Method for preserving pods in spaceflight breeding of Chinese orchid

Abstract

The invention discloses a Chinese orchid space breeding pod preservation method, and belongs to the field of Chinese orchid space breeding. The method comprises the following steps: firstly, taking seven-percent mature Chinese orchid capsules, and performing disinfection and sterilization treatment in a clean bench to obtain sterile pods; then, the sterile condition is kept, and the sterile fruit pods are dehydrated to obtain complete dry fruit pods; and finally, putting the dried pods into a modified atmosphere bag under a sterile condition, and storing at low temperature. Before warehousing, the pods are taken out of the air-conditioning bag under the sterile condition, and the pods are completely immersed in medical liquid paraffin, so that the surfaces of the pods are uniformly covered with the paraffin. The method greatly improves the germination rate of seeds after spaceflight breeding, shortens the germination time, reduces the induced pollution rate, and improves the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of space breeding of Cymbidium goeringii, and relates to the preservation of Cymbidium goeringii pods, specifically to a method for preserving Cymbidium goeringii pods bred in space. Background Technology

[0002] Crop space breeding, also known as space mutation breeding or space breeding, refers to a breeding method that uses space vehicles such as spacecraft, recoverable satellites, and high-altitude balloons to carry crop seeds to a space environment of 200-400km. The special environment of space (such as cosmic rays, high-energy particles, microgravity, high vacuum, and weak magnetic fields) induces mutations in the crop seeds, which are then returned to Earth for selection and breeding of new germplasm, new materials, and new varieties. It has the advantages of large variation range, high variation frequency, many variation types, and good stability, which can significantly shorten the breeding cycle and obtain outstanding characteristics.

[0003] A single pod of Cymbidium orchids contains 100,000 to 1 million seeds. These seeds are tiny and poorly developed, lacking the endosperm that stores nutrients like beans or rice. They cannot provide the nutrients needed for germination and early growth. In nature, they require symbiotic fungi to infect them and provide the nutrients needed for germination and growth.

[0004] Currently, artificial breeding of Cymbidium goeringii mainly employs a non-symbiotic germination approach. This involves using a sterile culture medium in a tissue culture room to provide nutrients for seed germination, eliminating the need for symbiotic fungi. Research indicates that factors influencing Cymbidium goeringii seed germination primarily include seed maturity and the degree of seed contamination. Seeds at about 70% maturity germinate easily, as the seed pod provides a sterile environment with the lowest contamination rate, making induction easier and resulting in the highest germination rate. Fully mature seeds typically form a highly lignified layer of dead cells, which is robust and impermeable to water, causing physical difficulties in germination. Furthermore, mature orchid seeds form a "chemical lock" of hormones such as abscisic acid (ABA) or other phenolic compounds that inhibit germination, protecting the tiny and fragile embryo inside from damage and preventing dehydration, thus making induced germination difficult.

[0005] Currently, space breeding is mainly used for grains, vegetables, and fruits, with a small amount used for forest trees and flowers. Only a handful of organizations, such as Shenzhen Agricultural Science Plant Cloning Seedling Co., Ltd., Fujian Provincial Forestry Science and Technology Experiment Center, and Beijing Forestry University Forestry Science and Technology Co., Ltd., have reportedly used it for orchid breeding. When Cymbidium seeds are used for space breeding, the seeds undergo multiple transfers before launch, requiring long storage times and complex environments, making it difficult to maintain a sterile environment. Furthermore, considering economic costs and safety, there are strict weight restrictions on the plant material carried, making tissue culture containers or liquid nitrogen preservation methods unsuitable. The preferred method is to open mature pods and place the seeds in self-sealing bags for preservation.

[0006] The conventional method of preserving Cymbidium seeds in resealable bags for space breeding has the following problems: When using 70% mature seeds, the immature seed coat has not yet formed lignified dead cells, making the seeds prone to dehydration and reducing their viability. When using mature seeds, the presence of hormones that inhibit germination, such as abscisic acid, or other phenolic compounds, makes germination difficult, leading to failure in space breeding and extremely low breeding efficiency. Furthermore, conventional space breeding also faces challenges in maintaining the aseptic state of Cymbidium seeds after ex vivo, resulting in severe contamination during tissue culture upon return to Earth, further contributing to germination failure. Given these realities, it is urgent to research methods for preserving Cymbidium seeds during space breeding. Summary of the Invention

[0007] To improve the germination rate of Cymbidium goeringii seeds after space breeding and enhance the efficiency of space-bred Cymbidium goeringii, a method for preserving Cymbidium goeringii seed pods after space breeding is proposed, which enables the intact preservation of Cymbidium goeringii seed pods under sterile conditions and maintains seed viability.

