A method for inoculating and raising seedlings for tissue culture and rapid propagation of raspberry seeds

Abstract

The invention discloses an inoculation and seedling raising method for tissue culture and rapid propagation of raspberry seeds, and belongs to the technical field of plant tissue culture and seed seedling raising. The invention sterilizes raspberry seeds with alcohol having a mass concentration of 75% in a clean bench, rinses with sterile water, soaks in sterile water, cuts off part of the seed coat, soaks in a gibberellin solution, inoculates in a culture medium, and cultured under light. During the culture period, the seeds are free of pollution, browning, and brown death, and have high white exposure rate, germination rate, and seedling rate. The invention uses seeds as explants to inoculate and raise seedlings using a tissue culture method, omitting water washing before disinfection and disinfection with mercuric chloride or sodium hypochlorite, is simple to operate, saves time, labor, water, space, cost, safety, and environmental protection, and solves the problem of difficult germination and difficult seedling formation caused by hard and thick raspberry seed coats, small seeds, and low nutrition; compared with field seedling raising, the seedling raising time is shortened, management is more convenient, and the quantity and quality of seed germination and seedling formation are improved, and the seed germination and seedling formation can be used for hybrid breeding offspring seedling formation, molecular breeding, and gene function verification.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture and seed seedling raising, and more particularly to an inoculation and seedling raising method for tissue culture and rapid propagation of raspberry seeds. Background Art

[0002] Raspberry, also known as raspberry, tray, marlin, etc., is a perennial medicinal and edible plant of the genus Rubus in the Rosaceae family. Its roots, leaves and fruits are rich in medicinal active ingredients and have antibacterial, anti-tumor and antioxidant effects. Its fruits can be eaten fresh, and are also made into juice, fruit wine, jam, candy, cake, biscuits and other foods; therefore, raspberry has a high economic value.

[0003] In nature, raspberries can reproduce through root sprouts, so researchers currently focus on asexual reproduction of raspberries, and there are few reports on the technology of raspberry seed seedlings. However, in the process of raspberry hybrid breeding, it is necessary to make the hybrid F1 generation seeds develop into plants, so the research on raspberry seed seedling technology is very necessary.

[0004] Raspberry seeds are only a few millimeters in diameter, small in size, and contain little nutrition; the embryo is covered by a hard, thick, gear-shaped seed coat, which is composed of a large number of fibrous thick-walled cells and has poor water and air permeability. The above factors are the key factors that lead to the slow germination and low germination rate of raspberry seeds; existing production technologies use a variety of means to overcome the obstacles to raspberry seed germination, but the results are not ideal:

[0005] The low temperature stratification method for germinating raspberry seeds is time-consuming and labor-intensive and has a low germination rate (less than 1%);

[0006] The use of concentrated sulfuric acid, hypochlorite and other chemical reagents that damage the seed coat to treat seeds poses safety risks, and the germination speed and germination rate (less than 65%) cannot meet the requirements of practical application;

[0007] Treating seeds with hormones or nutrients has poor absorption effect, cannot effectively promote germination and wastes reagents;

[0008] Therefore, how to process seeds to effectively improve germination efficiency is a technical problem that needs to be solved in this field.

[0009] Furthermore, the existing field sowing method of raspberry seeds has a low germination rate and a low seedling rate. In addition to the factors that the seed coat is difficult to break through and the embryo has low nutrition, it is also related to the difficulty in controlling the field cultivation environment and the pests and diseases in the soil.

