Method for hardening and transplanting of Castanopsis chinensis tissue culture seedlings

Abstract

The invention provides a method for transplanting seedlings of tung oil tree tissue culture seedlings, and relates to the field of plant tissue culture rapid propagation technology. The method comprises the following steps: rooting culture of aseptic seedlings of tung oil tree, cleaning culture medium after rooting, planting in a transparent plastic cup to which a matrix is added, cultivating in a light culture room for 10-15d, opening the bottle cap in the evening or on a rainy day, continuing to cultivate for about 10d, transplanting in a matrix with a volume ratio of 2:1:1 added with organic nutrient soil, perlite and vermiculite, and achieving a transplant survival rate of up to 96%. The present invention overcomes the production problem that traditional tissue culture bottles need to open the bottle cap for 3-7d to harden seedlings, and have a low survival rate after transplanting. The present invention has the characteristics of high rooting rate, simple seedling hardening and transplanting, safe and reliable, high survival rate, shortened seedling raising period, and can carry out factory-based seedling cultivation of tung oil tree, providing a new approach for the tissue culture factory-based seedling cultivation of tung oil tree.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of Castanopsis schrenkiana, in particular to a method for rooting, hardening and transplanting Castanopsis schrenkiana tissue culture seedlings. Background Art

[0002] Idesia polycarpa Maxim is a deciduous tree belonging to the genus Idesia in the family Salicaceae. It is also known as mountain sycamore, half-frost red, and oil grape. It is widely distributed in my country, spanning 17 provinces and autonomous regions. The tree produces a large amount of fruit with a high oil content, earning it the nickname "the oil depot on the tree." Idesia polycarpa oil is primarily composed of unsaturated fatty acids, with the fruit containing over 82% unsaturated fatty acids, including over 73% linoleic acid. It can effectively improve blood lipids and thrombosis, and also has antioxidant and immune-enhancing properties. Idesia polycarpa oil also contains a large number of naturally active ingredients, making it a pure, natural, high-quality woody edible oil. Its oil is also used in various industries, including industry, fertilizers, healthcare, health products, and cosmetics. Idesia polycarpa grows rapidly, is highly adaptable, and is cold-resistant, barren, and drought-resistant. Therefore, Idesia polycarpa combines economic, ecological, and social value, possessing broad application prospects and enormous development potential.

[0003] At present, the seeds of Fructus Castanopsis are tiny, with a waxy layer on the surface, and have winter dormancy characteristics. Under natural conditions, the reproduction rate is extremely low, and its propagation method is subject to seasonal restrictions, and cutting propagation requires a large amount of maternal parents, which is difficult to meet the growing market demand. In addition, there are fewer reports on Fructus Castanopsis tissue culture, and mainly focus on callus induction culture of Fructus Castanopsis, with no detailed reports on transplanting and hardening. The present invention, by studying the influence of different sucrose concentrations, different types of plant growth regulators on the rooting of Fructus Castanopsis tissue culture seedlings, and the influence of different matrix ratios and container types on transplanting survival rate and seedling growth, shows that the rooted seedlings cultivated by this method have a large number of root systems, a strong root system, a high survival rate after transplanting, and rapid growth with strong adaptability. Simultaneously, seed germination or one-step seedling formation with budding stem segments are also used, greatly shortening the seedling formation cycle. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for hardening and transplanting of Castanopsis schrenkiana tissue culture seedlings to improve the seedling survival rate. Compared with previous related studies, this method significantly improves the transplant survival rate, shortens the seedling cycle, and enhances the stress resistance of seedlings, laying the foundation for the future industrialization of Castanopsis schrenkiana.

[0005] A method for hardening and transplanting tung oil tree tissue culture seedlings comprises the following steps:

[0006] (1) Selection of sterile seedlings: Cut the sterile seedlings of Castanopsis chinensis with basically uniform growth and robustness as materials;

[0007] (2) Rooting culture: The sterile seedlings obtained in step (1) were cut flat at the base of the hypocotyl and inoculated into a medium containing MS + 0.5-0.7 mg / L IBA, MS + 1.0 mg / L IAA, or MS + 0.3 mg / L IBA + 0.5 mg / L NAA for rooting culture;

[0008] (3) Washing the seedlings: directly wash the rooted seedlings to remove the residual culture medium from the roots, and try to keep the root system intact;

[0009] (4) Dipping the seedlings: Dip the roots of the rooted seedlings into the rooting powder solution, remove them, and drain the water;

[0010] (5) Transplanting seedlings: Transplant the rooted seedlings into a transparent plastic cup with a substrate added; after culturing in a light culture room for 10-15 days, place them in a greenhouse for further cultivation, and further acclimate them by reducing the number of waterings and appropriately lowering the substrate humidity, maintaining the substrate moisture content at 40-60%. After continuing to cultivate for 7-15 days, transplant them into seedling bags, continue to cultivate in the greenhouse for 7-10 days, and place them in a nursery covered with a shade net on rainy days.

