Method for inoculating embryos in green-skinned fruits of thin-skinned walnuts

Abstract

The invention discloses an inoculation method for seed embryos in green-skinned fruits of thin-skinned walnuts, and belongs to the technical field of plant tissue culture. Healthy green-skinned fruits of thin-skinned walnuts are selected, washed and disinfected with alcohol with a mass concentration of 75%, and in a clean bench, part of the green skin is cut off to expose the seed shell, and the seed embryos without cotyledons are inoculated into the culture medium after prying. On the 28th day of culture, the contamination rate, browning rate, and brown death rate are all 0, the survival rate is 100%, and the seed embryos grow well. The invention does not use highly toxic reagents such as mercuric chloride and irritating sodium hypochlorite for disinfection, the disinfection reagent does not contact the seed embryo, and after disinfection, it is not rinsed with sterile water, and it is not necessary to remove all the green skin and clamp the seed shell during inoculation; it saves time, labor, water, reagents, and costs, is safe, environmentally friendly, high-quality and efficient, is suitable for scientific research and factory-scale tissue culture seedlings, and lays a technical foundation for walnut subculture proliferation and the establishment of a leaf regeneration system for genetic transformation and gene editing to cultivate new varieties, and can also be used for the inoculation of seed embryos of other plant fruits.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and more particularly to a method for inoculating embryos in green-skinned fruits of thin-skinned walnuts. Background Art

[0002] Plant tissue culture technology is an important technology for rapid plant propagation, germplasm preservation and genetic improvement of new varieties. Explant inoculation is the first step to achieve rapid tissue culture propagation. Seed embryos are commonly used explants, especially for hybrid breeding offspring.

[0003] At present, in production and research, the explants are often rinsed with running water, then disinfected with a combination of alcohol and mercuric chloride, or alcohol and sodium hypochlorite, and then rinsed with sterile water. The disinfected explants are then inoculated on the culture medium. However, mercuric chloride is highly toxic, and improper use can easily lead to the death of the explants. Improper treatment of waste liquid containing heavy metal mercury ions can cause environmental pollution. The use of highly toxic drugs requires reporting and there are safety hazards during storage and use, so it is inconvenient to use. Sodium hypochlorite is safer than mercuric chloride, but because of its strong irritation, corrosiveness and oxidizing properties, it is easy to damage the surface of the explants and brown them when it comes into contact with them, which is particularly obvious for young and tender tissues. Inhalation of chlorine gas also harms the health of the operator.

[0004] For walnuts, there is a seed shell outside the embryo, and there is green skin outside the seed shell. According to the traditional inoculation method, the steps are as follows: washing with water → disinfection with mercuric chloride or sodium hypochlorite solution → rinsing with sterile water → disinfection with alcohol solution → rinsing with sterile water → removing all green skin → clamping the seed shell → taking out the embryo without cotyledons → disinfection with mercuric chloride or sodium hypochlorite solution → rinsing with sterile water → disinfection with alcohol solution → rinsing with sterile water → inoculation. There are the following problems: (1) The traditional disinfection method is to place the walnuts with green skin in a high-pressure sterilized beaker in an ultra-clean bench for disinfection. Since the diameter of the walnuts with green skin can reach about 5 cm, only a few walnuts can be disinfected at a time using a 1000 mL beaker, and the disinfection efficiency is low. (2) It is laborious and time-consuming to remove the green skin with a knife, and even with latex gloves, the hands are easily stained with walnut phenol, which is oxidized to form walnut quinone and becomes brown, which is difficult to wash off; (3) Since the green skin is not easy to remove completely, it is not easy to be disinfected with disinfectants or cleaned clean, which can easily cause contamination, bringing operational inconvenience to the inoculators; (4) The method of using a nutcracker to crack the walnut shell and then take out the embryo for inoculation is cumbersome, difficult and easy to contaminate; (5) Rinsing with sterile water after disinfection is water-consuming and time-consuming and increases the risk of embryo contamination. In summary, the traditional embryo inoculation method is difficult to operate for walnuts, time-consuming, labor-intensive, water-consuming, reagent-consuming, and costly. It can also cause pollution to the environment and the embryo itself, resulting in the loss of materials and time, resulting in the inability to efficiently and effectively complete the inoculation goals, affecting the subsequent links of tissue culture and rapid propagation, affecting genetic transformation or gene editing to cultivate new varieties, affecting scientific research and production plans, and causing economic losses.