[0010] Therefore, there is an urgent need to provide a method that is simple and convenient to operate, safe and efficient, has a fast germination speed of raspberry seeds, a high germination rate, and a good seedling effect. Summary of the invention

[0011] In view of this, the present invention provides an inoculation and seedling raising method for tissue culture and rapid propagation of raspberry seeds, which is simple and convenient to operate, has a high and fast seedling rate, is safe, environmentally friendly and efficient, lays a solid material foundation for the establishment of a raspberry seed tissue culture and rapid propagation system, seed seedling formation of hybrid breeding and subsequent research, and is suitable for the field of scientific research and industrialized tissue culture.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] A method for inoculating and raising seedlings for tissue culture and rapid propagation of raspberry seeds, comprising the following specific steps:

[0014] (1) Preparing raspberry seeds as tissue culture materials;

[0015] (2) In a clean bench, disinfect with 75% alcohol and rinse with sterile water;

[0016] (3) Soak in sterile water;

[0017] (4) Cut off part of the seed coat;

[0018] (5) Soaking in gibberellin solution;

[0019] (6) Inoculate into culture medium and culture under light.

[0020] The present invention realizes raspberry seed seedling formation through a tissue culture method. Compared with the existing field sowing and seedling raising method, the culture environment is simple, stable and easy to control, and daily management such as pest control, watering, fertilizing and weeding is not required; and the culture medium in the tissue culture bottle can provide sufficient nutrition for seed germination and seedling formation, and the germination speed is fast, the germination rate is high (the germination rate reaches 100.0% after culturing for 18 to 24 days), and the seedling rate is high (the seedling rate reaches 90.0% after culturing for 30 days).

[0021] First of all, for tissue culture, the first problem that needs to be solved is how to disinfect and avoid contamination; the traditional tissue culture disinfection method is to rinse with running water → disinfect with mercuric chloride solution or sodium hypochlorite solution → rinse with sterile water → disinfect with alcohol solution → rinse with sterile water; however, mercuric chloride is highly toxic, sodium hypochlorite is irritating and corrosive, it is unsafe to use, and the disinfection operation is cumbersome. The theoretical basis of the technical solution of the present invention is that the inside of healthy seeds without mechanical damage is sterile, so the surface of the seeds can be disinfected without contamination. Therefore, the technical solution of the present invention only uses an alcohol solution with a mass concentration of 75% to disinfect the seeds. There is no need to wash with water before disinfection, and there is no need to use a strong alcohol solution plus mercuric chloride solution or alcohol solution plus sodium hypochlorite solution disinfection method, which saves time, labor, water, reagents, costs, and is safe and environmentally friendly.

[0022] Secondly, the existing method for processing raspberry seeds is time-consuming and labor-intensive, has safety risks and has low germination efficiency. After the raspberry seeds are disinfected, they are first soaked in sterile water, which, on the one hand, provides the seeds with moisture required for germination, and on the other hand, softens the raspberry seed coat, which is convenient for the subsequent operation of cutting off part of the seed coat; after soaking in sterile water, part of the seed coat is cut off, which is simple to operate and can effectively remove the seed coat obstacle; after cutting off part of the seed coat, the seeds are soaked in a gibberellin solution, which replenishes moisture while allowing the gibberellin to be fully absorbed by the seeds, thereby promoting rapid germination of the seeds.

[0023] Generally speaking, the standard for successful seedling inoculation is to see the pollution, browning, brown death, white exposure, germination, and seedling growth of the seeds. The method of the present invention is used to carry out the rapid propagation inoculation and seedling raising of raspberry seeds, which can solve the problems of poor water absorption and air permeability, large germination resistance, and small germination force caused by the hard and thick seed coat, small seeds, and less nutrition of raspberry seeds. During the seedling raising process, the seeds are free of pollution, browning, and brown death, and the white exposure rate, germination rate, and seedling rate are high. The germination and seedling speed is fast and of good quality, and the loss of raspberry seed materials in the field sowing and seedling raising process can be effectively avoided, shortening the seedling raising time, and laying a solid foundation for establishing a raspberry seed tissue culture system, promoting hybrid seed germination and seedling raising, and saving hybrid breeding embryo abortion, using seed germination seedlings to induce callus tissue for transient expression and identification of gene function, transgenic breeding, and gene editing to cultivate new germplasm, etc.; and the tissue culture seedlings cultivated using the method of the present invention do not carry pests and diseases, and can be used as a higher-quality seedling resource compared to field sowing seedlings and root seedlings.