[0011] In step (1), the length of the sterile seedlings is 4 to 7 cm, and the number of leaves is 4 to 7.

[0012] In step (2), the rooting medium is supplemented with 10 g / L sucrose and 7 g / L agar, and the pH value is 5.7-6.0.

[0013] The light intensity of the rooting culture in step (2) is 1600-1800 lx, the light culture temperature is 26±2° C., the light duration is 12 h / d, and the rooted seedlings of Castanopsis chinensis are obtained after culturing for 25-35 days.

[0014] Step (3) Pour the rooted seedlings out of the culture bottle together with the culture medium, quickly immerse the entire seedling in a basin of water, and gently wash away the residual culture medium on the roots, trying to keep the root system intact.

[0015] Step (4) Dipping the seedlings: Wash the residual culture medium on the roots with clean water, then dip the roots into a rooting powder solution with a concentration of 10g / L-15g / L for 5-15s, pick them up and drain the water;

[0016] Step (5) transplanting seedlings: After dipping the seedlings, transplant the rooted seedlings of Castanopsis tung oil directly into a transparent plastic cup with a substrate in the seedling hardening room. When transplanting the seedlings, be careful to be gentle to avoid damaging the roots. The roots must be completely dense with the substrate, and the leaves must be kept as clean as possible to avoid affecting the normal growth of the plants.

[0017] In step (5), the transplanting medium is organic nutrient soil (BALTIC PEAT), perlite and vermiculite are mixed in a volume ratio of 2:1:1, and the rooted seedlings of Castanopsis chinensis are planted in transparent plastic cups, 1 to 3 plants per cup, and the lid is covered. A small hole of 3-6 mm in diameter is opened on the lid and the side at a height of 0.5-1 cm from the bottom to maintain the moisture content of the substrate above 70%. After culturing in a light culture room for 10-15 days, the light intensity is 1600-1800 lx, the light culture temperature is 26±2°C, and the light time is 12h / d. Then, the seedlings are placed in a greenhouse for further cultivation at a temperature of 20-35°C. The substrate humidity is further acclimated by reducing the number of waterings and maintaining the substrate moisture content at 40-60%. After further cultivation for 7-15 days, the seedlings are transplanted into a seedling bag added with organic nutrient soil, perlite and vermiculite in a volume ratio of 2:1:1, and continue to be cultivated in the greenhouse for 7-10 days. On rainy days, the seedlings are placed in a nursery covered with a shade net.

[0018] After transplanting the Castanopsis chinensis seedlings into seedling bags and placing them in the nursery, add water in time and spray 0.1% urea every 30 days to promote the growth of the Castanopsis chinensis seedlings.

[0019] The specifications of the transparent plastic cup in step (5) are: bottom diameter 8 cm, height 14.5 cm, bottle mouth diameter 11.5 cm, and the volume of the transplanting matrix is 1 / 5 of the volume of the transparent plastic cup.

[0020] In step (5), the transplanting substrate is moistened with water and stirred with tweezers until no water is precipitated. Then, the rooted Castanopsis chinensis seedlings that have been washed with the culture medium and dipped in rooting powder are transplanted into a transparent plastic cup with the substrate added.

[0021] During greenhouse cultivation in step (5), the bottle cap is opened every 5 days or so, and the leaf surface is sprayed with water, preferably with water dripping. After transplanting to the seedling bag and continuing to cultivate in the greenhouse for 7-10 days, it is appropriate to place the seedlings in a nursery covered with a shade net on a rainy day. The need for water supplementation is determined based on the color of the substrate and the weather. Spray 0.1% urea every 30 days to promote the growth of the Castanopsis chinensis seedlings.

[0022] The sterile seedlings obtained in step (1) are sterile seedlings germinated from seeds; preferably, the obtaining steps include:

[0023] (1) Seed pretreatment: Dry the freshly picked tung oil fruits in the sun, polish the fruits with sandpaper to separate the pulp from the seeds inside, then soak the removed seeds in clean water, rinse them, and place them in a ventilated place to dry in the shade. After drying in the shade, wash the seeds with laundry detergent to remove the surface wax before inoculation;

[0024] (2) Explant screening: Soak the seeds of Castanopsis chinensis in warm water at 30-40°C, then remove the seeds floating on the water surface. Repeat this operation at least twice. Then soak the seeds in distilled water, let it stand for at least 24 hours, and remove the seeds floating on the water surface.

[0025] (3) Explant disinfection: Under sterile conditions, soak the Castanopsis chinensis seeds in a 0.1% HgCl2 solution for 8-12 min, preferably 10 min. Shake occasionally during the soaking process to ensure that the HgCl2 can fully disinfect the seeds. Finally, rinse with sterile water 3-5 times.

