A tissue culture rapid propagation method of Ostryopsis nobilis

Abstract

The application provides a tissue culture and rapid propagation method of Ostryopsis nobilis, and comprises the following steps: sequentially performing shell disinfection treatment and shell removal disinfection treatment on Ostryopsis nobilis seeds to obtain explants; inoculating the explants into a primary culture medium to perform primary culture and obtain seedlings; sequentially performing first seedling strengthening culture and second seedling strengthening culture on the seedlings to obtain second seedlings; transferring the second seedlings into an adventitious bud induction culture medium to perform adventitious bud induction culture and obtain adventitious bud seedlings; cutting the adventitious buds and transferring them into a proliferation culture medium to perform proliferation culture and obtain proliferation seedlings with proliferation buds of more than 3 cm; cutting the proliferation buds to perform second seedling strengthening culture and obtain seedlings to be rooted with a seedling height of 3.5-5 cm; transferring the seedlings to be rooted into a rooting culture medium to perform rooting culture and obtain rooted seedlings; and then performing seedling raising and transplanting to obtain transplanted seedlings; the method can significantly improve the propagation coefficient of Ostryopsis nobilis and realize rapid propagation of Ostryopsis nobilis.

Description

Technical Field

[0001] This invention relates to a rapid tissue culture propagation method for hazelnut, belonging to the field of biotechnology. Background Technology

[0002] Chinese hazelnut is an excellent tree species for producing dried fruits, woody oil crops, and timber. It is a superior native tree species in the subtropics. Developing Chinese hazelnut resources is of great significance for increasing my country's supply of dried fruits, woody oil crops, and timber, increasing forest vegetation, and increasing forest carbon sequestration.

[0003] However, due to excessive deforestation, its distribution area is shrinking and its numbers are declining sharply, putting it at risk of being replaced by other broad-leaved tree species and becoming endangered. Therefore, it is urgent to increase the cultivation and afforestation of Chinese hazelnut seedlings. Currently, the common propagation methods for Chinese hazelnut are traditional methods such as division, layering, grafting, cutting, and sowing. However, traditional propagation methods have low propagation coefficients, and further exploration of propagation methods with high propagation coefficients is needed to achieve large-scale factory seedling production. Summary of the Invention

[0004] This invention provides a rapid tissue culture propagation method for hazelnut, which can significantly improve the propagation coefficient of hazelnut and realize large-scale seedling production in factories.

[0005] This invention provides a method for rapid tissue culture propagation of hazelnut, comprising the following steps:

[0006] 1) The seeds of *Corylus heterophylla* with shells were subjected to sterilization treatment with the shells on and sterilization treatment after the shells were removed, in order to obtain explants;

[0007] 2) The explants were inoculated into the primary culture medium for primary culture to obtain seedlings with a height of not less than 5 cm;

[0008] 3) The seedlings were cultured using the first seedling culture medium to obtain the first seedlings with axillary buds;

[0009] 4) Transfer the first seedling to the second seedling culture medium for second seedling cultivation to obtain a second seedling with a height of 6-8cm;

[0010] 5) Transfer the second seedling to an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious bud seedlings with adventitious buds with a bud length of 3-5cm;

[0011] 6) Cut the adventitious buds from the adventitious buds and transfer them to the proliferation medium for proliferation culture to obtain proliferation seedlings with proliferation buds of more than 3 cm;

[0012] 7) Cut off the proliferating buds and transfer them to the third seedling culture medium for third seedling culture to obtain seedlings with a height of 3.5-5cm that are ready to develop roots;

[0013] 8) Transfer the seedlings to be rooted into a rooting medium for rooting culture to obtain rooted seedlings;

[0014] 9) The rooted seedlings are subjected to hardening-off culture and transplanting culture to obtain transplanted seedlings with new roots and new leaves;

[0015] The shelling and disinfection process includes: shelling the hazelnut seeds after the shelled disinfection process to obtain shelled hazelnut seeds, and disinfecting the shelled hazelnut seeds with 0.1% mercuric chloride for 4-6 minutes to obtain the explants;

[0016] The primary culture medium includes MS medium, 2 g / L GA3, 30 g / L sugar, and 6 g / L agar;

[0017] The first and second seedling culture media consist of MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar.

[0018] In the tissue culture rapid propagation method described above, in step 1), the sterilization time for the shell removal and sterilization process is 5 minutes.

[0019] In the tissue culture rapid propagation method described above, step 1) of the shell sterilization treatment includes:

[0020] Soak the shelled hazelnut seeds in 60°C hot water for 24 hours. Then, soak the shelled hazelnut seeds in a 0.5% laundry detergent solution for 10 minutes, rinse with running water for 1 hour, soak in 75% alcohol for 30 seconds, disinfect with 0.1% mercuric chloride for 15 minutes, and finally rinse with sterile water 5-6 times.

[0021] The tissue culture rapid propagation method described above includes an adventitious bud induction medium comprising MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar.