[0005] Therefore, it is an urgent problem for technicians in this field to provide a method for inoculating walnut embryos that does not require disinfection in a clean bench, does not require disinfection with mercuric chloride and sodium hypochlorite, does not require thorough removal of green peel, does not require nutcrackers to open the walnuts, and does not require sterile water rinsing after disinfection. Summary of the invention

[0006] In view of this, the present invention provides a method for inoculating seed embryos in green-skinned fruits of thin-skinned walnuts, which is a method for effectively completing walnut inoculation without disinfection in a clean bench, without disinfection with mercuric chloride and sodium hypochlorite, without direct contact of disinfectant with seed embryos, without thorough removal of green skin and seed shell, without inoculation of cotyledons, and without rinsing with sterile water after disinfection. With the method of the present invention, the seed embryos have zero pollution, zero browning, zero brown death after inoculation, and sprout and grow normally, achieving the goal of saving time, labor, water, reagents, and costs, being safe and environmentally friendly, and completing the inoculation link of walnut seed embryos as explants with high quality and efficiency, laying a solid technical foundation for the subsequent walnut subculture proliferation and establishment of a leaf regeneration system for genetic transformation and gene editing to cultivate new varieties, and can also be used for the inoculation of seed embryos in fruits of other plants.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for inoculating embryos in green-skinned fruits of thin-skinned walnuts, comprising the following specific steps:

[0009] (1) Select healthy thin-skinned walnuts with green skin that are plump, free of pests and diseases, and without obvious mechanical damage;

[0010] (2) Wash the fruits and set aside;

[0011] (3) Disinfect the washed fruits with an alcohol solution having a mass concentration of 75%;

[0012] (4) Transfer the sterilized fruit with alcohol into a clean bench, cut off part of the green peel, pry open the green peel and seed shell, take out the embryo and inoculate it into the culture medium for cultivation.

[0013] Generally speaking, the standard for successful inoculation is whether the embryo is contaminated, browned, browned and alive. The theoretical basis of the technical solution of the present invention is: ① The embryo inside the plump and healthy green-skinned fruit is sterile; ② The appearance of the green skin is oval and smooth and easy to disinfect; ③ The seed shell of the thin-skinned walnut is thin and the suture line is shallow and easy to pry open; ④ The disinfectant only uses volatile alcohol and contacts the green skin, and the green skin is not used for inoculation. The embryo will not be poisoned by the disinfectant if it does not contact the disinfectant, and it does not need to be rinsed after alcohol disinfection. For this reason, the technical solution of the present invention firstly adopts the method of disinfecting green-skinned walnuts outside the clean bench with only an alcohol solution with a mass concentration of 75%. Compared with the method of disinfecting with a strong alcohol solution plus a mercuric chloride solution or an alcohol solution plus a sodium hypochlorite solution, the risk of disinfectant harming the embryo or even killing the embryo is avoided, and the safety risks brought to the environment and the operator by using mercuric chloride and sodium hypochlorite are also avoided; and the green-skinned walnuts can be sealed and disinfected in a large-volume plastic box outside the clean bench, which is simple to operate, and the number of green-skinned walnuts disinfected each time is large, and the disinfection efficiency is high. Secondly, the present invention transfers the disinfected fruits with alcohol into the clean bench for processing, and utilizes the characteristics of alcohol that can continue to disinfect and is volatile, eliminating the steps of disinfection in the clean bench and rinsing with sterile water after disinfection, saving effort and time. Thirdly, the present invention only cuts off part of the green skin at the fruit stalk to expose the seed shell, and the shell opener is inserted from here to pry open the green skin and seed shell to expose the seed embryo, eliminating the trouble of cutting and peeling all the green skin, then using a nutcracker to open the seed shell, and inoculating the entire walnut seed embryo. In short, this method breaks through the shackles of the inherent thinking and achieves the inoculation effect of saving time, labor, water, reagents, cost, safety, environmental protection, high efficiency and high quality.

[0014] Furthermore, the green-skinned fruit in step (1) is a green-skinned fruit with a hardened or semi-hardened seed shell.

[0015] Furthermore, the cleaning in step (2) needs to be dried.

[0016] Furthermore, the disinfection with the alcohol solution having a mass concentration of 75% in step (3) is a closed disinfection for 5 to 20 minutes, with appropriate shaking during the period.

[0017] Furthermore, the specific operation of step (4) is as follows: the sterilized fruit is fished out from the alcohol solution, and the fruit is directly transferred from the entrance of the clean bench to a sterile tray in the clean bench with the alcohol, and part of the green skin at the fruit stalk is cut off to expose the seed shell, and the green skin and seed shell are pried open with a sterile shell opener, and plump, white, disease-free, and cotyledon-free embryos are selected for inoculation.

[0018] The above-mentioned shell opener has been disclosed in patent number CN201410825986.6, patent name walnut opener, and the specific usage method can be found in the disclosed patent document.