[0024] Furthermore, the seeds in step (1) are mature, plump, free of mechanical damage, pests and diseases, and healthy. When selecting seeds, water selection, wind selection, hand selection and other means can be used to screen out seeds that do not meet the requirements, thereby ensuring the efficiency of subsequent inoculation and seedling raising.

[0025] Furthermore, the specific operation described in step (2) is: at the entrance of the clean bench, the seeds are placed in a high-pressure sterilized bottle in the clean bench, sealed and sterilized in the bottle with a 75% alcohol solution for 3 to 5 minutes, shaken upside down, and rinsed with sterile water for 3 to 5 times.

[0026] Furthermore, the sterile water immersion in step (3) is carried out at 20 to 30° C. and in a sealed sterile water immersion for 20 to 48 hours.

[0027] Furthermore, step (4) is to cut off the seed coat at both ends of the seed with a scalpel.

[0028] Since raspberry seeds are too small, it is difficult and laborious to completely peel off the seed coat. The present invention only cuts off the seed coat at both ends of the seeds after soaking the seeds in sterile water. The operation can be completed in more than ten seconds, with low difficulty, high speed and good subsequent germination effect.

[0029] Furthermore, in step (5), the gibberellin solution is soaked at 20-30°C with 0.05-0.2 mg·L -1 Soak in GA3 solution for 12 to 18 hours.

[0030] In the prior art, the seed coat is not cut off, even if the concentration of gibberellin is as high as 800 mg·L -1 The invention relates to a method for treating raspberry seeds with a solution of gibberellin, which can still fail to obtain a faster germination speed and a high germination rate. The invention cuts off part of the seed coat of the seeds to remove the barrier of the seed coat, which is beneficial for gibberellin to enter the seeds and act on the embryo. The invention uses a gibberellin solution with a concentration thousands of times lower than that of the prior art to treat raspberry seeds to obtain an excellent germination effect, and reduces the amount of gibberellin used, thereby saving costs.

[0031] Furthermore, the culture medium in step (6) is supplemented with 0.1 to 0.4 mg·L -1 GA3 culture medium.

[0032] Since the soaking time of gibberellins is limited, in order to ensure that the seeds receive sufficient growth stimulation during the seedling raising process, gibberellins are added to the culture medium to further increase the growth rate and promote germination and seedling formation.

[0033] Furthermore, the culture medium is a MS culture medium with the following components added thereto: sucrose 20-30 g·L -1 and agar 5.5~6.0g·L -1 , pH value 5.8~6.2.

[0034] More preferably, the GA3 concentration in the culture medium is 0.1-0.2 mg·L -1 .

[0035] Furthermore, the illumination culture in step (6) is performed with an illumination time of 10 h to 16 h / day, an illumination intensity of 500 to 2000 lx, and a temperature of 25±2°C.

[0036] Furthermore, during the cultivation period, the seed contamination rate, browning rate, brown death rate, whitening rate, germination rate, and seedling rate were counted, and the growth of the seeds was observed;

[0037] The calculation formulas for the contamination rate, browning rate, brown death rate, whitening rate, germination rate, and seedling rate are as follows:

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses an inoculation and seedling raising method for tissue culture and rapid propagation of raspberry seeds, which uses seeds as explants for tissue culture, saves the trouble of daily management in field sowing and seedling raising technology, greatly shortens the seedling raising time and improves the seed germination rate compared with field sowing and seedling raising which takes several months; only 75% alcohol is used for disinfection, avoiding the use of highly toxic reagents such as mercuric chloride and irritating sodium hypochlorite for disinfection, and no water washing is required before disinfection, which is simple to operate, safe, environmentally friendly and efficient; through the method of soaking in sterile water, cutting off part of the seed coat, soaking in a gibberellin solution, and then culturing in a culture medium, the use of The invention solves the safety risk of damaging the embryo and the operator when destroying the seed coat with concentrated sulfuric acid, and solves the problems of poor water absorption and air permeability, large germination resistance, small germination force, and urgent need for supplementary nutrition to strengthen the seedlings after germination, otherwise the seedlings are prone to wilt and die due to the hard and thick seed coat, small seeds and less nutrition of raspberries. In summary, the invention is simple to operate, saves time, labor, water, space and cost, is safe and environmentally friendly, shortens the time for raspberry seeds to germinate and seedleave, improves the quantity and quality of germinated seedlings, can be used to save embryo abortion in hybrid breeding offspring and promote hybrid seeds to germinate and seedleave, the obtained seedlings can be directly used for tissue culture rapid propagation to obtain more seedlings, and can also be used for molecular breeding and gene function verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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.

[0046] Figure 1 The accompanying drawings show the germination and growth of raspberry seeds of the present invention;

[0047] Among them, A. raspberry seeds before inoculation; B. seeds inoculated after incision; C. white seeds; D. germinated seeds; E. plants in treatment 2; F. plants in treatment 3; G. plants in treatment 8;

[0048] Figure 2 The attached figure shows the whitening rate and germination rate of the seeds of treatment 1 of Example 1 of the present invention on the 3rd to 30th day of cultivation;

[0049] Figure 3 The attached figure shows the whitening rate and germination rate of seeds treated with 2 in Example 1 of the present invention on the 3rd to 30th day of cultivation;

[0050] Figure 4The attached figure shows the whitening rate and germination rate of seeds treated 3 in Example 1 of the present invention on the 3rd to 30th day of cultivation;

[0051] Figure 5 The attached figure shows the whitening rate and germination rate of seeds treated 4 in Example 1 of the present invention on the 3rd to 30th day of cultivation;

[0052] Figure 6 The attached figure shows the whitening rate and germination rate of seeds treated with 5 in Example 1 of the present invention on the 3rd to 30th day of cultivation;

[0053] Figure 7 The attached figure shows the white exposure rate and germination rate of seeds treated with 8 in comparative example 1 of the present invention on the 3rd to 30th day of cultivation. DETAILED DESCRIPTION

[0054] 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.

[0055] Example 1

[0056] (1) Select the plump, mechanically undamaged, disease-free and insect-free, healthy seeds of ripe red raspberries by water selection, wind selection, hand selection, etc. Figure 1 -A). At the entrance of the clean bench, the seeds were placed in a high-pressure sterilized bottle in the clean bench, and sterilized in the bottle with a 75% alcohol solution for 4 minutes, during which they were shaken upside down, rinsed with sterile water for 3 times, and then immersed in sterile water at 25±2℃ for 24 hours.

[0057] (2) After soaking, remove the seeds, cut off the seed coat at both ends with a scalpel, and soak them in 0.1 mg / L gibberellin solution at 25±2°C for 12 hours. Then divide them into different treatment groups for cultivation, with a light exposure time of 16 hours / day, a light intensity of 2000 lx, and a temperature of 25±2°C. Use 60 seeds for each treatment:

[0058] Treatment 1: Seeds were inoculated into the culture medium (the culture medium was MS medium with the following mass concentrations of components added: sucrose 30 g L -1 and agar 5.5 g·L -1 , pH 5.8) Figure 1 -B),

[0059] The seeds of treatments 2 to 5 were inoculated into the culture medium (the culture medium was MS medium with the following mass concentrations of components added: GA30.1 mg·L -1(Treatment 2), GA30.2 mg·L -1 (Treatment 3), GA30.3 mg·L -1 (Treatment 4), GA30.4 mg·L -1 (Treatment 5), 30 g·L sucrose was added to treatments 2 to 5 -1 and agar 5.5 g·L -1 , pH 5.8) Figure 1 -B).