[0026] (4) Primary seed culture: Place the sterilized seeds on sterile filter paper to absorb moisture, and then inoculate the seeds into a culture medium consisting of 1 / 2MS + 1.0mg / L GA3 + 1.0-3.0g / L activated carbon or 1 / 2MS + 2.0mg / L GA3 + 1.0-3.0g / L activated carbon; the culture medium is supplemented with 30g / L sucrose and 8g / L agar, and the pH is adjusted to 5.7-5.9; culture under light conditions for 40-50 days as the original material for rooting seedlings, the culture temperature is 26±2℃, the light culture intensity is 1600-1800lx, and the light culture time is 12-14h / d.

[0027] The sterile seedlings obtained in step (1) are derived from sterile seedlings induced from stem segments with buds; preferably, the obtaining steps include:

[0028] (1) Acquisition of bud-bearing stem segments of Castanopsis chinensis: The experimental materials were derived from potted Castanopsis chinensis seedlings. Tender shoots and semi-lignified bud-bearing stem segments that sprouted in April and May were used as explant materials.

[0029] (2) Explant pretreatment: Cut the side branches of the Castanopsis chinensis potted seedlings from the base and rinse them in running water for 30-40 minutes; then cut the upper part of the lateral branches and the semi-lignified stem segments into 3-4 cm long stem segments, leaving 1-2 axillary buds on each stem segment, wash them 1-2 times with detergent, clean the remaining impurities in the stem segments with a soft brush, and then rinse them under running water for at least 1 hour;

[0030] (3) Explant disinfection: Under sterile conditions, the stem segments of the Chinese tallow tree with buds were disinfected with 75% alcohol for 1 minute. During the soaking process, the stem segments were shaken from time to time to ensure that the alcohol fully disinfected the stem segments with buds. After 1 minute, the stem segments were soaked again with fresh 75% alcohol for 1 minute. The stem segments were rinsed with sterile water 3-5 times. The shoots were then disinfected with 0.1% HgCl2 solution for 25 or 35 minutes and the semi-lignified stem segments for 50 minutes. During the soaking process, the stem segments were shaken from time to time to ensure that the mercuric chloride solution fully disinfected the stem segments with buds. Finally, the stem segments were rinsed with sterile water 3-5 times.

[0031] (4) Primary culture of stem segments with buds: Place the sterilized stem segments with buds on sterile filter paper to absorb moisture; use a sterile knife to cut off the brown part of the top and tail of the stem segments, and then inoculate the stem segments with buds into a culture medium containing 1 / 2MS+1.0-2.0mg / L6-BA+0.5-1.0mg / LNAA; add 30g / L sucrose and 8g / L agar to the culture medium, and adjust the pH to 5.5-5.7; culture under light conditions for 30-40 days, the culture temperature is 26±2℃, the light culture intensity is 1600-1800lx, and the light culture time is 12-14h / d.

[0032] Compared with the prior art, the advantages of the present invention are:

[0033] 1. The acquisition of sterile seedlings of Castanopsis chinensis is not restricted by season;

[0034] 2. The rooting rate of the rooting culture method of the present invention is maintained at above 96%, and the transplanting survival rate reaches 96%, which is about 10% higher than the transplanting survival rate in the existing literature (Li Xuyou, 2014); one month after transplanting, the height of the Castanopsis chinensis seedlings can reach 27 cm, which is 11 cm higher than the Castanopsis chinensis seedlings transplanted in seedling trays in the control of the present invention.

[0035] 3. The seedling hardening and transplanting method of the present invention is to directly transplant the seedlings from the tissue culture bottle into a transparent plastic cup for cultivation for 17-25 days and then plant them in a seedling bag, thereby eliminating the traditional 3-7 days of hardening the seedlings in the tissue culture bottle. This greatly increases the growth of the Castanopsis truncatula tissue culture seedlings after transplanting, shortens the seedling raising cycle, reduces the maintenance cost, improves the seedling raising efficiency, and allows the seedlings to grow neatly.

[0036] 4. The present invention effectively alleviates the high contamination rate of bud-bearing stem segments during tissue culture. These stem segments carry a large number of bacteria and endophytes. During primary culture, these endophytes and naturally occurring bacteria can lead to high contamination rates, severely impacting the germination and subsequent proliferation of axillary buds. The present invention pre-treats the bud-bearing stem segments, using detergent and a soft-bristled brush for cleaning, and disinfecting them with a combination of alcohol and a 0.1% HgCl₂ solution, effectively reducing the contamination rate and increasing the germination rate of axillary buds.