[0022] The tissue culture rapid propagation method described above includes a proliferation medium comprising 1 / 3 MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, 6 g / L agar, and 1.0 g / L GA3.

[0023] As described above, the third seedling culture medium comprises 1 / 3 MS medium, 0.5 mg / L 6-BA, 0.3 mg / L IBA, 1.0 g / L GA3, 30 g / L sugar, and 6 g / L agar.

[0024] The tissue culture rapid propagation method described above includes a rooting medium comprising 1 / 4 MS medium, 0.1 mg / L IBA, 0.3 mg / L NAA, 0.5 g / L AC, 30 g / L sugar, and 6 g / L agar.

[0025] In the tissue culture rapid propagation method described above, step 9) includes: placing the rooted seedlings in a greenhouse at 15-25℃, opening the bottle cap for cultivation, and the time is 1 week.

[0026] In the tissue culture rapid propagation method described above, step 9) includes the transplanting culture:

[0027] After hardening off, the rooted seedlings are cut, cleaned, and sterilized in sequence. Then, the rooted seedlings are placed in a seedling pot containing substrate to cultivate transplanted seedlings with new roots and new leaves.

[0028] The matrix is ​​prepared by mixing perlite, peat, and vermiculite in a volume ratio of 1:2:1.

[0029] As described above, the conventional culture method for rapid tissue culture involves a culture temperature of 23-25℃, a culture humidity of 75-85%, a light intensity of 2000-2500 lx, a light exposure of 14-16 h / d.

[0030] The conventional culture includes the primary culture, the first strong seedling culture, the second strong seedling culture, the adventitious bud induction culture, the proliferation culture, the third strong seedling culture, the rooting culture, and the transplanting culture.

[0031] The tissue culture method for rapid propagation of hazelnut provided by this invention uses shelled hazelnut seeds as explants. Seedlings are obtained through primary culture of the seeds. Subsequently, a series of culture processes are carried out, including first strong seedling culture, second strong seedling culture, adventitious bud induction culture, proliferation culture, third strong seedling culture, and rooting culture. The culture medium and culture conditions are optimized to achieve rapid propagation of hazelnut, improve the propagation coefficient, shorten the propagation time, and lay the foundation for large-scale seedling production in factories. Attached Figure Description

[0032] Figure 1 Photographs of seedlings obtained in Example 1;

[0033] Figure 2 Photographs of the adventitious buds obtained in Example 1;

[0034] Figure 3 Photographs of the adventitious buds obtained in Example 1;

[0035] Figure 4 Photographs of the propagated seedlings obtained in Example 1;

[0036] Figure 5 Photographs of the rooted seedlings obtained in Example 1;

[0037] Figure 6 This is a photo of the rooted seedlings obtained in Example 1 after hardening off and awaiting transplantation. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] This invention provides a method for rapid tissue culture propagation of hazelnut, comprising the following steps:

[0040] 1) The seeds of *Corylus heterophylla* with shells were subjected to sterilization treatment with the shells on and sterilization treatment after the shells were removed, in order to obtain explants;

[0041] 2) Inoculate the explants into the primary culture medium for primary culture to obtain seedlings with a height of not less than 5cm;

[0042] 3) The seedlings were cultured in the first seedling culture medium to obtain the first seedlings with axillary buds;

[0043] 4) Transfer the first seedling to the second seedling culture medium for second seedling cultivation to obtain second seedlings with a height of 6-8cm;

[0044] 5) Transfer the second seedling to the adventitious bud induction medium for adventitious bud induction culture to obtain adventitious bud seedlings with adventitious buds with a bud length of 3-5cm;

[0045] 6) Cut adventitious buds from the adventitious buds and transfer them to the proliferation medium for proliferation culture to obtain proliferating seedlings with proliferating buds of more than 3 cm;

[0046] 7) Cut off the proliferating buds and transfer them to the third seedling culture medium for the third seedling culture to obtain seedlings with a height of 3.5-5cm that are ready to develop roots;

[0047] 8) Transfer the seedlings to rooting medium for rooting culture to obtain rooted seedlings;

[0048] 9) Harden-off seedlings and transplant them to obtain transplanted seedlings with new roots and leaves;

[0049] The shelling and disinfection process includes: shelling the hazelnut seeds after the shell disinfection process to obtain shelled hazelnut seeds, and then disinfecting the shelled hazelnut seeds with 0.1% mercuric chloride for 4-6 minutes to obtain explants.

[0050] The primary culture medium consisted of MS medium, 2 g / L GA3, 30 g / L sugar, and 6 g / L agar;

[0051] The first and second seedling culture media consist of MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar.

[0052] The tissue culture rapid propagation method provided by this invention establishes a sterile rapid propagation system using shelled hazelnut seeds as explants and adventitious buds to induce proliferation. Through a series of culture processes and optimized culture media and culture conditions, the rapid propagation of hazelnuts is achieved, thereby increasing the propagation coefficient of hazelnuts.