[0060] On the 3rd, 6th, 9th, 12th, 15th, 18th, 21st, 24th, 27th and 30th days of culture, the seed contamination rate, browning rate, brown death rate, whitening rate and germination rate were counted. On the 30th day of culture, the seedling rate was counted and the seed growth was observed.

[0061]

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[0065]

[0066]

[0067] Results Figures 1 to 6 The results showed that the contamination rate, browning rate and brown death rate of raspberry seeds in treatments 1 to 5 were all 0 during the 30-day culture period; the whitening rate of seeds in treatment 1 was 96.7% on the 21st day of culture and did not increase thereafter; the whitening rate of raspberry seeds in treatments 2 to 5 was 96.7% on the 21st day of culture (the white embryo was elongated and exposed outside the seed coat), and the whitening rate was 96.7% on the 21st day of culture. The whitening rate did not increase thereafter. The whitening rate of seeds in treatments 2 to 5 was 96.7% on the 21st day of culture, and ... the whitening rate was 96.7% on the 21st day of culture. The whitening rate of seeds in Figure 1 -C) rate reached 100.0%, and the germination of treatments 1 to 5 (the cotyledons exposed outside the seed coat unfolded and turned green, Figure 1 -D) rates were 80.0%, 100.0%, 100.0%, 96.7% and 88.3% on the 24th, 24th, 18th, 18th and 18th days, respectively. The seedling rates of treatments 1 to 5 were 80.0%, 90.0%, 90.0%, 88.3% and 86.7% on the 30th day, respectively. The seedlings of treatments 1 to 5 had good growth conditions, had multiple green leaves and were taller than 1 cm. That is, more than 90.0% of the seeds germinated into seedlings after inoculation by the methods of treatments 2 to 3 ( Figure 1 -E and Figure 1-F) can enter the stages of subculture proliferation, leaf regeneration, gene function verification, transgenic breeding, and gene editing to create new germplasm.

[0068] The invention does not use concentrated sulfuric acid to destroy the seed coat, does not need water washing before disinfection, does not use mercuric chloride and sodium hypochlorite for disinfection, and does not require daily care, effectively completes the inoculation and seedling raising links of raspberry seed tissue culture, is simple to operate, saves time, labor, water, space, cost, is efficient, safe, and environmentally friendly, and ensures that 90.0% of the seeds are normally whitened and germinated into seedlings, laying a foundation for subsequent subculture proliferation, leaf regeneration, gene function verification, transgenic breeding, gene editing to create new germplasm, etc. of raspberries, and is suitable for scientific research and factory-based seedling raising.

[0069] Comparative Example 1

[0070] (1) Select healthy seeds of ripe red raspberry fruits that are plump, free of mechanical damage, free of pests and diseases by water selection, wind selection, hand selection, etc., put the seeds into autoclaved bottles in the clean bench at the entrance of the clean bench, and then divide them into different treatment groups, using 60 seeds for each treatment:

[0071] Treatment 6 is to disinfect with a sodium hypochlorite solution with an effective chlorine concentration of 1% for 1 minute, rinse with sterile water three times, and then disinfect with a 75% alcohol solution for 4 minutes, then rinse with sterile water three times, and then soak in sterile water at 25±2℃ for 24 hours.

[0072] Treatment 7 was to disinfect with 75% alcohol solution for 4 minutes, then rinse with sterile water for 3 times, and then immerse in sterile water at 25±2°C for 24 hours.

[0073] Treatment 8 was to disinfect with a 75% alcohol solution for 4 minutes, then rinse with sterile water for 3 times, and then soak in sterile water at 25±2°C for 24 hours; after soaking, the seeds were taken out, the seed coats at both ends were cut off with a scalpel, and then soaked in sterile water at 25±2°C for 12 hours.

