A method for obtaining regenerated plants of benincasa hispida by embryo rescue

Abstract

The application provides a method for obtaining winter melon regenerated plants by embryo rescue, comprising the following steps: (1) obtaining winter melon seeds to be rescued, and placing the seeds on an embryo rescue medium and culturing in a normal temperature environment with 14-18 h light and 6-10 h darkness; (2) screening the seeds to be germinated, and transferring the seeds to a growth medium for growth and development and rooting culture after the appearance of embryoid; (3) transferring the seeds to a substrate for seedling culture after rooting, and obtaining winter melon regenerated plants. The application provides a method for obtaining winter melon regenerated plants by embryo rescue, which effectively overcomes the problem of hybridization failure caused by poor growth and insufficient seed vigor in the traditional breeding process of winter melon.

Description

Technical Field

[0001] This invention belongs to the field of plant in vitro culture technology, and in particular relates to a method for obtaining regenerated winter melon plants by embryo rescue. Background Technology

[0002] Embryo rescue technology plays a crucial role in rescuing aborted embryos, preventing early embryonic degeneration, and cultivating new triploid and polyploid germplasm resources. There are three main methods for plant embryo rescue: in vivo embryo transfer, in vivo embryo culture, and in vitro embryo culture. For in vitro embryo culture, depending on the developmental stage of the embryo to be rescued, three culture methods are applicable: in vitro ovary culture, ovule culture, and immature embryo culture. Numerous studies have shown that embryo age, culture medium type and phase, culture conditions, osmotic pressure, growth regulators, exogenous additives, and genotype all affect the success or failure of plant embryo rescue.

[0003] Winter melon is one of my country's major vegetable varieties and an ideal raw material for modern agricultural product processing, as well as an important vegetable crop for regulating and balancing market supply. It is characterized by wide adaptability, good storage and transportation resistance, and a long shelf life. Besides domestic sales, some products are processed and exported to earn foreign exchange. However, due to the presence of EMS mutants in some severely mutated winter melon plants, these plants suffer from defects such as poor growth, difficulty in fruit setting, or insufficient seed viability. Furthermore, a significant portion of winter melon resources are from short-day plants. In Hunan province, flowering and pollination, or hybridization with medium-day plants for resource creation, must be carried out during the autumn period when daylight hours shorten. However, the rapid drop in autumn temperature and insufficient accumulated temperature during the growing season can also lead to decreased or non-viable seed viability, making it impossible to obtain usable winter melon resources. In such cases, assisted breeding methods are needed to allow embryos that cannot develop normally under natural conditions to produce offspring.

[0004] Assisted breeding methods require specialized laboratory equipment and technicians, and necessitate the culture and regeneration of plant tissues. However, tissue culture and regeneration of winter melon are difficult to perform, requiring research and technological improvements tailored to the specific characteristics of winter melon. Therefore, there is currently no technical solution using embryo rescue technology to assist in winter melon breeding, and related research is needed to fill this gap. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that it is difficult to obtain winter melon resources under natural conditions. To overcome the deficiencies and defects mentioned in the background art above, a method for obtaining winter melon regenerated plants by embryo rescue is provided.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A method for obtaining regenerated winter melon plants through embryo rescue includes the following steps:

[0008] (1) Obtain the winter melon seeds to be rescued, place the seeds on the embryo rescue medium, and culture them at room temperature under 14-18h light and 6-10h darkness.

[0009] (2) Select seeds to germinate, and after the seeds develop embryoids, transfer them to a growth medium for growth, development and rooting culture.

[0010] (3) After the seeds have taken root, they are transferred to a substrate to harden off and obtain winter melon regenerated plants.

[0011] Winter melon seeds typically have a high water content and a short shelf life, making them more susceptible to rot and damage. Therefore, the temperature and humidity requirements for winter melon seeds during preservation and rescue differ from those for other plants.

