Method for doubling haploid plants of watermelon

Abstract

The present application discloses a method for doubling the haploid plants of watermelon. The method for doubling the haploid plants of watermelon aims to solve the problems that the use of the induction doubling agent will pollute the environment and the doubling effect of the existing method for obtaining watermelon polyploids by culturing unpollinated ovaries is not obvious. The method includes: inoculating the haploid plants obtained by in vitro culture of watermelon anthers into a medium containing MS, 0.15 - 0.25 mg / L kinetin KT, 80 - 120 ml / L fresh coconut milk, 25 - 35 g / L sucrose, 6.0 - 7.0 g / L agar, and with a pH value of 5.80 - 5.90, and then placing them in an incubator with different red and blue light quality ratios for culture. The present application can greatly improve the doubling rate of watermelon haploids by using the light quality conditions of red and blue ratio in combination with the improved medium (MS medium + exogenous substances); the in vitro development of haploid plants into diploid plants by photocatalysis not only has higher culture efficiency and is more environmentally friendly, but also has simple operation and low cost.

Description

Technical Field

[0001] This invention application relates to the field of doubling plant haploids, and specifically relates to a method for doubling watermelon haploid plants. Background Art

[0002] Watermelon belongs to the annual herbaceous plant of the genus Citrullus in the Cucurbitaceae family. Its pulp is juicy and refreshing, making it one of the best-selling fruits in summer, with rich nutritional value and considerable economic value. At present, the genetic basis of watermelons in China is narrow, and excellent germplasm resources are lacking. Moreover, traditional breeding methods are mainly used, resulting in a lag in the update of high-quality new varieties. Therefore, breeders need to accelerate the process of watermelon breeding. And through haploid breeding, factors such as the long cycle, large workload, and unstable genetic traits of conventional breeding can be effectively solved, thus promoting the progress of breeding.

[0003] Approaches such as anther culture in vitro, unpollinated ovary or ovule culture in vitro, and pollen irradiation are all effective ways to obtain haploid plants. Watermelon haploid plants can be obtained through anther culture in vitro of watermelon, and haploid plants need to be doubled into double haploid plants to be applied in breeding and genetic research. The probability of doubling under natural conditions is relatively low, and artificial induction is needed to increase the doubling rate. Among them, chemical agents such as colchicine, trifluralin, and oryzalin are commonly used doubling agents. These inducing agents are used for doubling through methods such as seed soaking, root soaking, bud soaking, and dripping on the growth point. The doubling effect is difficult to be satisfactory, the doubling results are uneven, the harm and lethality to plants are relatively high, and these agents have certain toxic effects on operators and also have a certain degree of pollution to the environment.

[0004] The patent document CN111837948A known to the inventor discloses a method for obtaining double haploid plants by culturing unpollinated ovaries of watermelon, but the process and efficiency of doubling its haploid to diploid cannot meet the current large demand of consumers for polyploid watermelons with advantages such as disease resistance, storage resistance, and convenient consumption.

[0005] The information disclosed in this background art section is only used to deepen the understanding of the background art of this disclosure, and should not be regarded as an admission or any form of implication that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] The inventor's research found that the existing methods for doubling watermelon haploids mainly involve using doubling agents and culturing unpollinated ovaries to obtain haploid doubling; the use of existing doubling agents will pollute the environment, and the doubling effect of the existing method of obtaining watermelon polyploids by culturing unpollinated ovaries is not obvious; while the research of this application found that using specific culture conditions (red-blue light quality ratio 4 - 1.5:1, MS medium + certain exogenous additives) can induce the doubling of watermelon haploids, with high efficiency and no pollution.

[0007] According to one aspect of the present disclosure, a method for doubling watermelon haploid plants is provided, including the following steps:

[0008] (1) Obtaining haploid plants by in vitro culture of watermelon anthers;

[0009] (2) Inoculating the obtained haploid plants into a culture medium for culture and subculture. The light quality conditions for culture are: the ratio of the light intensity of red light to blue light is 4 - 1.5:1. The culture medium contains MS, 0.15 - 0.25 mg / L kinetin KT, 80 - 120 ml / L fresh coconut juice, 25 - 35 g / L sucrose, 6.0 - 7.0 g / L agar, and the pH value is 5.80 - 5.90.

[0010] In some embodiments of the present disclosure, in the step (2), the light cycle during the culture process is 12h - 16h•d -1 .

[0011] In some embodiments of the present disclosure, in the step (2), the total light intensity during the culture process is 4000 LX - 5000 LX.

[0012] In some embodiments of the present disclosure, in the step (2), the daytime light temperature during the culture process is 26 - 28 °C, and the nighttime dark temperature is 22 - 23 °C.

[0013] In some embodiments of the present disclosure, in the step (2), the culture medium contains MS medium, 0.2 mg / L kinetin KT, 100 ml / L fresh coconut juice, 30 g / L sucrose, 6.5 g / L agar, and the pH value is 5.8 - 5.85.

[0014] In some embodiments of the present disclosure, in the step (2), the culture conditions are: red light and blue light with a ratio of the light intensity of red light to blue light of 4 - 1.5:1, the light cycle is 16h•d -1 , the total light intensity is 4000 LX - 5000 LX, the temperature under light is 26 - 28 °C, the temperature in the dark is 22 - 23 °C, and the culture lasts for 6 weeks.

[0015] One or more technical solutions provided in the embodiments of the present application have at least any one of the following technical effects or advantages:

[0016] (1) Adopting the light quality conditions of red - blue ratio in combination with the improved culture medium (MS medium + exogenous substances) can greatly improve the doubling rate of watermelon haploids;

[0017] (2) Using photocatalysis to develop haploid plants in vitro into diploid plants on a specific culture medium is not only more efficient and greener in culture, but also simple in operation and low in cost. Description of the Drawings

[0018] Figure 1 Schematic diagram of ploidy identification of plants in the control group and the experimental group after subculture. Detailed implementation manners

[0019] In the following examples, the instrument and equipment involved are all conventional instrument and equipment unless otherwise specified; the light sources and light quality components involved are all provided by conventional equipment.