[0053] In step 1), the shelled hazelnut seeds are subjected to shell disinfection treatment and shell removal disinfection treatment in sequence to obtain explants.

[0054] This invention does not limit the specific process of sterilization with shells or the reagents used; common sterilization processes in the field can be selected.

[0055] After sterilizing the shelled hazelnut seeds, the hard outer shell was removed using sterilized hazelnut-specific pruning shears to obtain shelled hazelnut seeds. Subsequently, the shelled hazelnut seeds were sterilized with 0.1% mercuric chloride for 4-6 minutes, rinsed with sterile water, and finally dried with sterile filter paper to obtain explants.

[0056] Understandably, healthy, disease-free, mature, shelled hazelnut seeds can be selected using common seed selection methods. For example, seeds can be soaked in tap water first, and empty, shriveled seeds floating on the surface can be sieved out. Then, the seeds that sink to the bottom can be selected a second time, choosing those that are disease-free, pest-free, and large. To further reduce the contamination rate, refrigerated seeds can also be used.

[0057] By sequentially treating the seeds of *Hazelnut* with and without shells with disinfection, explants were obtained, reducing the explant contamination rate and ensuring that the seeds could be cultured into seedlings from the primary culture medium. This provided a good foundation for the rapid tissue culture propagation of *Hazelnut*.

[0058] In step 2), the sterilized explants were aseptically transferred to the primary culture medium for primary culture. The primary culture medium included MS medium, 2 g / L GA3, 30 g / L sugar, and 6 g / L agar. During the primary culture, the Hazelnut seeds, which served as explants, germinated, sprouted, and grew into robust seedlings with dark green leaves, reaching a height of at least 5 cm.

[0059] This invention does not limit the initial cultivation time. As long as the seeds can successfully germinate and grow into seedlings with dark green leaves and robust growth, with a height of not less than 5cm, it is acceptable. For example, it can be cultivated for 30 days.

[0060] After initial culture on the primary culture medium, the seeds successfully germinated into seedlings, preparing them for rapid propagation through tissue culture in the later stages.

[0061] In step 3), the seedlings are transferred as a whole to the first seedling culture medium under aseptic conditions for the first seedling culture. As the first seedling culture proceeds, under the influence of nutrients and hormones in the first seedling culture medium, axillary buds grow from the leaf axils of the seedlings, thus obtaining the first seedlings.

[0062] The first seedling culture medium of this invention comprises MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar. The MS medium formulation of this invention is disclosed. 6-BA (6-benzylaminopurine) is mainly used to promote shoot formation. NAA (naphthaleneacetic acid) is a common plant growth regulator. Common sucrose can be used as the sugar. Sugar provides the carbon skeleton for cells to synthesize new compounds, provides substrates and energy for cellular respiration and metabolism, and can effectively regulate the osmotic pressure of the culture medium. Agar can convert the liquid culture medium into a solid culture medium.

[0063] This invention does not limit the time for cultivating the first strong seedling, as long as axillary buds can grow from the leaf axils of the seedling.

[0064] In step 4), the first seedling is aseptically transferred to the second seedling culture medium for second seedling cultivation, resulting in a second seedling with a height of 6-8 cm.

[0065] The second seedling culture medium of the present invention is the same as the first seedling culture medium described above, and will not be repeated here.

[0066] This invention does not limit the time for cultivating the second seedling, as long as a second seedling with a height of 6-8cm can be obtained.

[0067] After the first and second seedling cultivation, robust second seedlings with axillary buds can be obtained, which prepares the seedlings for later cultivation.

[0068] In step 5), the black substance at the base of the second seedling is removed using sterile instruments. Then, the second seedling is inoculated into the adventitious bud induction medium under sterile conditions for adventitious bud induction culture. During the adventitious bud induction culture, multiple adventitious buds will grow from the leaf axils of the second seedling. After a certain period of cultivation, adventitious bud seedlings with adventitious buds with a bud length of 3-5cm are obtained.

[0069] In this invention, the adventitious bud length of 3-5cm refers to the adventitious buds that grow from the adventitious bud induction culture, which have an adventitious bud length of 3-5cm. It is understood that the adventitious buds that grow from the adventitious bud induction culture may also have adventitious buds with a length of less than 3cm.

[0070] This invention does not limit the time for inducing and culturing adventitious buds, as long as adventitious buds with a bud length of 3-5 cm can be obtained. It is understood that multiple adventitious buds with a bud length of 3-5 cm can be obtained through a longer culture time, such as 30 days.

[0071] As mentioned above, in step 5), there may be multiple adventitious buds with a length of 3-5cm in the adventitious seedlings. In step 6), adventitious buds with a length of 3-5cm are cut from the adventitious seedlings and transferred to the proliferation medium for proliferation culture. Each adventitious bud corresponds to a single proliferation medium, resulting in a proliferation seedling with a length of more than 3cm.

[0072] The proliferating buds of this invention refer to a series of adventitious buds that are induced to grow from adventitious buds inoculated onto the proliferation culture medium during the proliferation culture process.