[0012] Winter melon seeds have a relatively small number of endosperm cells, so it is necessary to preserve as many original cells as possible during embryo rescue. Furthermore, winter melon seeds have a high content of stored protein, which may affect cell differentiation and proliferation during embryo rescue. Therefore, the culture of winter melon embryos requires more specific approaches, and the hormone formulation and culture conditions used in the embryo rescue process need to be adjusted to suit its unique biological characteristics to ensure the effective cultivation of normal plants.

[0013] Preferably, the winter melon seeds to be rescued are winter melon embryos that cannot develop normally after hybridization pollination.

[0014] Preferably, the method for obtaining the winter melon seeds to be saved specifically includes:

[0015] Different male and female winter melons were cross-pollinated after entering their full flowering period; the pollinated winter melons were disinfected and seeds were obtained under sterile conditions.

[0016] Preferably, the hybridization pollination specifically includes:

[0017] To cultivate the male and female plants of winter melon into flowering stage, after the female flowers of the female plant bloom, cover the male flowers of the male plant and the female flowers of the female plant with bags. The next day, remove the male flowers of the male plant from the bags, remove the corolla, and apply the pollen to the open female flowers covered with bags so that the stigma of the female flowers is covered with pollen.

[0018] Preferably, the method for disinfecting the winter melon includes: 15 days after pollination, rinsing the winter melon with clean water and then transferring it to a clean bench to wipe and disinfect its skin with sodium hypochlorite; the method for obtaining the seeds includes cutting the winter melon open with a sterile blade and picking out the seeds.

[0019] Preferably, the seed to be germinated has the characteristic that the middle part of the seed changes from milky white to light green.

[0020] Preferably, the embryo rescue medium in step (1) comprises: MS + 25-35 g / L sucrose + 0.3-0.5 mg / L 6-BA + 0.3-0.5 mg / L 2,4-D + 5-8 g / L agar, and the pH of the embryo rescue medium is 5.8-6.0.

[0021] The components of the embryo rescue culture medium have the following functions:

[0022] 1. The role of sucrose:

[0023] Sucrose, as a carbon source, provides energy for cell division and growth during the winter melon embryo rescue process.

[0024] The concentration is set at 25-35 g / L, which provides sufficient energy support for the cells without inhibiting cell growth due to excessive concentration.

[0025] The role of 2,6-BA (6-benzylaminopurine):

[0026] 6-BA is a cytokinin that can promote cell division and proliferation, and accelerate the growth and development of winter melon embryos.

[0027] The concentration is set at 0.3-0.5 mg / L, which can effectively promote cell division while avoiding abnormal growth caused by excessive concentration.

[0028] The role of 4,2,4-D (2,4-dichlorophenoxyacetic acid):

[0029] 2,4-D is an auxin analog that can promote cell elongation and differentiation, and helps the regeneration and development of winter melon embryos.

[0030] When used in combination with 6-BA at a concentration of 0.3-0.5 mg / L, 2,4-D can coordinate cell division and elongation, promoting the complete regeneration of winter melon embryos.

[0031] 5. The function of agar:

[0032] Agar acts as a solidifying agent, making the culture medium solid, which facilitates the fixation and growth of winter melon embryos.

[0033] A concentration of 5-8 g / L can provide sufficient solidification while avoiding excessively hardened culture medium due to high concentration.

[0034] In summary, the embryo rescue culture medium formula provides a stable and nutrient-rich environment for the rescue of winter melon embryos, and promotes embryo regeneration and development by adjusting the concentration of different hormones. This formula has high application value in the rescue of winter melon embryos.

[0035] Preferably, the growth medium in step (2) comprises: MS + 25-35 g / L sucrose + 0.3-0.5 mg / L BA + 0.3-0.5 mg / L GA3 + 5-8 g / L agar, and the pH of the growth medium is 5.8-6.0.

