear lysate in the CyStain UVPrecise P kit was added around the sample, and it was chopped with a sharp blade to fully extract the complete nuclei. The extraction time was 60 seconds. The liquid in the culture dish was filtered through a 50-μm celltrics filter into a sample tube. 2000 μl of the DAPI fluorescent staining solution in the CyStain UV Precise P kit was added to the sample tube, and it was stained for 2 minutes in the dark. The filtered supernatant was detected on the flow cytometer to determine the doubling rate.

[0096] The comparison results of the tetraploid grape plants and diploid grape plants are as Figure 1 , Figure 2 and Figure 3 shown. From the perspective of plant morphology, the tetraploid plants are more robust and have larger leaves than the diploid plants; from the comparison of the root systems, the root system of the tetraploid grapes is more developed, the number of roots is more than that of the diploid, and the root length is longer than that of the diploid; from the comparison of the leaves, the leaves of the tetraploid grapes are significantly larger than those of the diploid grapes; from the overall growth state, the growth of the stem segments of the tetraploid grapes in the culture medium is better.

[0097] The flow cytometer detection results of the tetraploid grapes and diploid grapes are as Figure 4 shown. The peak of the tetraploid grapes is located on the right side of the peak of the diploid grapes, which indicates that the fluorescence intensity of the tetraploid grape cells is higher; the distribution widths of the two are similar, indicating that the fluorescence intensity distribution ranges of the cells of the two kinds of grapes in the FL4 channel are similar; the peak height of the tetraploid grapes is slightly higher than that of the diploid grapes, which may mean that the fluorescence intensity distribution of the tetraploid grape cells in the FL4 channel is more concentrated.

[0098] Test Example 2

[0099] The tetraploid grapes cultured in the liquid culture media with different concentrations of colchicine in Examples 1 - 3 were compared, and the results are shown in Table 1.

[0100] The tetraploid grapes cultured on solid media with different colchicine concentrations in Examples 6 - 8 were compared, and the results are shown in Table 2.

[0101] Table 1. Doubling effects of colchicine solutions with different concentrations

[0102] Colchicine concentration mg / L Soaking time (h) Survival rate (%) Mortality rate (%) Doubling rate (%) 6000 48 31.11 68.89 17.78 4000 48 53.33 46.67 5.56 2000 48 91.11 8.89 0

[0103] Table 2. Doubling effects of solid media with different colchicine concentrations

[0104]

[0105] According to the results, it was found from the data comparison in Table 1 that among the solution agent treatments, the induction quantity was the most significant at 6000 mg / L for 48 h, with a doubling rate of 17.78%; from the data comparison in Table 2, it was found that among the solid media treatments with colchicine, the induction quantity was the most significant at 400 mg / L for 30 d, with a doubling rate of 8.89%. Based on the number of doublings for selection, the overall doubling quantity of the colchicine solution treatment was higher than that of the solid media with colchicine. From the data in Table 1 and Table 2, it can be seen that the concentration of the agent treatment has a certain influence on the doubling rate of grape chromosomes.

[0106] Test Example 2

[0107] The doubling effects of Examples 1, 4, and 5 after being induced with the same colchicine solution for different times were compared, and the results are shown in Table 3.

[0108] The tetraploid grapes of Examples 1, 4, and 5 cultured on solid media with the same colchicine concentration for different times were compared, and the results are shown in Table 4.

[0109] Table 3. Doubling effects of colchicine solution treatment for different times

[0110]

[0111] Table 4. Doubling effects of solid media with colchicine treatment for different times

[0112]

[0113] According to the results, it was found from the data comparison in Table 3 that when the colchicine treatment concentration was 6000 mg / L, the induction effect was the best after 48 h; from the data comparison in Table 4, it was found that when the solid media agent treatment concentration was 400 mg / L, the induction effect of tetraploid grapes was the best after 30 d. From the data in Table 3 and Table 4, it can be seen that the treatment time of the agent has a certain influence on the survival rate, mortality rate, and mutation rate of grape chromosomes.

