Tissue culture and rapid propagation method of tectona grandis clone and application

Abstract

This invention discloses a method and application for rapid propagation of teak clonal lines through tissue culture, belonging to the field of plant tissue culture technology. The method uses teak segments as explants and detoxifies them using solutions of alcohol, mercuric chloride, penicillin, and streptomycin. This method effectively removes bacteria and endophytes from the surface of the explants, filling a gap in current methods for detoxifying teak explants. After obtaining tissue culture seedlings, they are propagated using a bud proliferation medium, yielding a large number of plants in a short period. By adding plant growth regulators to the medium, rooting of the tissue culture seedlings is promoted. A rooting medium formula for teak tissue culture seedlings has been developed, enabling rooting within the tissue culture bottle, solving the long-standing problem of difficult rooting of teak tissue culture seedlings, and significantly improving the survival rate after transplanting.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method and application for rapid propagation of teak clonal lines through tissue culture. Specifically, it relates to the establishment of a rapid propagation system for teak tissue culture, with the main innovations being the explant detoxification method and the formulation of the bud proliferation and rooting culture medium. Background Technology

[0002] Teak (Tectona grandis Lf) is a tall tree belonging to the genus Tectona in the family Verbenaceae. It is an important introduced and valuable timber species in my country, possessing excellent characteristics such as rapid growth, resistance to decay and insects, and is now widely planted in Yunnan, Hainan, Guangxi, Fujian, Guangdong, and other regions. Currently, conventional propagation methods cannot meet the huge market demand for teak seedlings. Tissue culture technology can achieve large-scale asexual propagation of superior strains in a short period while maintaining the superior traits of the parent plant, greatly improving the teak propagation process. Tissue culture rapid propagation is one of the important methods of asexual propagation of teak. Its main processes include explant collection and detoxification, regeneration and growth of buds, rooting of tissue culture seedlings, and transplanting and hardening off. Among these, explant detoxification and rooting of tissue culture seedlings are the technical bottlenecks limiting the rapid propagation of teak through tissue culture. Explants carry various surface bacteria and endophytic bacteria; the type of detoxification agent and the detoxification method directly affect the detoxification effect and further affect the success of tissue culture. Due to the current lack of effective detoxification methods, the asexual varieties of teak propagated through tissue culture are relatively limited. Furthermore, rooting of tissue-cultured seedlings is crucial for the rapid propagation of teak through tissue culture. Tissue-cultured seedlings that have rooted during the tissue culture stage can significantly improve the survival rate after transplanting. Currently, asexual propagation of teak often employs a two-step rooting method: transplanting first, followed by rooting in soil. This method has a low transplant survival rate because rootless tissue-cultured seedlings are highly susceptible to death during transplanting. However, to date, no systematic research has been found on the detoxification of teak explants and the rooting of tissue-cultured seedlings.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a method and application for rapid propagation of teak clonal line 7029 through tissue culture. This invention focuses on investigating the effects of different disinfectants and disinfection times on the detoxification of teak explants, exploring a detoxification method that reduces contamination without affecting explant germination. By investigating the effects of different plant growth regulators on the rooting of teak tissue culture seedlings, the optimal rooting culture medium was obtained, increasing the rooting rate to 92.22%, laying a solid foundation for improving transplant survival rates.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for rapid propagation of teak clone 7029 via tissue culture includes the following steps:

[0007] (1) Pretreatment of explants: Take stem segments with only one pair of axillary buds as explants, rinse the explants with running water, clean the surface hairs, and then soak them in ascorbic acid solution to obtain pretreated explants;

[0008] (2) Detoxification of explants: The pretreated explants were disinfected sequentially with 70% to 75% (preferably 75%) alcohol, mercuric chloride, and penicillin and streptomycin solutions to obtain detoxified explants;

[0009] (3) Sprouting and bud proliferation culture of explants: The virus-free explants were inoculated into MS basic medium to germinate buds. After the buds elongated, the buds were cut off and used as new explants to be inoculated into bud proliferation medium for culture.

[0010] (4) Rooting culture: After subculture, the tissue culture seedlings are inoculated into the rooting medium for rooting culture.

[0011] To better achieve the technical solution of this invention, it also includes:

[0012] (5) Hardening and transplanting: Harden and sterilize the rooted seedlings before transplanting.

[0013] Preferably, in step (1),

[0014] The rinsing time with running water is 30–50 minutes; more specifically, 30 minutes.

[0015] The concentration of the ascorbic acid solution is 0.1–0.2 g / L; more specifically, 0.1 g / L.

[0016] The soaking time is 45–60 min; more specifically, 60 min.

[0017] Preferably, in step (2),

[0018] The alcohol disinfection time is 15±1 s;

[0019] The conditions for mercuric chloride disinfection are: 0.1-0.2% mercuric chloride disinfection for 3-9 min; further, 0.1-0.2% mercuric chloride disinfection for 3-6 min; and even further, 0.2% mercuric chloride disinfection for 6 min.

[0020] The penicillin and streptomycin solution is a solution containing 90–100 mg / L penicillin and 90–100 mg / L streptomycin; more specifically, it is a solution containing 100 mg / L penicillin and 100 mg / L streptomycin.