[0008] A method for preserving pods from Cymbidium goeringii bred in space, comprising the following steps:

[0009] Step 1: Take the 70% mature Cymbidium goeringii capsules and sterilize them in a clean bench to obtain sterile pods;

[0010] The specific process of disinfection and sterilization is as follows: First, the capsules of Cymbidium goeringii are disinfected in 75% alcohol for 20 minutes. After being removed, they are soaked in 0.1% sodium hypochlorite solution for 15 minutes. Finally, they are rinsed 2-3 times with sterile water and dried with gauze to obtain sterile pods.

[0011] Step 2: Maintain sterile conditions, place the sterile pods treated in Step 1 into a desiccator, and use color-changing silica gel to dehydrate them, reducing the moisture content of the pods to 8%-10% to obtain intact dried pods, and test the seed viability.

[0012] The method for calculating the moisture content of fruit pods is as follows:

[0013] Pod moisture content (%) = (weight of pods before drying - weight of pods after drying) / weight of pods before drying x 100%.

[0014] Seed viability was assessed using the TTC staining method on the dried pods.

[0015] Step 3: Under sterile conditions, place the intact, dried pods from Step 2 into a modified atmosphere bag. Fill the bag with a mixture of oxygen, carbon dioxide, and nitrogen (volume ratio 0.6:12:87.4), filtered through a 0.22μm microporous membrane. This preserves seed viability and slows down aging. Furthermore, placing the pods in a modified atmosphere bag effectively prevents them from being crushed during storage.

[0016] Step 4: Store the modified atmosphere bags containing Cymbidium goeringii seed pods prepared in Step 3 at low temperature.

[0017] Step 5: Before entering the warehouse, remove the fruit pods from the modified atmosphere bag under sterile conditions and completely immerse the fruit pods in medical liquid paraffin to evenly cover their surface with paraffin.

[0018] After the above treatment, the orchid pods are placed in resealable bags and stored in the warehouse.

[0019] The advantages of this invention are:

[0020] (1) The present invention provides a method for preserving pods of Cymbidium goeringii in space breeding. Unlike the method of putting mature seeds into a self-sealing bag, this method uses pods that are 70% mature, dehydrates them, and preserves them in a dormant state. More mature seeds do not contain germination inhibitors, which greatly improves the germination rate after landing.

[0021] (2) The present invention provides a method for preserving pods of Cymbidium goeringii in space breeding. Unlike the method of removing seeds, the present invention uses pods to preserve seeds, creating a sterile environment for the seeds, overcoming the unfavorable conditions of complex storage and transportation environment before Cymbidium goeringii seeds are launched into space, and greatly reducing the induced contamination rate after landing. Attached Figure Description

[0022] Figure 1 These are images of Cymbidium goeringii pods preserved using the method of this invention, after space breeding.

[0023] Figure 2 These are images of mature Cymbidium seeds bred on a spacecraft. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0025] This invention employs a method of collecting 70% mature Cymbidium goeringii pods, disinfecting them, and then drying them under aseptic conditions in a silica gel container until the moisture content is reduced to approximately 8%–10%, ensuring the pods remain intact and do not crack. Under aseptic conditions, the pods are then placed in a modified atmosphere bag for low-temperature storage. Before entering the spacecraft, the pods are soaked in medical paraffin wax under aseptic conditions, ensuring a uniform coating. Using 70% mature Cymbidium goeringii seeds eliminates the presence of germination inhibitors, significantly increasing the germination rate after return. Furthermore, the seeds are in a dormant state before launch and are preserved under aseptic conditions with the protection of the pods, greatly reducing seed contamination. Compared to current methods for preserving Cymbidium goeringii seeds bred in space, this method significantly reduces the contamination rate of seeds after space breeding, increases the germination rate, shortens the germination time, and improves breeding efficiency.

[0026] A method for preserving pods from Cymbidium goeringii bred in space, comprising the following steps:

[0027] Step 1: Take 70% mature Cymbidium goeringii capsules to ensure a high seed germination rate. Disinfect them in 75% alcohol for 20 minutes, then soak them in 0.1% sodium hypochlorite solution for 15 minutes. Finally, rinse them 2-3 times with sterile water, wipe them dry with gauze, and perform the procedure in a clean bench to achieve the effect of disinfecting the pods.