[0073] In this invention, a propagation seedling with a propagation bud of 3 cm or more refers to a propagation seedling that has a propagation bud of 3 cm or more after propagation culture. It is understood that there may also be propagation buds of less than 3 cm in the propagation seedling.

[0074] This invention does not limit the propagation culture time, as long as propagation seedlings with propagation buds of 3cm or more can be obtained. It is understood that propagation seedlings with multiple propagation buds of 3cm or more can be obtained through a longer culture period, such as 30 days.

[0075] In step 7), select the relatively robust propagation seedlings from step 6), separate the propagation seedlings into individual buds, cut the separated individual buds into stem segments with a length of 3cm or more and a terminal bud, and transfer them into the third seedling culture medium for third seedling culture under aseptic conditions to obtain seedlings with a height of 3.5-5cm that are ready to grow roots.

[0076] This invention does not limit the time for cultivating the third seedling. The purpose of cultivating strong seedlings can be achieved by appropriately extending the cultivation time of the third seedling, for example, by cultivating for 30 days.

[0077] The seedlings that are ready to take root after the third seedling culture are more robust than the proliferating buds, which improves the success rate of subsequent rooting culture.

[0078] In step 8), the seedlings that have undergone the third seedling culture are transferred to the rooting culture medium for rooting culture. Under the action of various components in the rooting culture medium, the seedlings gradually grow roots and obtain rooted seedlings.

[0079] This invention does not limit the rooting culture time; rooted seedlings with a higher rooting rate and longer root length can be obtained through a longer culture time, such as 30 days.

[0080] In step 9), the rooted seedlings are hardened off and transplanted to obtain transplanted seedlings with new roots and leaves.

[0081] Among them, the purpose of hardening-off cultivation is to enable rooted seedlings to quickly adapt to the adverse environmental conditions of actual external cultivation, shorten the seedling recovery time, and enhance the resistance of rooted seedlings to harsh environments such as low temperature and strong wind.

[0082] Common seedling hardening methods in this field can be used, such as wind protection, cooling, and appropriate water control.

[0083] Transplanting cultivation refers to transferring rooted seedlings from the culture medium to a substrate such as soil, allowing the seedlings to gradually adapt to the growth environment in the soil.

[0084] This invention does not limit the specific steps of transplanting culture, as long as the success rate after transplanting culture can be guaranteed.

[0085] The rapid propagation method of Haworthia truncata tissue culture provided by this invention uses Haworthia truncata seeds as explants. Through a series of culture steps, including shell sterilization, shell removal sterilization, primary culture, first strong seedling culture, second strong seedling culture, adventitious bud induction culture, proliferation culture, rooting culture, hardening culture, and transplanting culture, the types of primary culture medium, first strong seedling culture medium, and second strong seedling culture medium are optimized, which realizes the rapid propagation of Haworthia truncata, improves the propagation coefficient of Haworthia truncata, and is conducive to the large-scale seedling production of Haworthia truncata.

[0086] Furthermore, in a specific embodiment of the present invention, in the above-mentioned rapid tissue culture propagation method, step 1), the sterilization time for the shelling and sterilization treatment is 5 minutes.

[0087] Through numerous creative experiments, the inventors discovered that when the disinfection time for the dehulling and disinfection process is 5 minutes, it can not only control the contamination rate of seedlings, but also significantly improve the survival rate of explants during cultivation. This is because when the disinfection time is controlled at 5 minutes, pathogens can be killed as much as possible without damaging the seed structure.

[0088] The contamination rate of shelled hazelnut seeds was calculated by dividing the number of contaminated seeds by the total number of seeds by 100%.

[0089] Furthermore, in a specific embodiment of the present invention, in step 1) of the above-mentioned rapid tissue culture propagation method, the shell-on sterilization treatment includes:

[0090] Soak the shelled hazelnut seeds in 60℃ hot water for 24 hours. Then, soak the shelled hazelnut seeds in a 0.5% laundry detergent solution for 10 minutes, rinse with running water for 1 hour, soak in 75% alcohol for 30 seconds, disinfect with 0.1% mercuric chloride for 15 minutes, and finally rinse with sterile water 5-6 times.

[0091] Soaking whole hazelnut seeds in 60℃ hot water for 24 hours not only serves as a preliminary disinfection method but also helps break dormancy and accelerates the cultivation process. Subsequently, treating the whole hazelnut seeds with laundry detergent solution, alcohol, and mercuric chloride in sequence can eliminate pathogens in the hazelnut shell as much as possible, preventing contamination of the shelled hazelnut seeds after subsequent shelling.

[0092] Furthermore, in a specific embodiment of the present invention, the adventitious bud induction medium in the above-mentioned tissue culture rapid propagation method includes MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar.