[0037] 5. The present invention uses sterile seedlings germinated from seeds, eliminating seasonal restrictions on experimental materials. Both new and old Castanopsis chinensis seeds can be used as explants. Castanopsis chinensis seeds have a waxy outer layer, which significantly affects germination. This method improves germination by rubbing and washing with detergent, then soaking in warm water and removing seeds that float on the water surface. The disinfectant used is only 0.1% HgCl2, resulting in a near-zero contamination rate. The seed germination rate is high, approaching 100%, and the germination cycle is short, with an average germination time of only 7 days. The sterile seedlings germinated from seeds have robust root systems and can be directly hardened and transplanted without seedling hardening. This solves the problems of slow and inefficient traditional seedling cultivation. This method can better implement factory-based Castanopsis chinensis seedling cultivation, increase Castanopsis chinensis yields, and meet market demand for Castanopsis chinensis seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 、 2 These are photos of rooted seedlings and root systems after 30 days of rooting at an IBA concentration of 0.7 mg / L;

[0039] Figure 3 、 4 The photos of rooted seedlings and root systems after 30 days of rooting at an IAA concentration of 1.0 mg / L;

[0040] Figure 5 、 6 The photos of rooted seedlings and root systems after 30 days of rooting at an NAA concentration of 0.05 mg / L;

[0041] Figure 7 、 8 The photos of rooted seedlings and root systems after 30 days of rooting with MS+0.3mg / L IBA+0.5mg / L NAA;

[0042] Figure 9 This is a photo taken 15 days after the cells were transplanted directly from the tissue culture bottle into a transparent plastic cup;

[0043] Figure 10 This is a photo of the plant transplanted into a transparent plastic cup 25 days ago;

[0044] Figure 11 Photo taken 25 days after transplanting into a transparent plastic cup with a substrate ratio of organic nutrient soil: perlite: vermiculite = 2:1:1;

[0045] Figure 12 This is a photo of the plant 30 days after transplanting into the seedling bag;

[0046] Figure 13 、 14 These are photos of the seeds transplanted into transparent plastic cups and seedling trays respectively;

[0047] Figure 15 This is the overall schematic diagram after planting.

[0048] Figure 16 This is the growth status of Castanopsis chinensis seeds in Example 5 after 10 days of inoculation into the culture medium;

[0049] Figure 17 This is the growth status of Castanopsis truncatula seeds in Example 5 after 20 days of inoculation into the culture medium;

[0050] Figure 18 This is the growth status of Castanopsis truncatula seeds in Example 5 after 30 days of inoculation into the culture medium;

[0051] Figure 19 This is the growth status of Castanopsis chinensis seeds in Example 5 when the culture medium is not added with activated carbon;

[0052] Figure 20 This is an overall picture of Castanopsis chinensis seeds in Example 5 in a culture medium without added activated carbon;

[0053] Figure 21 This is an overall diagram of the germination of Castanopsis chinensis seeds in Example 5;

[0054] Figure 22 The growth status of the axillary buds of the bud-bearing stem segment of Castanopsis truncatula in Example 6 10 days after germination;

[0055] Figure 23 This is the growth status of the axillary buds of the bud-bearing stem segment of Castanopsis truncatula in Example 6 20 days after germination. DETAILED DESCRIPTION

[0056] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0057] Example 1

[0058] Perform the following operations in sequence:

[0059] (1) Sterile seedlings of Castanopsis chinensis with a length of about 5 cm and basically the same growth were cut, the base of the hypocotyl was cut flat, and the seedlings were inserted into a rooting medium of MS + 0.5 mg / L IBA + 10-30 g / L sucrose + 7 g / L agar, with a pH of 5.7-6.0, a light intensity of 12 h / d, a light intensity of 1600-1800 lx, and a culture temperature of 26±2°C to induce rooting. After culturing for 30 days, the average root length, average number of roots, and rooting rate were calculated. Then, the seedlings were transplanted according to step (2) of Example 4 and the growth of the transplanted materials was observed (Table 1).

[0060] Table 1 Effects of different sucrose concentrations at the same hormone concentration (IBA = 0.5 mg / L) on rooting of Castanopsis chinensis tissue culture seedlings

[0061]

[0062] As can be seen from Table 1, when the sucrose concentration is 10 g / L, the rooting effect is the best.

[0063] Example 2

[0064] (1) Sterile seedlings of about 5 cm in length with basically the same growth were cut, the base of the hypocotyl was cut flat, and the seedlings were respectively inserted into the rooting medium MS + 0.1-0.9 mg / L IBA + 10 g / L sucrose + 7 g / L agar, MS + 0.05-1.5 mg / L IAA + 10 g / L sucrose + 7 g / L agar, and MS + 0.01-1.0 mg / L NAA + 10 g / L sucrose + 7 g / L agar, with a pH value of 5.7-6.0, a light duration of 12 h / d, a light intensity of 1600-1800 lx, and a culture temperature of 26±2°C to induce rooting. After culturing for 30 days, the average root length, average number of roots, and rooting rate were calculated. Then, the seedlings were transplanted according to step (2) of Example 4 and the growth of the transplanted materials was observed (Table 2).