[0019] Furthermore, the culture medium in step (4) is a medium in which the following components are added to the DKW culture medium: 6-BA 0.3-0.8 mg·L -1 、IBA 0.1~0.3mg·L -1 , sucrose 25~30g·L -1 and agar 5.5~6.0g·L -1 , the pH of the culture medium is 5.8-6.2.

[0020] Furthermore, the culture conditions in step (4) are as follows: after 5 days of dark culture, the culture is transferred to natural light for culture, with a light exposure time of 14 to 18 h / d and a culture temperature of 25±2°C.

[0021] Furthermore, during the culture period, the contamination rate, browning rate, brown death rate, and survival rate of the embryos were counted, and the growth of the embryos was observed.

[0022] Furthermore, the calculation formulas for the contamination rate, browning rate, brown death rate and survival rate are as follows:

[0023]

[0024]

[0025]

[0026]

[0027] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for inoculating embryos in green-skinned fruits of thin-skinned walnuts, and the beneficial effects achieved are as follows: (1) The present invention is based on the theoretical basis that "the interior of plump, pest-free, mechanically damaged, and healthy green-skinned fruits is free of bacteria". The surface of the green skin is disinfected with only an alcohol solution of 75% by mass, eliminating the use of highly toxic reagents such as mercuric chloride and irritating, corrosive, and highly oxidizing sodium hypochlorite for disinfection and the rinsing step after disinfection, reducing the pollution of the environment by chemical reagents and the harm to the operator, and also reducing the use of chemical reagents and water, thereby reducing costs; (2) The present invention is based on the fact that "the seed shell of thin-skinned walnuts is thin, the suture line is shallow, and the seed shell is easy to open". Only part of the green skin is cut off at the fruit stalk to expose the seed shell, and then the green skin and seed shell are pried open with a shell opener, and the embryo without cotyledons is directly pinched for inoculation, eliminating the need to use the green skin with a shell opener. The steps of completely cutting off the peel with a knife and opening the hard shell with a nutcracker save time and effort; (3) when the fruit is transferred to the clean bench, the fruit is brought with alcohol. Taking advantage of the alcohol's ability to continue disinfection and its volatility, the steps of disinfection in the clean bench and rinsing with sterile water after disinfection are omitted. In addition, the alcohol disinfects the green peel, and the disinfectant does not directly contact the embryo, thus avoiding the risk of contamination caused by rinsing, saving water, time and no damage to the embryo; (4) the present invention uses a method that is simple to operate, saves time, effort, water and reagents, saves costs, is safe and environmentally friendly, and has high quality and efficiency to complete the inoculation link in the walnut tissue culture rapid propagation technology, greatly reducing the technical difficulty and complexity, improving the inoculation quality and efficiency, and being suitable for scientific research and factory-based tissue culture seedling fields, laying a solid technical foundation for the subsequent walnut subculture proliferation and establishment of a leaf regeneration system for genetic transformation and gene editing to cultivate new varieties, and can also be used for the inoculation of embryos in other plant fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 The attached figure shows the morphology of the green-skinned walnut of 'Green Ridge' and the germination and growth of 1 embryo after inoculation in Example 1 of the present invention;

[0030] Among them, A is the appearance of the green-skinned walnut of 'Green Ridge'; B is the longitudinal section of the green-skinned walnut of 'Green Ridge'; C is the embryo without cotyledons; D is the embryo inoculated at 0 days; E is the embryo inoculated at 14 days; F is the embryo inoculated at 28 days;

[0031] Figure 2 The accompanying drawing shows the survival rate of embryo cultured on days 0 to 28 in treatment 1 of Example 1 of the present invention;

[0032] Figure 3 The accompanying figure shows the survival rate of embryos cultured on days 0 to 28 in treatment 2 of Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] (1) Select the 'Green Ridge' walnuts with hardened or semi-hardened husks, plump, free of pests and diseases, and free of mechanical damage, with green skin.

[0036] (2) Wash the fruits with a 1% detergent solution, rinse with tap water, wipe dry, and place in a plastic box.

[0037] (3) Add 75% alcohol solution into the plastic box and seal it for disinfection for 5 minutes, shaking it appropriately during the process (treatment 1). 100 embryos were used in this treatment.

[0038] A sodium hypochlorite solution with an effective chlorine concentration of 1% was added to the plastic box, and the box was sealed and disinfected for 15 minutes, with appropriate shaking during the period. After the disinfection, the sodium hypochlorite solution was poured out, and the box was rinsed with sterile water for 3 times, and then an alcohol solution with a mass concentration of 75% was added, and the box was sealed and disinfected for 5 minutes, with appropriate shaking during the period (treatment 2). 100 embryos were used in this treatment.

[0039] (4) Take out the green-skinned walnuts from the alcohol solution and transfer them directly from the entrance of the clean bench to a sterile tray inside the clean bench with the alcohol. Cut off part of the green skin at the fruit stalk to expose the seed shell. Use a sterile shell opener to pry open the green skin and seed shell. Use tweezers to take out the plump, white, disease-free, and cotyledon-free embryos and inoculate them into the culture medium.