[0093] Through extensive creative experiments, the inventors discovered that when the above-mentioned culture medium is used for adventitious bud induction culture, culturing 30 robust second seedlings with a height of 6-8cm for 30 days results in an average adventitious bud length of 5.5cm, an adventitious bud induction rate of 97%, normal bud growth, and green leaves.

[0094] Among them, the adventitious bud induction rate refers to the ratio between the second seedling that induced adventitious buds to grow adventitious buds and all the second seedlings that participated in the adventitious bud induction culture.

[0095] It is evident that the adventitious bud induction medium prepared according to the above formula can better induce cell division and induce the growth of adventitious buds under the synergistic effect of each component, laying the foundation for subsequent proliferation culture.

[0096] Furthermore, in a specific embodiment of the present invention, the proliferation medium in the above-mentioned tissue culture rapid propagation method includes 1 / 3 MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, 6 g / L agar, and 1.0 g / L GA3.

[0097] Among them, 1 / 3MS medium refers to the medium prepared according to 1 / 3 of the components of MS medium, and its formula is also known. GA3 (gibberellin) in this invention is a common plant growth hormone that can promote cell division and cell growth.

[0098] When the above-mentioned proliferation culture medium is selected, under the same conditions, the proliferation seedlings obtained by proliferation culture have a higher bud elongation rate and proliferation bud induction rate, and the leaves are dark green and free from diseases and pests, which prepares them for subsequent third seedling culture and rooting.

[0099] Furthermore, in a specific embodiment of the present invention, the third seedling culture medium in the above-mentioned rapid tissue culture propagation method includes 1 / 3 MS medium, 0.5 mg / L 6-BA, 0.3 mg / L IBA, 1.0 g / L GA3, 30 g / L sugar, and 6 g / L agar.

[0100] The inventors discovered that, under the same culture time and conditions, when the above-mentioned third seedling culture medium was used for the third seedling culture, the resulting seedlings had dark green leaves and grew vigorously.

[0101] Furthermore, in a specific embodiment of the present invention, the rooting medium in the above-mentioned tissue culture rapid propagation method includes 1 / 4 MS medium, 0.1 mg / L IBA, 0.3 mg / L NAA, 0.5 g / L AC, 30 g / L sugar, and 6 g / L agar.

[0102] Among them, 1 / 4MS medium refers to the medium prepared according to 1 / 4 of the MS medium components, and its formula is also known. The AC (activated carbon) in this invention can adsorb harmful substances in the medium, prevent browning and accumulation of harmful substances, and promote root induction and root growth.

[0103] A 30-day rooting culture experiment was conducted on 30 seedlings of the above-mentioned seedlings with a height of 3.5-5cm. The rooting rate of the above-mentioned rooting culture medium can reach 98%, the number of primary roots reaches 5.6, the root length reaches 25cm, and the roots are relatively thick.

[0104] It is evident that the aforementioned rooting medium exhibits superior rooting induction properties, thereby improving the survival rate of hazelnut tissue culture during rapid propagation.

[0105] Furthermore, in a specific embodiment of the present invention, in the above-mentioned tissue culture rapid propagation method, step 9), the seedling hardening culture includes: placing the rooted seedlings in a greenhouse at 15-25℃, opening the bottle cap for cultivation, and the time is 1 week.

[0106] After the above-mentioned hardening-off cultivation, the ability of rooted seedlings to adapt to adverse environments can be improved, and the survival rate of Chinese hazelnut transplanted seedlings can be increased when transplanted to the external environment in the later stage.

[0107] Furthermore, in a specific embodiment of the present invention, in the above-mentioned rapid propagation method of tissue culture, step 9), the transplanting culture includes:

[0108] After hardening off, the rooted seedlings are cut, cleaned, and sterilized in sequence. Then, the rooted seedlings are placed in seedling trays filled with substrate to cultivate transplanted seedlings with new roots and new leaves.

[0109] The matrix is ​​composed of perlite, peat, and vermiculite in a volume ratio of 1:2:1.

[0110] In detail, after the rooted seedlings have been hardened off, the leaves and callus tissue at the base are cut off using sterile tools, leaving 2-3 leaves. Then, the culture medium adhering to the rooted seedlings is washed off with clean water, and the rooted seedlings are sterilized with carbendazim. The sterilized rooted seedlings are then planted into seedling pots containing substrate, which is prepared by perlite, peat moss and vermiculite in a volume ratio of 1:2:1.

[0111] This invention does not limit the specific cultivation operations in transplanting; conventional transplanting methods in the field can be used. For example, water thoroughly on the first day, and water only when the soil is dry to the touch, and sprinkle water on the leaves daily to maintain humidity.

[0112] This invention determines survival by whether new leaves unfold and whether the plant grows normally after 3 weeks of cultivation. When transplanted and cultivated using the above-mentioned substrate, it has a high survival rate.