[0065] Table 2 Effects of different hormone concentrations on rooting of Castanopsis chinensis tissue culture seedlings

[0066]

[0067]

[0068] Example 3

[0069] (1) Sterile seedlings of about 5 cm in length with basically the same growth were cut, the base of the hypocotyl was cut flat, and the seedlings were inoculated on a rooting medium containing MS + 0.1-0.5 mg / L IBA + 0.1-0.5 mg / L NAA + 10 g / L sucrose + 7 g / L agar, with a pH of 5.7-6.0, a light intensity of 12 h / d, a light intensity of 1600-1800 lx, and a culture temperature of 26±2°C to induce rooting. After 30 days of culture, the average root length, average number of roots, and rooting rate were calculated. The seedlings were then transplanted according to step (2) of Example 4 and the growth of the transplanted material was observed (Table 3).

[0070] Table 3 Effects of different hormone combinations on the rooting of Castanopsis chinensis

[0071]

[0072] The optimal rooting medium based on Example 2 and Example 3 is MS + 1.0 mg / L IAA + 10 g / L sucrose + 7 g / L agar, MS + 0.5-0.7 mg / L IBA + 10 g / L sucrose + 7 g / L agar, and MS + 0.3 mg / L IBA + 0.5 mg / L NAA + 10 g / L sucrose + 7 g / L agar.

[0073] Example 4

[0074] (1) Cut the sterile seedlings of Castanopsis chinensis with basically the same growth, about 5 cm in length, and strong, cut the base of the hypocotyl, and inoculate them in MS+0.7 mg / L IBA+10 g / L sucrose+7 g / L agar, pH 5.7-6.0, light duration of 12 h / d, light intensity of 1600-1800 lx, and culture temperature of 26±2℃. Induce roots for 30 days and calculate the average root length, average number of roots, rooting rate, and observe the growth of the materials.

[0075] (2) Select the rooted seedlings with well-developed root system and good growth, wash away the residual culture medium from the roots with clean water, and transplant them into the soil containing organic nutrient soil (BALTIC 1:1:1, 2:1:1, and 1:2:1, respectively, of a matrix of organic soil, perlite, and vermiculite. 1 to 3 plants were sown in each cup with a lid. A small hole with a diameter of 3-6 mm was made on the lid and on the side 0.5 cm above the bottom to maintain a moisture content of the matrix above 70%. After 15 days of cultivation in a light culture room with a photoperiod of 12 h / d, a light intensity of 1600-1800 lx, and a culture temperature of 26±2°C, the plants were placed in a greenhouse for a further 10 days. During the greenhouse cultivation, the bottle caps were opened every 5 days or so and the leaves were sprayed with water, preferably with water dripping. The plants were then transplanted into seedling bags containing organic nutrient soil, perlite, and vermiculite in a volume ratio of 2:1:1. The survival rate was calculated and the plants were cultured in the greenhouse for a further 10 days. On rainy days, they were placed in a nursery covered with a shade net (Table 4).

[0076] Table 4 Effects of different substrate ratios on the growth of Castanopsis chinensis seedlings

[0077]

[0078] The results showed that the most suitable matrix for seedling growth was organic nutrient soil, perlite, and vermiculite in a volume ratio of 2:1:1.

[0079] (3) Cut off the sterile seedlings of Castanopsis chinensis with basically the same growth, about 5 cm in length, and strong, cut the base of the hypocotyl flat, inoculate in the medium of MS+0.7mg / L IBA+10g / L sucrose+7g / L agar, screen the rooted Castanopsis chinensis seedlings with basically the same seedling height and growth, and transplant them into transparent plastic cups (follow the subsequent operations according to step (2)) and seedling trays with a mixed matrix of organic nutrient soil, perlite and vermiculite in a volume ratio of 2:1:1. The seedlings in the seedling trays were tissue culture seedlings that had been closed and opened in a greenhouse for 7 days. When transplanting, wash off the culture medium, then dip in a rooting powder solution with a concentration of 10g / L-15g / L for 5-15s, and directly transplant them into the seedling trays. Keep the humidity basically the same, place them in the greenhouse for 25 days, and calculate the transplant survival rate, average seedling height and growth of the observed materials (Table 5).

[0080] Table 5 Effects of different containers on the growth of Castanopsis chinensis seedlings

[0081]

[0082] Example 5

[0083] (1) Seed pretreatment: Use mature seeds of Castanopsis chinensis as explant materials; sun-dry freshly picked Castanopsis chinensis fruits, polish the fruits with sandpaper to separate the pulp from the seeds inside, then soak the removed seeds in clean water, rinse them thoroughly, and place them in a ventilated place to dry in the shade. Wash the shade-dried seeds with laundry detergent to remove surface wax before inoculation. Then soak the Castanopsis chinensis seeds in warm water at 30-40℃, remove the seeds floating on the water surface, and repeat this operation at least twice; then soak the seeds in distilled water, let them stand for at least 24 hours, and remove the seeds floating on the water surface; finally, inoculate them into 1 / 2MS culture medium containing 1.0 mg / L GA3.