[0020] Example: Verification test on the method for doubling haploid plants of watermelon

[0021] 1. Test materials: The haploid plants obtained by anther culture in vitro of the small watermelon Sweet were used as test materials, provided by the Melon and Watermelon Research Group of the Horticultural Institute of Henan Academy of Agricultural Sciences.

[0022] 2. Test methods

[0023] (1) Identification of haploid plants: The plants obtained by anther culture in vitro of watermelon were subjected to ploidy identification by flow cytometry, and the identified haploid plants were inoculated into the improved medium NMS (MS + 0.2 mg / L kinetin KT + 100 ml / L coconut milk + 30 g / L sucrose + 6.5 g / L agar, pH value 5.8 - 5.85) for culture; Control group: The haploid plants were inoculated into the blank medium with only MS for culture.

[0024] (2) Subculture: The inoculated haploid plants were placed in a light quality incubator for three weeks and then subcultured once. The subculture medium was the improved medium NMS (MS + 0.2 mg / L kinetin KT + 100 ml / L coconut milk + 30 g / L sucrose + 6.5 g / L agar, pH value 5.8 - 5.85). Control group: The haploid plants were inoculated into the medium with only MS for culture. After subculturing twice, the ploidy of the cultured plants was identified (the ploidy of the doubled plants was identified by flow cytometry).

[0025] (3) Culture conditions

[0026] Light quality conditions: The light intensity ratios were respectively set as white light (W), white light (W): red light (R) = 2:1 or 1:2 (denoted as 2W1R or 1W2R), white light (W): blue light (B) = 3:1 or 1:3 (denoted as 3W1B or 1W3B), red light (R): blue light (B) = 4:1 or 1:4 or 3:2 or 2:3 (denoted as 4R1B or 1R4B or 3R2B or 2R3B); The light cycles were respectively set as 12 h·d -1 and 16 h·d -1; The total light intensity is controlled at 4000 LX - 5000 LX, the light temperature during the day is controlled at 26 - 28 °C, and the dark temperature is 22 - 23 °C.

[0027] 3. Results and Analysis:

[0028] Thirty-six treatments were carried out on the doubling culture conditions of watermelon haploid plants (Table 1). The doubling results of the Svet haploid are shown in Table 2 and Figure 1 It can be seen that the medium composition, light quality, and light cycle are closely related to the doubling effect of haploid plants, and the results obtained from different treatments are significantly different.

[0029] Analyzing from the additive components of the medium, in treatments 1 - 18, KT and fresh coconut juice were added to the conventional MS medium, and in treatments 19 - 36, it was only the conventional MS medium without any additives. As shown in Table 2, the highest probability of obtaining DH plants in treatments 1 - 18 was 80%, and the highest doubling rate of single and double haploids in treatments 19 - 36 was only 10%. The probability of obtaining DH plants in treatments 1 - 18 was significantly higher than that in treatments 19 - 36, indicating that the exogenous additives KT and coconut juice play a certain role in the doubling of plants.

[0030] Analyzing from the light quality, different light qualities have a promoting effect on the doubling rate of double haploid plants. In treatments 1 - 18, double haploid plants were obtained with light qualities of different red and blue light ratios. Among them, the light quality of 4R1B obtained the highest doubling rate of double haploids of 80%, which was significantly higher than other treatments. The study also found that the probability of obtaining double haploids with a light quality combination with a higher proportion of red light than blue light was significantly higher than that with a low red light and high blue light ratio combination. For example, the probability of obtaining DH plants with 4R1B and 3W2B was significantly higher than that with 1R4B and 2W3B, indicating that different ratios of red and blue light qualities have a significant impact on the doubling rate of single and double haploids, and the impact of red light is greater than that of blue light.

[0031] From the analysis results of the light cycle, it can be seen that the light cycle has an impact on the doubling rate of plants. Under the same conditions, the doubling rate of DH plants under the 16 h•d -1 light cycle treatment was significantly higher than that under the 12 h•d -1 light cycle treatment, indicating that the light cycle also has a limiting effect on the doubling rate of haploids.

[0032] Overall, watermelon haploid plants are cultured in the improved NMS medium, with the ratio of the light intensity of red and blue light being 4 - 1.5:1, the light cycle being 16 h•d -1 , the light intensity being 4500 LX, the temperature under light being 26 - 28 °C, and the temperature in the dark being 22 - 23 °C. Cultured for six weeks under these conditions, the maximum doubling rate of single and double haploids can reach 80%.

[0033] Table 1 Doubling culture conditions of watermelon haploid plants

[0034] 。

[0035] Table 2 Doubling rate of watermelon haploids

[0036] 。

Claims

1. A method for doubling the haploid plants of watermelon, characterized in that, It includes the following steps: (1) Obtaining haploid plants by in vitro culture of watermelon anthers; (2) The obtained haploid plants were subcultured in the medium for 2 times, with each culture lasting for three weeks. The culture conditions were as follows: red and blue light with a light intensity ratio of red to blue light of 4 - 1.5:1, a light cycle of 16 h•d -1 , a total light intensity of 4500 LX, a temperature of 26 - 28 °C under light, and a temperature of 22 - 23 °C in the dark; the composition of the medium was: MS medium, 0.2 mg / L kinetin KT, 100 ml / L coconut milk, 30 g / L sucrose, 6.5 g / L agar, and a pH value of 5.80 - 5.85.

Images