[0113] Furthermore, in a specific embodiment of the present invention, the above-mentioned rapid propagation method of tissue culture includes a conventional culture temperature of 23-25℃, a culture humidity of 75-85%, a light intensity of 2000-2500 lx, a light exposure of 14-16 h / d, and a light intensity of 2000-2500 lx. The conventional culture includes primary culture, first strong seedling culture, second strong seedling culture, adventitious bud induction culture, proliferation culture, third strong seedling culture, rooting culture, and transplanting culture.

[0114] The above-mentioned culture conditions can provide a suitable culture environment for each stage of rapid tissue culture propagation of Huazhen, and the above methods are easy to control, reducing the difficulty of controlling the culture conditions at each stage of rapid tissue culture propagation.

[0115] The following detailed description of the rapid propagation method for hazelnut tissue culture provided by the present invention is provided through specific embodiments.

[0116] The hazelnut seeds used in this invention are all selected from healthy, disease-free hazelnut seeds that have been stored at low temperature for five years by the Yangtze River Rare Plant Research Institute.

[0117] Example 1

[0118] (1) Seed disinfection treatment with and without hulls

[0119] 360 healthy, disease-free, whole-shelled hazelnut seeds that have been stored at low temperature for five years were selected. The whole-shelled hazelnut seeds were soaked in 60℃ hot water for 24 hours, then soaked in 0.5% laundry detergent solution for 10 minutes, rinsed with running water for 1 hour, then soaked in 75% alcohol for 30 seconds on a clean bench, then disinfected with 0.1% mercuric chloride for 15 minutes, and finally rinsed 6 times with sterile water. The whole-shelled hazelnut seeds after disinfection were placed on a sterilization tray.

[0120] The special pruning shears for hazelnut were sterilized and used to cut off the hard outer shell of the hazelnut seeds to obtain shelled hazelnut seeds. The shelled hazelnut seeds were then sterilized with 0.1% mercuric chloride for 4 min, 5 min, and 6 min respectively. The number of seeds in each sterilization treatment group was 120. After that, they were rinsed 6 times with sterile water and the excess water was absorbed with sterile filter paper to obtain explants.

[0121] (2) Primary culture of explants

[0122] Explants were inoculated onto primary culture medium, which included MS medium, 2 g / L GA3, 30 g / L sugar, and 6 g / L agar. The culture was carried out for 30 days at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity to obtain seedlings with a height of not less than 5 cm.

[0123] The seedlings were tested, and the contamination rate, survival rate and mortality rate of the seedlings after disinfection treatment for 4 min, 5 min and 6 min were statistically obtained. The specific results are shown in Table 1.

[0124] Table 1

[0125]

[0126] Single-factor experimental analysis revealed the following: Regarding contamination rate, 0.1% mercuric chloride disinfection for 5 minutes resulted in the lowest contamination rate, significantly lower than disinfection times of 5 and 6 minutes, while there was no significant difference between 5 and 6 minutes. Regarding survival rate, 0.1% mercuric chloride disinfection for 5 minutes achieved 100% survival, significantly higher than disinfection times of 4 and 6 minutes, while there was no significant difference between 4 and 6 minutes. Regarding mortality rate, 0.1% mercuric chloride disinfection for 5 minutes resulted in the lowest mortality rate, significantly lower than disinfection times of 4 and 6 minutes, while there was no significant difference between 4 and 6 minutes. In conclusion, disinfection with 0.1% mercuric chloride for 5 minutes is the optimal disinfection time.

[0127] (3) Cultivation of robust seedlings from seedlings

[0128] The seedlings obtained after the above treatment were transferred to the first seedling culture medium for first seedling cultivation. The first seedling culture medium included MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar. The seedlings were cultured at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity until axillary buds grew from the leaf axils, thus obtaining the first seedlings.

[0129] The first seedlings were transferred to a second seedling culture medium for further cultivation. This second seedling culture medium consisted of MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar. The culture was carried out at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity until the first seedlings reached a height of 6-8 cm. This yielded second seedlings with a height of 6-8 cm. Figure 1 As shown.

[0130] (4) Adventitious bud induction culture

[0131] Remove the black substance from the base of the second seedlings. Select 180 relatively robust second seedlings and inoculate them onto six different adventitious bud induction media numbered A, B, C, D, E, and F. One seedling is inoculated onto each medium, and each medium is replicated 30 times. Culture the seedlings for 30 days at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity to obtain adventitious buds. Figure 2 As shown.

[0132] The adventitious bud induction media in all 6 groups contained MS medium, 30 g / L sugar, 6 g / L agar, and the contents of 6-BA and NAA varied. The specific contents of 6-BA and NAA are shown in Table 2.

[0133] Adventitious buds were observed and measured, and data such as bud length, growth status, and number of adventitious buds were recorded. Statistical analysis was performed using SAS software to obtain the average length of adventitious buds, the average number of adventitious buds, the average elongation of axillary buds, and the induction rate of adventitious buds. The specific results are shown in Table 2.