[0084] (2) Seed disinfection: Under sterile conditions, soak the treated seeds in 0.1% HgCl2 solution for 8 min, 10 min, and 12 min, shaking them occasionally during the process to ensure that the seeds are fully disinfected by HgCl2. Finally, rinse with sterile water 3-5 times and inoculate them into a culture medium containing 1 / 2MS + 1.0 mg / L GA3 + 1.0 g / L activated carbon. The culture temperature was 26 ± 2°C, the light culture intensity was 1600-1800 lx, and the light culture time was 14 h / d. To prevent cross-contamination of explants, 20 bottles were inoculated in each treatment, with 10 seeds in each bottle, and this was repeated 3 times (Table 6);

[0085] Table 6 Effects of different disinfection methods on seed germination

[0086]

[0087] Experiments have shown that disinfection with 0.1% HgCl₂ for 10 minutes can eliminate contamination during the sterile germination of Castanopsis truncatum seeds. However, increasing the duration of mercuric chloride disinfection can actually reduce the germination rate of Castanopsis truncatum seeds. Ultimately, the preferred disinfection method of the present invention is to disinfect with 0.1% HgCl₂ for 10 minutes, followed by rinsing with sterile water 3-5 times.

[0088] (3) Effects of different basal culture media and activated carbon dosages on seed germination: The basal culture media used for seed germination of Castanopsis chinensis included MS and 1 / 2MS + 1.0 g / L GA3, with activated carbon added at 0 g / L, 1.0 g / L, 2.0 g / L, and 3.0 g / L, respectively. The culture temperature was 26 ± 2°C, the light intensity was 1600–1800 lx, and the light duration was 14 h / d. Seed germination was analyzed after 30 days of culture. Twenty bottles of each medium formulation were prepared, and 10 seeds were seeded into each medium. The experiment was repeated three times (Table 7).

[0089] Table 7 Effects of different basal culture media and activated carbon dosage on seed germination

[0090]

[0091] It can be concluded that the optimal culture medium for inducing seed germination of Castanopsis chinensis and improving the seed germination rate is 1 / 2MS medium, and the optimal amount of activated carbon added to the culture medium is 1.0 g / L activated carbon.

[0092] (4) Seed germination was performed by inoculating the sterilized seeds in 1 / 2 MS medium containing 0, 1.0, 1.5, and 2.0 mg / L GA3 at 1.0 g / L activated carbon. The incubation temperature was 26 ± 2°C, the light intensity was 1600–1800 lx, and the light duration was 14 h / d. Seed germination was recorded after 30 days of incubation. Twenty bottles of each medium formulation were prepared, and 10 seeds were inoculated into each medium. The experiment was repeated three times (Table 8).

[0093] Table 8 Effects of different plant growth regulators on seed germination

[0094]

[0095] The experiment found that the germination rate of Castanopsis chinensis seeds was relatively low when gibberellins were not added, but the germination rate of seeds was significantly improved after adding a certain concentration of gibberellins. It can be concluded that the optimal plant growth regulator for inducing seed germination is 1.0 mg / LGA3.

[0096] The optimal culture medium formula for primary culture of Castanopsis chinensis seeds was finally obtained as 1 / 2MS+1.0mg / L GA3+1.0g / L activated carbon.

[0097] The sterile seedlings obtained in Example 5 were cut flat at the base of the hypocotyl and subjected to rooting culture according to the method of the present invention.

[0098] Example 6

[0099] (1) Acquisition of bud-bearing stem segments of Castanopsis chinensis: The experimental materials were potted Castanopsis chinensis seedlings from the tree building nursery of Central South University of Forestry and Technology in Changsha, Hunan Province. Tender shoots and semi-lignified stem segments with buds that sprouted in April and May were used as explant materials.

[0100] (2) Explant pretreatment: Cut the side branches of the Castanopsis chinensis potted seedlings from the base and rinse them in running water for 30-40 minutes; then cut the upper part of the lateral branches (remove the terminal buds and leave 3-5 segments below the terminal buds) and the semi-lignified stem segments into 3-4 cm long stem segments, leaving 1-2 axillary buds on each stem segment, wash them 1-2 times with detergent, clean the remaining impurities in the stem segments with a soft brush, and then rinse them in running water for at least 1 hour;

[0101] (3) Explant disinfection: Under sterile conditions, the stem segments with buds of Castanopsis chinensis were disinfected with 75% alcohol for 1 min. During the soaking process, the stem segments should be shaken from time to time to ensure that the alcohol can fully disinfect the stem segments with buds. After 1 min, the stem segments were soaked again with new 75% alcohol for 1 min. The stem segments were rinsed 3-5 times with sterile water. The tender branches were disinfected with 0.1% HgCl2 solution for 25 or 35 min, and the semi-lignified stem segments were disinfected for 50 min. During the soaking process, the stem segments should be shaken from time to time to ensure that the mercuric chloride solution can fully disinfect the stem segments with buds. Finally, the stem segments were rinsed 3-5 times with sterile water (Tables 9 and 10).