[0040] The above culture medium is prepared by adding the following components in mass concentration to DKW culture medium: 6-BA 0.5 mg·L -1 、IBA0.1mg·L -1 , sucrose 30g·L -1 and agar 5.5 g·L -1 , pH 5.8.

[0041] (5) After 5 days of dark culture, the cells were cultured under natural light with a light intensity of 16 h / d and a culture temperature of 25 ± 2°C.

[0042] On the 3rd, 7th, 14th, 21st and 28th days of culture, the contamination rate, browning rate, brown death rate and survival rate of the embryos were counted, and the growth of the embryos was observed.

[0043]

[0044]

[0045]

[0046]

[0047] Results Figures 1 to 3 . The results showed that the contamination rate, browning rate and brown death rate of the embryos of the 'Green Ridge' green-skinned walnut in treatments 1 and 2 were all 0 on the 3rd, 7th, 14th, 21st and 28th days of culture, and the survival rate of the embryos was 100%; the embryos began to germinate on the 3rd day of culture, the embryos began to elongate on the 7th day of culture, the embryos obviously elongated and turned green on the 14th day of culture, and the embryos grew well on the 28th day of culture. That is, through the two inoculation methods of treatments 1 and 2, 100% of the embryos grew well after inoculation and could enter the subsequent links such as subculture proliferation and leaf regeneration. Treatment 1 omitted the steps of sodium hypochlorite disinfection and sterile water rinsing compared with treatment 2, so treatment 1 was better.

[0048] The present invention can effectively complete the disinfection and inoculation links of walnut embryos as explants without the need for disinfection with mercuric chloride and sodium hypochlorite, without disinfection in a clean bench, without direct contact of disinfection reagents with embryos, without thorough removal of green skin and seed shells, without inoculation of cotyledons, and without rinsing with sterile water after disinfection, and ensures that the embryos are zero-polluting, zero-browning, zero-brown dead, and germinate and grow normally after inoculation. The method is simple to operate, saves time, labor, water, reagents, and costs, is safe and environmentally friendly, efficient, and high-quality, and ensures that 100% of the embryos grow normally, laying a solid foundation for subsequent walnut subculture proliferation, establishing a leaf regeneration system for genetic transformation, and cultivating new varieties by gene editing technology, and is suitable for scientific research and factory-based seedling cultivation.

[0049] The technical solution of the present invention is also applicable to the seed embryos of other plants with peels for explant inoculation, and those skilled in the art may refer to it according to actual conditions. It should be noted that the step of taking the seed embryos for inoculation in the clean bench of the present invention should be strictly aseptic, and scientific researchers should also strictly operate when trying to use this method for tissue culture of other plants with smooth peels. The present invention will help to say goodbye to the methods of disinfection of seed embryos by direct contact with disinfectants, disinfection with mercuric chloride and sodium hypochlorite, disinfection in the clean bench, and rinsing with sterile water after disinfection, making the explant disinfection method simpler, saving water, labor, reagents, and costs, high quality, high efficiency, safety, and environmental protection.

[0050] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for inoculating embryos in green-skinned fruits of thin-skinned walnuts, characterized in that: The specific steps are as follows: (1) Select the walnuts of the 'Green Ridge' variety that have hardened or semi-hardened husks, are plump, free of pests and diseases, and have green skins without mechanical damage; (2) Wash the fruits with 1% detergent solution, rinse with tap water, dry them, and place them in a plastic box; (3) Add 75% alcohol solution into the plastic box and seal it for disinfection for 5 minutes, shaking it appropriately during the process; (4) The green-skinned walnuts were removed from the alcohol solution and transferred directly from the entrance of the clean bench to a sterile tray in the clean bench with the alcohol, and part of the green skin at the fruit stalk was cut off to expose the seed shell. The green skin and seed shell were pried open with a sterile shell opener, and plump, white, disease-free, insect-free, and cotyledon-free embryos were taken out with tweezers and inoculated into a culture medium; the culture medium was a DKW medium with the following mass concentrations of components added: 6-BA 0.5 mg·L -1 、IBA 0.1mg·L -1 , sucrose 30g·L -1 and agar 5.5 g·L -1 , the pH of the culture medium is 5.8; (5) After 5 days of dark culture, the cells were cultured under natural light with a light intensity of 16 h / d and a culture temperature of 25 ± 2°C. During the culture period, the contamination rate, browning rate, brown death rate, and survival rate of the embryos were counted, and the growth of the embryos was observed; The calculation formulas for the contamination rate, browning rate, brown death rate and survival rate are as follows:

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