[0134] Table 2

[0135]

[0136] In terms of adventitious bud length, the adventitious buds cultured in adventitious bud induction medium No. 1 reached a length of 5.5 cm, significantly better than other formulations. Regarding the number of adventitious buds, the average number of adventitious buds cultured in adventitious bud induction media No. 1 and No. 2 was 6.2 and 6.5 respectively, significantly better than other formulations, with no significant difference between the two. In terms of axillary bud elongation, the axillary bud elongation in adventitious bud induction medium No. 1 was 1.0 cm, significantly better than other formulations. In terms of adventitious bud induction rate, formulation No. 1 had an induction rate of 97%, significantly better than other formulations. Finally, considering the overall growth status, it can be concluded that adventitious bud induction medium No. 1 has superior performance.

[0137] (5) Proliferation culture

[0138] From the adventitious shoots obtained above, 270 robust adventitious shoots with a length of 3-5 cm were cut and transferred to 9 groups of proliferation media numbered 1-9 for proliferation culture, with one adventitious shoot corresponding to one proliferation medium. Each group of media was repeated 30 times. The culture was carried out at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity for 30 days to obtain proliferated seedlings. Figure 3 , Figure 4 As shown.

[0139] All nine proliferation media contained 1 / 3 MS medium, 30 g / L sugar, 6 g / L agar, 1.0 g / L GA3, and 0.1 mg / L NAA. The proliferation media also contained 6-BA or KT. The specific types and concentrations are shown in Table 3.

[0140] The propagation seedlings were observed and measured, and data such as the length of adventitious buds and growth status were recorded. Statistical analysis was performed using SAS software to obtain the adventitious bud elongation rate, adventitious bud elongation length, propagation induction rate, and adventitious bud length of the propagation seedlings. The specific results are shown in Table 3.

[0141] Table 3

[0142]

[0143]

[0144] The analysis in Table 3 shows that the No. 2 proliferation medium is significantly higher than other formulations in terms of shoot elongation rate, average shoot elongation, and proliferation shoot induction rate. Therefore, the No. 2 proliferation medium has superior performance.

[0145] (6) Cultivation of the third seedling

[0146] Select the robust propagation seedlings mentioned above, and separate the seedlings into individual buds. Cut the separated individual buds into stem segments with a length of 3 cm or more and a terminal bud. A total of 270 stem segments were cut. After aseptic operation, they were transferred into three groups of third-strength seedling culture media numbered 1, 2, and 3 for third-strength seedling culture. One stem segment corresponds to one third-strength seedling culture medium, and each group of third-strength seedling culture media was repeated 90 times.

[0147] All three groups of third-stage seedling culture media contained 1 / 3 MS medium, 0.5 mg / L 6-BA, 1.0 g / L GA3, 30 g / L sugar, and 6 g / L agar. Different third-stage seedling culture media also included IBA; specific concentrations are shown in Table 4. Seedlings were cultured for 30 days at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity to obtain rooted seedlings.

[0148] The seedlings to be rooted were observed and measured, and data such as bud length were recorded. Statistical analysis was performed using SAS software to obtain the average bud length and bud vigor of the seedlings to be rooted. The specific results are shown in Table 4.

[0149] Table 4

[0150]

[0151] The intuitive analysis in Table 4 shows that the No. 2 third seedling culture medium has a stronger seedling strengthening ability than other third seedling culture media.

[0152] (7) Rooting culture

[0153] Following the above cultivation process, the seedlings to be rooted were vertically inoculated into nine groups of rooting media numbered 1, 2, 3, 4, 5, 6, 7, 8, and 9 for rooting culture. One rooting seedling corresponded to one rooting medium, and each group of rooting media was repeated 30 times. Each group of rooting media contained 0.5 g / L LAC, 30 g / L sugar, and 6 g / L agar. Different numbers of rooting media also included different proportions of MS medium as the basal medium, as well as IBA and NAA (specific types are shown in Table 5). The seedlings were cultured for 30 days at 23-25℃, 75%-85% humidity, 14-16 h / d light, and 2000-2500 lx light intensity to obtain rooted seedlings. Figure 5 As shown.

[0154] The rooted seedlings were observed and measured, and data such as the number of roots, length, and growth status were recorded. Statistical analysis was performed using SAS software to obtain the rooting rate, number of roots, and root length of the rooted seedlings. The specific results are shown in Table 5.

[0155] Table 5

[0156]

[0157] The analysis in Table 5 shows that rooting medium No. 5 has a stronger ability to induce rooting than other rooting media.

[0158] (8) Hardening off seedlings and transplanting

[0159] The rooted seedlings obtained by the above culture process were taken out of the laboratory and placed in a greenhouse at 15-25℃ for a week. The bottle mouth was opened and the surface was sprayed with water every day.