[0102] (4) Primary culture of stem segments with buds: Place the sterilized stem segments with buds on sterile filter paper to absorb moisture; use a sterile knife to cut off the browned part of the top and tail of the stem segments, and then inoculate the stem segments with buds into a culture medium containing 1 / 2MS + 1.0-2.0 mg / L 6-BA + 0.5-1.0 mg / L NAA; the culture medium is supplemented with 30 g / L sucrose and 8 g / L agar, and the pH is adjusted to 5.5-5.7; culture is carried out under light conditions for 30-40 days, the culture temperature is 26±2℃, the light culture intensity is 1600-1800 lx, and the light culture time is 12-14 h / d (Table 11).

[0103] Table 9 Effects of different disinfection treatments on the contamination rate and axillary bud induction rate of Castanopsis chinensis tender branches with buds

[0104]

[0105]

[0106] Table 10 Effects of different disinfection treatments on the contamination rate and axillary bud induction rate of semi-lignified stem segments with buds in Castanopsis chinensis

[0107]

[0108] The final conclusion was: disinfect the stem segments of Castanopsis chinensis with buds with 75% alcohol for 1 minute, shake them from time to time during the soaking process to ensure that the alcohol can fully disinfect the stem segments with buds. After 1 minute, replace them with new 75% alcohol and soak them again for 1 minute. Rinse with sterile water 3-5 times, and then use 0.1% HgCl2 solution to disinfect the tender branches for 25 or 35 minutes and the semi-lignified stem segments for 50 minutes. Shake them from time to time during the soaking process to ensure that the mercuric chloride solution can fully disinfect the stem segments with buds. Finally, rinse with sterile water 3-5 times.

[0109] Table 11 Effects of different culture media on the induction rate of axillary buds in the stem segments of Castanopsis sylvestris with buds

[0110]

[0111] It was finally determined that after disinfection, the stem segments with buds of Castanopsis chinensis were cut into 1-2 cm segments with 1-2 axillary buds on a sterile operating table and inoculated into 1 / 2MS+2.0mg / L6-BA+0.5mg / LNAA culture medium to better induce the growth of axillary buds in the stem segments with buds.

Claims

1. A method for hardening and transplanting of Castanopsis chinensis tissue culture seedlings, characterized in that: The following steps are involved: (1) Selection of sterile seedlings: Cut the sterile seedlings of Castanopsis chinensis with basically uniform growth and robustness as materials; (2) Rooting culture: The sterile seedlings obtained in step (1) were cut flat at the base of the hypocotyl and inoculated into a medium containing MS + 0.5-0.7 mg / L IBA, MS + 1.0 mg / L IAA, or MS + 0.3 mg / L IBA + 0.5 mg / L NAA for rooting culture; (3) Washing the seedlings: directly wash the remaining culture medium on the roots of the rooted seedlings, and try to keep the root system intact; (4) Dipping the seedlings: Dip the roots of the rooted seedlings into the rooting powder solution, remove them, and drain the water; (5) Transplanting: Transplant the rooted seedlings into a transparent plastic cup with a substrate; after culturing in a light culture room for 10-15 days, place them in a greenhouse for further cultivation. Reduce the moisture content of the substrate by reducing the number of waterings and further acclimate them, maintaining the substrate moisture content at 40-60%. After culturing for 7-15 days, transplant them into a seedling bag and continue to cultivate in the greenhouse for 7-10 days. On rainy days, place them in a nursery covered with a shade net. The sterile seedlings obtained in step (1) are from sterile seedlings germinated from seeds; the obtaining steps include: 1) Seed pretreatment: Freshly picked Castanopsis chinensis fruits were sun-dried and sanded to separate the pulp from the seeds. The removed seeds were then soaked in clean water, rinsed, and placed in a well-ventilated area to dry in the shade. The shade-dried seeds were then washed with laundry detergent to remove surface wax before inoculation. 2) Explant screening: Soak the seeds of Castanopsis chinensis in warm water at 30-40°C, then remove any seeds floating on the water surface. Repeat this process at least twice. Then, soak the seeds in distilled water for at least 24 hours, and remove any seeds floating on the water surface. 3) Explant disinfection: Under sterile conditions, soak the Castanopsis chinensis seeds in a 0.1% HgCl2 solution for 10 minutes, shaking them occasionally during the soaking process to ensure that the HgCl2 fully disinfects the seeds. Finally, rinse with sterile water 3-5 times; 4) Primary Seed Culture: Sterilized seeds were placed on sterile filter paper to absorb moisture. The seeds were then inoculated into a medium consisting of 1 / 2 MS + 1.0 mg / L GA3 + 1.0-3.0 g / L activated carbon or 1 / 2 MS + 2.0 mg / L GA3 + 1.0-3.0 g / L activated carbon. The medium was supplemented with 30 g / L sucrose and 8 g / L agar, and the pH was adjusted to 5.7-5.