[0074] (2) The seeds of the above treatments were inoculated into a culture medium (the culture medium was MS culture medium with the following mass concentrations of components added: sucrose 30 g / L -1 and agar 5.5 g·L -1 , pH 5.8), with a lighting time of 16 h / day, a light intensity of 2000 lx, and a temperature of 25±2°C. The subsequent observation and statistical methods were the same as those in Example 1.

[0075] The results showed that the contamination rate, browning rate and brown death rate of raspberry seeds in treatments 6 to 8 were all 0 during the 30-day cultivation period; the whitening rate, germination rate and seedling rate of seeds in treatments 6 and 7 were all 0 during the 30-day cultivation period, and the whitening rate, germination rate and seedling rate of seeds in treatment 8 were 90.0%, 71.7% and 68.3% on the 21st, 24th and 30th days of cultivation respectively; the growth condition of seedlings germinated in treatment 8 was better, but the leaves were yellowish and the plants were smaller than those in treatments 1 to 5 ( Figure 1 -G); that is, no seeds germinated into seedlings through the methods of treatments 6 and 7, and the inoculation loss was large. In treatment 8, 68.3% of the seeds germinated into seedlings after inoculation and could enter the stages of subculture proliferation, leaf regeneration, gene function verification, transgenic breeding, and gene editing to create new germplasm. The seedling rate was lower than that of treatments 1 to 5, and the seedling growth condition was poor.

[0076] The technical solution of the present invention is also applicable to the inoculation and seedling raising of other plant seeds with thick and hard seed coats and small seeds, eliminating the trouble of destroying the seed coat with sulfuric acid, washing with water before disinfection, disinfection with mercuric chloride or sodium hypochlorite, and daily management such as fertilizer, water bug disease, light and temperature in the field during seed germination and seedling formation. The inoculation method is also applicable to the inoculation and seedling raising of explants of other plants with full and healthy seeds, and those skilled in the art can refer to it according to actual conditions. It should be noted that the steps from disinfection to inoculation in the clean bench of the present invention should be strictly aseptically operated, and scientific researchers should also strictly operate when trying to use this method for inoculation and cultivation of other plant seeds with thick and hard seed coats and small seeds. The present invention will help to say goodbye to the method of washing with water before disinfection and disinfecting seeds with mercuric chloride or sodium hypochlorite, making the seed disinfection method simpler, more efficient, safer and more environmentally friendly.

[0077] 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 and raising seedlings for tissue culture and rapid propagation of raspberry seeds, characterized in that: The specific steps are as follows: (1) preparing raspberry seeds as tissue culture materials; the seeds are mature, plump, free of mechanical damage, pests and diseases, and healthy; (2) At the entrance of the clean bench, the seeds were placed in a high-pressure sterilized bottle in the clean bench, sealed and sterilized with a 75% alcohol solution for 4 min, shaken upside down, and rinsed with sterile water three times; (3) Soak in sterile water at 25±2℃ for 24h; (4) Use a scalpel to cut off the seed coat at both ends of the seed; (5) At 25±2℃, use 0.1mg·L -1 Soak in GA3 solution for 12h; (6) Inoculate into culture medium and culture under illumination; illumination time is 10 h to 16 h / day, light intensity is 500 to 2000 lx, and temperature is 25 ± 2 °C; The culture medium is a MS culture medium with the following components added thereto: GA3 0.1-0.4 mg·L -1 , sucrose 30g·L -1 and agar 5.5 g·L -1 , pH 5.8; During the cultivation period, the seed contamination rate, browning rate, brown death rate, whitening rate, germination rate, and seedling rate were counted, and the growth of the seeds was observed; The calculation formulas for the contamination rate, browning rate, brown death rate, whitening rate, germination rate, and seedling rate are as follows: The whitening refers to: the white embryo elongates and is exposed outside the seed coat; The germination is as follows: the cotyledons exposed outside the seed coat unfold and turn green; The mature seedlings have a plurality of green leaves and a plant height greater than 1 cm.

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