[0036] The components of the growth medium have the following functions:

[0037] 1. BA (6-Benzylaminopurine): BA is a cytokinin that promotes the division and proliferation of plant cells. In the rescue growth of winter melon embryos, an appropriate amount of BA can stimulate cell division and accelerate the rescue growth of winter melon embryos.

[0038] 2. GA3 (gibberellin): GA3 is a plant growth hormone that promotes plant growth and development. In the rescue growth of winter melon embryos, GA3 can stimulate the growth of winter melon embryos, promote root elongation and leaf unfolding.

[0039] 3. Agar: As a solidifying agent, agar can maintain the solid state of the culture medium, allowing cells to grow and differentiate within it. In the rescue growth of winter melon embryos, the presence of agar provides a stable growth environment, which is conducive to cell attachment and growth.

[0040] In summary, the growth medium formula can provide a balanced, stable and nutrient-rich growth environment for the rescue growth of winter melon embryos, in which each component plays an important role and works together to promote the rescue growth of winter melon embryos.

[0041] Preferably, the temperature for growth, development and rooting culture in step (2) is 20-25 degrees Celsius.

[0042] Winter melon requires suitable temperature and light conditions for rooting and growth, with 20-25 degrees Celsius being the most suitable temperature for rooting and growth. In addition, it is necessary to control the development time; the rooting and growth process should generally take place within a suitable timeframe to ensure that the plant develops healthy roots and above-ground parts at the appropriate time.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] (1) This invention provides a method for obtaining winter melon regenerated plants by using embryo rescue. By using artificial assistance to rescue underdeveloped winter melon seeds, it effectively overcomes the problems of poor growth and insufficient seed vigor in the traditional breeding process of winter melon, which leads to hybridization failure.

[0045] (2) The embryo rescue method provided by the present invention can use incompletely developed seeds for tissue culture, and finally obtain a large number of hybrid seedlings. The method is simple, saves breeding time for winter melon, and avoids waste of resources. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This is a photograph of the winter melon cultivation process in Example 1, in which winter melon regenerated plants were obtained using embryo rescue. Detailed Implementation

[0048] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0049] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0050] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0051] Example 1

[0052] A method for obtaining regenerated winter melon plants using embryo rescue. Figure 1 These are photographs of the winter melon cultivation process in Example 1, where winter melon regenerated plants were obtained using embryo rescue. In this example, A is a winter melon fruit 15 days after pollination.

[0053] B represents seeds selected from fruits collected 15 days after pollination.

[0054] C represents the seed culture process on embryo rescue medium.

[0055] D represents the entire seed turning green.

[0056] E indicates that the bottom of the seed is green.

[0057] F represents the formation of green or white callus tissue from seeds.

[0058] G is the seed that remains unchanged.

[0059] H represents the germinating embryoid.

[0060] I represents the development of a plant from a germinating embryo.

[0061] J represents the regenerated plantlets cultured on rooting medium.

[0062] K is a photo of winter melon being domesticated in a growing room.

[0063] The method specifically includes the following steps:

[0064] Obtain winter melon seeds to be saved

[0065] Interspecific hybridization: After different winter melon materials enter the full flowering period, they are cross-pollinated. After the winter melon enters the flowering period, the male flowers of the male parent and the female flowers of the female parent are covered with bags. On the second day, the male flowers of the male parent covered with bags are removed, the corolla is removed, and the pollen is applied to the open female flowers covered with bags so that the stigma is covered with yellow pollen.

[0066] Aseptic seed acquisition: 15 days after pollination, the winter melon is harvested, rinsed thoroughly under running water, and then transferred to a clean bench where its surface is wiped and disinfected with sodium hypochlorite. It is then cut open with a sterile blade, and the seeds are gently removed from the pulp with tweezers.

[0067] This application selected 100 winter melon seeds for embryo rescue experiments, and conducted three groups, with 12 seeds cultured in each culture dish.

[0068] (a) Place the seeds on embryo rescue medium and culture them at room temperature under 16 hours of light and 8 hours of darkness.