[0114] Test Example 3

[0115] The tetraploid grapes obtained by subjecting Example 1, Comparative Example 1, and Comparative Example 2 to the same colchicine solution for the same induction time after different pretreatment times were compared, and the results are shown in Table 5.

[0116] Table 5. Doubling effects with different pre-culture days

[0117]

[0118] According to the test results, it can be seen that when the pre-culture days are reduced, the lethality rate of the chemical treatment will be higher; however, if the pre-culture days are too many, the doubling rate induced by the chemical treatment will decrease. Therefore, it is necessary to select an appropriate pre-culture time for treatment to increase its doubling rate.

[0119] Test Example 4

[0120] The tetraploid grapes obtained by subjecting Examples 1 - 3 and Comparative Examples 3 - 5 to the same colchicine solution for the same induction time using different stem segments were compared, and the results are shown in Table 6.

[0121] Table 6. Doubling effects with different stem segment types

[0122] Colchicine concentration mg / L Soaking time (h) Survival rate (%) Mortality rate (%) Doubling rate (%) 6000 48 46.67 53.33 13.33a 4000 48 75.56 24.44 3.33b 2000 48 91.11 5.56 0c

[0123] According to the test results, it can be seen from the comparison between Table 6 and Table 1 that the selection of mutagenic materials has a certain influence on the doubling rate, and the doubling effect of single-bud stem segments is better than that of double-bud stem segments; and the doubling rate is the best at 6000 mg / L in different mutagenic material treatments.

[0124] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for in vitro induction of tetraploid grapes, characterized in that, It includes the following steps: S1. Take young diploid grape tissue culture seedlings, remove the apical buds, cut them into single-bud stem segments, and conduct a pretreatment for 3 - 5 days. S2. Induce the pre-cultured stem segments with colchicine. After the induction ends, inoculate them into the first medium for the first culture. S3. After the first culture is completed, cut each tissue culture seedling into stem segments, transfer them to the second medium for the second culture, and finally obtain grape tetraploid tissue culture seedlings. S4. Identify tetraploid plants by flow cytometry. The specific method is as follows: Take 0.2 g of young leaves, lyse the cell nuclei with the CyStain UVPrecise P kit, detect them after DAPI staining for 2 minutes, and screen out the tetraploid tissue culture seedlings with the peak fluorescence intensity located on the right side of the diploid and more concentrated distribution.

2. The method for in vitro inducing tetraploid grapes according to claim 1, characterized in that, The diploid grape is Jintian Rose grape.

3. A method for in vitro induction of tetraploid grapes according to claim 1, characterized in that, The colchicine is a colchicine solution of 2000 mg / L - 6000 mg / L or a colchicine solid medium with a concentration of 200 mg / L - 400 mg / L.

4. A method for in vitro induction of tetraploid grapes according to claim 1, characterized in that, The composition of the first medium is a medium of 1 / 2MS + 5.0 g / L agar + 20 - 30 g / L sucrose.

5. A method for in vitro induction of tetraploid grapes according to claim 1, characterized in that, The second medium is a medium of 1 / 2MS + 5.0 g / L agar + 30 g / L sucrose + 0.1 - 0.5 mg / L IBA.

6. A method for in vitro induction of tetraploid grapes according to claim 3, characterized in that, The treatment time of the colchicine solution is 24 - 72 h.

7. A method for in vitro induction of tetraploid grapes according to claim 6, characterized in that, The treatment conditions are: temperature 24 - 26°C, under dark conditions, and the rotation speed of the constant temperature shaker is 180 rpm.

8. A method for in vitro induction of tetraploid grapes according to claim 3, characterized in that, The treatment time of the colchicine solid medium is 20 - 40 days.

9. A method for in vitro induction of tetraploid grapes according to claim 8, characterized in that, The culture conditions for the treatment are: temperature 23 - 25°C, 2000 lx light for 16 h, and dark conditions for 8 h.

10. A method for in vitro induction of tetraploid grapes according to claim 3, characterized in that, The morphological characteristics of the tetraploid plants in step S4 include: the stem diameter increases by 20% - 30%, the leaf area increases by 40% - 50%, and flow cytometry detection shows that the chromosome multiple doubles.

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