9. The seedlings were cultured for 40-50 days under illumination conditions at 26 ± 2°C, 1600-1800 lx, and 12-14 h / d as the starting material for rooted seedlings. Alternatively, the sterile seedlings obtained in step (1) are derived from sterile seedlings induced from stem segments with buds; the obtaining steps include: 1) Acquisition of bud-bearing stem segments of Castanopsis chinensis: The experimental materials were derived from potted Castanopsis chinensis seedlings. Tender shoots or semi-lignified bud-bearing stem segments that germinated in April or May were used as explant materials. 2) Explant Pretreatment: Cut the side branches of the potted Castanopsis truncatula seedlings from the base and rinse them under running water for 30-40 minutes. Then, cut the upper part of the lateral shoots or semi-lignified stem segments into 3-4 cm long segments, leaving 1-2 axillary buds on each segment. Wash them 1-2 times with detergent, clean the segments with a soft brush to remove any remaining impurities, and rinse them under running water for at least 1 hour. 3) Explant Disinfection: Under sterile conditions, the stem segments of the tung tree with buds were disinfected with 75% alcohol for 1 minute, shaking the immersion area from time to time to ensure that the alcohol fully disinfected the stem segments with buds. After 1 minute, the 75% alcohol was replaced and soaked again for 1 minute. The 75% alcohol was rinsed 3-5 times with sterile water. The shoots were then disinfected with 0.1% HgCl2 solution for 25 or 35 minutes, and the semi-lignified stem segments were disinfected for 50 minutes. The mercuric chloride solution was shaken from time to time during the soaking process to ensure that the mercuric chloride solution fully disinfected the stem segments with buds. Finally, the 75% alcohol solution was rinsed 3-5 times with sterile water. 4) Primary culture of stem segments with buds: Place the sterilized stem segments with buds on sterile filter paper to absorb moisture; use a sterile knife to cut off the top and brown part of the stem segments, and then inoculate the stem segments with buds into Culture in a medium containing 1 / 2MS, 1.0-2.0mg / L 6-BA, and 0.5-1.0mg / L NAA; supplement the medium with 30g / L sucrose and 8g / L agar, and adjust the pH to 5.5-5.7; culture for 30-40 days under illumination conditions at a temperature of 26±2°C, a light intensity of 1600-1800lx, and a light incubation time of 12-14h / d; In step (5), the transplanting matrix is a mixture of organic nutrient soil, perlite and vermiculite in a volume ratio of 2:1:

1. The rooted seedlings of the Chinese tallow tree are planted in a transparent plastic cup with a lid, 1 to 3 plants per cup, and the lid is covered. A small hole with a diameter of 3-6 mm is opened on the lid and the side at a height of 0.5-1 cm from the bottom. The moisture content of the matrix is kept above 70%. After culturing in a light culture room for 10-15 days, the light intensity is 1600-1800 lx and the light culture temperature is 200-300 °C. The temperature is 26±2℃, the light duration is 12h / d, and then placed in a greenhouse for further cultivation at a temperature of 20-35℃. The substrate humidity is further reduced by reducing the number of waterings to maintain a substrate moisture content of 40-60%. After further cultivation for 7-15 days, the seeds are transplanted into a seedling bag with organic nutrient soil, perlite, and vermiculite in a volume ratio of 2:1:

1. The seeds are further cultivated in the greenhouse for 7-10 days and placed in a nursery covered with a shade net on rainy days. The specifications of the transparent plastic cup in step (5) are: bottom diameter 8 cm, height 14.5 cm, bottle mouth diameter 11.5 cm, and the volume of the transplanting matrix is 1 / 5 of the volume of the transparent plastic cup.

2. The method according to claim 1, characterized in that In step (1), the length of the sterile seedlings is 4 to 7 cm, and the number of leaves is 4 to 7.

3. The method according to claim 1, characterized in that In step (2), the rooting medium is supplemented with 10 g / L sucrose and 7 g / L agar, and the pH value is 5.7-6.

0.

4. The method according to claim 1, wherein The light intensity of the rooting culture in step (2) is 1600-1800 lx, the light culture temperature is 26±2° C., the light duration is 12 h / d, and the rooted seedlings of Castanopsis chinensis are obtained after culturing for 25-35 days.

5. The method according to claim 1, wherein In step (4), the mixture is immersed in a rooting powder solution having a concentration of 10 g / L-15 g / L for 5-15 seconds.

6. The method according to claim 1, characterized in that During greenhouse cultivation in step (5), the bottle cap is opened every 5 days or so, and the leaf surface is sprayed with water, preferably with water dripping.

7. The method according to claim 6, characterized in that After transplanting the Castanopsis chinensis seedlings into seedling bags and placing them in the nursery, add water in time and spray 0.1% urea every 30 days to promote the growth of the Castanopsis chinensis seedlings.

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