[0069] Culture conditions: Seeds were extracted from the pulp and placed on embryo rescue medium (embryo rescue medium formula is MS + 30 g / L sucrose + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 6 g / L agar, pH = 5.80). The medium was cultured in a 24℃ incubator with 16 h light and 8 h darkness.

[0070] (ii) Select the seeds that have turned green at the bottom after cultivation, and after the seeds have developed embryoids, transfer them to the growth medium for growth, development and rooting culture.

[0071] During the selection of seeds with green bottoms obtained from cultivation, the development status was observed. After about 4-5 weeks, the winter melon seeds showed the following four situations: (1) the seeds gradually became plump and a large area gradually turned green; (2) the bottom of the seeds turned green; (3) the seeds formed callus tissue, some of which were initially green, but gradually lost their green color and died as the cultivation time increased; (4) the seeds did not change at all and eventually died. Situation (1) is a seed with green bottoms that meets the conditions.

[0072] Growth, development and rooting culture: Seeds in state (1) were cultured and transferred to growth medium (MS + 30 g / L sucrose + 0.5 mg / L BA + 0.5 mg / L GA3 + 6 g / L agar, pH = 5.8) for further growth, development and rooting culture of the plants. The temperature for growth, development and rooting culture was 20-25 degrees Celsius.

[0073] (3) After the seeds have rooted, the plants are transferred to a substrate and hardened off in a cultivation room. The cultivation temperature is between 20-30 degrees Celsius, the relative humidity is between 60-80%, and sufficient light and ventilation are maintained to obtain winter melon regenerated plants, which are then transferred to greenhouses or open fields for planting. The final survival rate of winter melon seeds is 40%.

Claims

1. A method for obtaining regenerated winter melon plants by embryo rescue, characterized in that, Includes the following steps: (1) After the male and female parents of winter melon enter the full flowering period, they are cross-pollinated. 15 days after pollination, the winter melon fruits are harvested. After disinfection, the winter melon embryos that cannot develop normally after cross-pollination are obtained under sterile conditions as winter melon seeds to be rescued. The winter melon seeds to be rescued are placed on embryo rescue medium and cultured at room temperature under 14-18h light and 6-10h darkness. The components of the embryo rescue medium are: MS + 25-35g / L sucrose + 0.3-0.5 mg / L 6-BA + 0.3-0.5 mg / L 2,4-D + 5-8g / L agar. The pH of the embryo rescue medium is 5.8-6.

0. (2) Selecting seeds to germinate, wherein the characteristics of the seeds to germinate are that the middle part of the seeds changes from milky white to light green. After the embryoids appear in the seeds, they are transferred to a growth medium for growth, development and rooting culture. The composition of the growth medium is: MS + 25-35 g / L sucrose + 0.3-0.5 mg / L BA + 0.3-0.5 mg / L GA3 + 5-8 g / L agar. The pH of the growth medium is 5.8-6.

0. The temperature for growth, development and rooting culture is 20-25 degrees Celsius. (3) After the seeds have taken root, they are transferred to a substrate to harden off and obtain winter melon regenerated plants.

2. The method for obtaining regenerated winter melon plants by embryo rescue as described in claim 1, characterized in that, The hybridization pollination specifically includes: To cultivate the male and female plants of winter melon into flowering stage, after the female flowers of the female plant bloom, cover the male flowers of the male plant and the female flowers of the female plant with bags. The next day, remove the male flowers of the male plant from the bags, remove the corolla, and apply the pollen to the open female flowers covered with bags so that the stigma of the female flowers is covered with pollen.

3. The method for obtaining regenerated winter melon plants by embryo rescue as described in claim 2, characterized in that, The method for disinfecting winter melon includes: 15 days after pollination, rinsing the winter melon with clean water and then transferring it to a clean bench to wipe and disinfect its skin with sodium hypochlorite; the method for obtaining seeds includes cutting the winter melon with a sterile blade and picking out the seeds.

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