[0028] Step 2: Under aseptic conditions, dry the pods treated in Step 1 in a silica gel container until their moisture content is approximately 8%–10%. During the drying process, ensure the pods do not deform or crack. Seed viability after treatment is assessed using the TTC staining method.

[0029] Step 3: Under sterile conditions, place the pods treated in Step 2 into a modified atmosphere bag that has undergone gas filtration, and fill it with modified atmosphere gas (oxygen, carbon dioxide, and nitrogen in a volume ratio of 0.6:12:87.4). Placing the pods in a modified atmosphere bag effectively prevents them from being crushed during storage. The modified atmosphere gas, while ensuring seed viability, promotes dormancy and delays seed aging.

[0030] Step 4: Store the prepared Cymbidium seed pods in a refrigerator at 4°C.

[0031] Step 5: Before entering the warehouse, remove the fruit pods from the modified atmosphere bag under sterile conditions and completely immerse the fruit pods in medical liquid paraffin to evenly cover their surface with paraffin.

[0032] After the above treatment, the orchid pods are placed in resealable bags and stored in the warehouse.

[0033] Example

[0034] To assess the effectiveness of the seed pod preservation method of this invention in space breeding, this embodiment selected mature Cymbidium seeds preserved using traditional breeding methods for a comparative breeding experiment. Figure 1These are pods of Cymbidium goeringii preserved using the method of this invention, after space breeding. Figure 2 These are seeds of mature Cymbidium goeringii seeds preserved using traditional methods, which have undergone space breeding. The contamination status and seed germination rate in the comparative germination experiment are shown in Tables 1 and 2.

[0035] As can be seen from the contamination rate data in Table 1, the induced contamination rate of Cymbidium seeds is greatly reduced by the preservation method of the present invention, thereby improving the seed germination rate.

[0036] As can be seen from the germination rate data in Table 2, the germination rate of Cymbidium seeds obtained by the method of the present invention is more than 3 times higher than that of mature seeds obtained by the traditional method. This fully demonstrates that the preservation method of the present invention can effectively improve the induced germination rate of seeds after space breeding, and increase the probability of breeding effective mutations in the limited space of flower seeds in space breeding.

[0037] Table 1. Comparison of disinfection and contamination between the present invention and traditional space breeding preservation methods;

[0038] Number of inoculation bottles Number of contaminated bottles Pollution rate This invention sowing 15 3 20% Sowing using mature seed preservation methods 15 9 60%

[0039] Table 2. Comparison of seed germination rates between the present invention and traditional space breeding preservation methods

[0040] Number of inoculation bottles Number of germination bottles Germination rate This invention 15 12 80% Sowing using mature seed preservation methods 15 4 26.7%

Claims

1. A method for preserving the pods of Cymbidium goeringii bred in space, characterized in that, Includes the following steps: First, select 70% mature Cymbidium goeringii capsules and sterilize them in a clean bench to obtain sterile pods; Then, the pods are dehydrated in a sterile environment to obtain whole, dried pods, which are then placed in modified atmosphere bags and stored at low temperature. Finally, before entering the space breeding chamber, the pods are removed from the modified atmosphere bag and completely immersed in medical liquid paraffin to evenly cover their surface with paraffin. The treated pods are then placed in a self-sealing bag and put into the chamber.

2. The method for preserving pods of Cymbidium goeringii bred in space according to claim 1, characterized in that, The specific process for disinfecting and sterilizing Cymbidium goeringii capsules is as follows: First, disinfect the Cymbidium goeringii capsules in 75% alcohol for 20 minutes. After removing them, soak them in 0.1% sodium hypochlorite solution for 15 minutes. Finally, rinse them 2-3 times with sterile water and wipe them dry with gauze to obtain sterile capsules.

3. The method for preserving pods of Cymbidium goeringii bred in space according to claim 1, characterized in that, The pods are dehydrated using color-changing silica gel, which reduces the moisture content of the pods to 8%–10%.

4. The method for preserving pods of Cymbidium goeringii bred in space according to claim 1, characterized in that, The gas in the modified atmosphere bag is a mixture of oxygen, carbon dioxide, and nitrogen in a volume ratio of 0.6:12:87.4 after filtration through a 0.22μm microporous membrane filter.

Images