[0160] One week after hardening off, open the bottle cap and remove the test-tube seedlings. Select 120 healthy, rooted seedlings with similar growth conditions, such as... Figure 6 As shown, cut off the leaves and callus tissue at the base, leaving 2-3 leaves. Wash the culture medium adhering to the test tube seedlings with clean water, and pot them into four different substrates numbered 1, 2, 3, and 4. Substrate 1 was prepared with a perlite to peat volume ratio of 1:2; Substrate 2 was prepared with a perlite, peat, and vermiculite volume ratio of 1:2:1; Substrate 3 was prepared with a perlite, peat, vermiculite, and fine sand volume ratio of 1:2:1:1. Substrate 3 was pure loam. One seedling per pot, 10 pots of each type, repeated three times. Water thoroughly on the first day, and then water when the soil is dry to the touch. Sprinkle water on the leaves daily to maintain humidity. After 30 days of cultivation, transplanted seedlings were obtained.

[0161] The survival rate was determined based on whether the new leaves had unfolded and whether the plant was growing normally. Statistics showed that 10 plants survived in substrate No. 1, 29 plants survived in substrate No. 2, 15 plants survived in substrate No. 3, and 12 plants survived in substrate No. 4.

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapid tissue culture propagation of hazelnut, characterized in that, Includes the following steps: 1) The seeds of *Corylus heterophylla* with shells were subjected to sterilization treatment with the shells on and sterilization treatment after the shells were removed, in order to obtain explants; 2) The explants were inoculated into the primary culture medium for primary culture to obtain seedlings with a height of not less than 5 cm; 3) The seedlings were cultured using the first seedling culture medium to obtain the first seedlings with axillary buds; 4) Transfer the first seedling to the second seedling culture medium for second seedling cultivation to obtain a second seedling with a height of 6-8cm; 5) The second seedling was transferred to an adventitious bud induction medium for adventitious bud induction culture to obtain adventitious bud seedlings with adventitious buds with a bud length of 3-5 cm; the adventitious bud induction medium was MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar; 6) Cut the adventitious buds from the adventitious buds and transfer them to the proliferation medium for proliferation culture to obtain proliferating seedlings with proliferating buds longer than 3 cm; the proliferation medium is 1 / 3 MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, 6 g / L agar, and 1.0 g / L GA3; 7) Cut off the proliferating buds and transfer them to the third seedling culture medium for third seedling culture to obtain seedlings with a height of 3.5-5cm that are ready to develop roots; the third seedling culture medium is 1 / 3 MS medium, 0.5mg / L 6-BA, 0.3mg / L IBA, 1.0g / L GA3, 30g / L sugar, and 6g / L agar; 8) The seedlings to be rooted are transferred to a rooting medium for rooting culture to obtain rooted seedlings; the rooting medium is 1 / 4 MS medium, 0.1 mg / L IBA, 0.3 mg / L NAA, 0.5 g / L AC, 30 g / L sugar, and 6 g / L agar; 9) The rooted seedlings are subjected to hardening-off culture and transplanting culture to obtain transplanted seedlings with new roots and new leaves; The shelling and disinfection process includes: shelling the hazelnut seeds after the shell disinfection process to obtain shelled hazelnut seeds, and disinfecting the shelled hazelnut seeds with 0.1% mercuric chloride for 4-6 minutes to obtain the explants; The primary culture medium consisted of MS medium, 2 g / L GA3, 30 g / L sugar, and 6 g / L agar. The first and second seedling culture media consist of MS medium, 0.5 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sugar, and 6 g / L agar.

2. The tissue culture rapid propagation method according to claim 1, characterized in that, In step 1), the disinfection time for the shelling and disinfection process is 5 minutes.

3. The tissue culture rapid propagation method according to claim 1 or 2, characterized in that, In step 1), the shell-on disinfection process includes: Soak the shelled hazelnut seeds in 60°C hot water for 24 hours. Then, soak the shelled hazelnut seeds in a 0.5% detergent solution for 10 minutes, rinse with running water for 1 hour, soak in 75% alcohol for 30 seconds, disinfect with 0.1% mercuric chloride for 15 minutes, and finally rinse with sterile water 5-6 times.

4. The tissue culture rapid propagation method according to claim 1, characterized in that, In step 9), the seedling hardening culture includes: placing the rooted seedlings in a greenhouse at 15~25℃, opening the bottle cap for cultivation, and the time is 1 week.

5. The tissue culture rapid propagation method according to any one of claims 1, 2, and 4, characterized in that, In step 9), the transplanting culture includes: After hardening off, the rooted seedlings are cut, cleaned, and sterilized in sequence. Then, the rooted seedlings are placed in a seedling pot containing substrate to cultivate transplanted seedlings with new roots and new leaves. The matrix is ​​prepared by mixing perlite, peat, and vermiculite in a volume ratio of 1:2:

1.

6. The tissue culture rapid propagation method according to any one of claims 1, 2, and 4, characterized in that, The conventional culture temperature is 23-25℃, the culture humidity is 75-85%, the light intensity is 2000-2500 lx, the light intensity is 14-16 h / d. The conventional culture includes the primary culture, the first strong seedling culture, the second strong seedling culture, the adventitious bud induction culture, the proliferation culture, the third strong seedling culture, the rooting culture, and the transplanting culture.

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