[0021] The sterilization time is 5 to 7 minutes; more specifically, 5 minutes.

[0022] Preferably, in step (3),

[0023] The MS basal medium was formulated as MS + 30±0.5 g / L sucrose and 7±0.5 g / L agar, pH 5.8–6.0;

[0024] The bud proliferation medium was formulated as follows: MS + 0.5±0.02 mg / L IAA + 0.3~0.5 mg / L 6-BA + 0.2~0.5 mg / L 2-iP + 30~80 mg / L inositol + 0.5±0.02 mg / L AgNO3 + 0.2±0.02 g / L water + casein + 30±0.5 g / L sucrose + 7±0.5 g / L agar, pH 5.8~6.0; or further as MS + 0.5 mg / L IAA + 0.5 mg / L 6-BA + 0.5 mg / L 2-iP + 80 mg / L inositol + 0.5±0.02 mg / L AgNO3 + 0.2±0.02 g / L water + casein + 30±0.5 g / L sucrose + 7±0.5 g / L agar, pH 5.8~6.0.

[0025] The culture time is 25–30 days; more specifically, 25 days.

[0026] The culture conditions were 25±2℃, 2400~2500 lux light intensity, and 16 h of light per day.

[0027] Preferably, in step (4),

[0028] The term "successful generation" refers to 3-4 generations.

[0029] The rooting medium formulation is MS + 0.5±0.02 mg / L 6-BA + 0.5~1.5 mg / L IBA + 0.1~0.3 g / L AC + 0.25~0.5 mg / L biotin + 30±0.5 g / L sucrose + 7±0.5 g / L agar, pH 5.8~6.0; further, MS + 0.5 mg / L 6-BA + 1.0 mg / L IBA + 0.1 g / L AC + 0.5 mg / L biotin + 30±0.5 g / L sucrose + 7±0.5 g / L agar, pH 5.8~6.0;

[0030] The rooting culture time is 30-35 days; more specifically, 30 days.

[0031] The conditions for rooting culture were 25±2℃, 2400~2500 lux light intensity, and 16 h of light per day.

[0032] Preferably, in step (5),

[0033] The hardening-off period for seedlings is 3 to 5 days.

[0034] The sterilization process involves immersing the roots in a carbendazim solution diluted 1800-2000 times for 1-2 minutes; further, it involves immersing the roots in a carbendazim solution diluted 2000 times for 2 minutes.

[0035] The substrate used for transplanting is a mixture of loess, peat moss, and vermiculite in a ratio of 3:3:1.

[0036] Application of the above method in the rapid propagation of teak clone 7029 through tissue culture.

[0037] The present invention has the following advantages and effects compared with the prior art:

[0038] The method of this invention uses teak segments as explants and detoxifies them using solutions of alcohol, mercuric chloride, penicillin, and streptomycin. This method can effectively remove bacteria and endophytes from the surface of the explants, filling the gap in current methods for detoxifying teak explants. After obtaining tissue culture seedlings, they are propagated using a bud proliferation medium, resulting in a large number of plants in a short period of time. By adding plant growth regulators to the medium, rooting of the tissue culture seedlings is promoted, and a rooting medium formula for teak tissue culture seedlings has been developed. This allows for rooting within the tissue culture bottle, solving the long-standing problem of difficult rooting of teak tissue culture seedlings and significantly improving the survival rate after transplanting. Attached Figure Description

[0039] Figure 1 It is an explant; where A: stem tip; B: segment.

[0040] Figure 2 It refers to the budding of explants; where A: budding of the shoot tip; B: budding of the segment.

[0041] Figure 3 These are the rooting of tissue culture seedlings; where A: a rooting diagram in a culture bottle; B and C: complete plants with stems and leaves. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0043] The teak clone 7029 used in the examples is disclosed in the literature “Huang Guihua, Liang Kunnan, Fu Qiang, et al. Genetic variation and selection of superior clones of 11-year-old teak clones [J]. Journal of Northeast Forestry University, 2023, 51(8):18-22.”

[0044] In the examples, IAA is indoleacetic acid, 6-BA is 6-benzylaminopurine, 2-iP is 2-isopentene adenine, KT is kinetin, NAA is naphthaleneacetic acid, IBA is indolebutyric acid, and AC is activated carbon.

[0045] Example 1

[0046] 1. Plant materials and culture medium formulation

[0047] Teak clone 7029 was used as the test material, and shoot tips and segments were collected as explants. MS medium supplemented with 30 g / L sucrose and 7 g / L agar was used as the basic medium. The pH of the medium was adjusted to 5.8 with 1M potassium hydroxide (KOH), and the medium was sterilized in a steam sterilizer at 121℃ for 20 min.

[0048] 2. Experimental Methods

[0049] 2.1 Collection and Pretreatment of Explants

[0050] On the morning of the fourth consecutive sunny day, teak stem segments were collected and brought back to the laboratory as soon as possible. Stem segments were taken from disease-free teak plants, with a cross-sectional diameter of 5-10 mm being optimal. The collected stem segments were divided into two types: stem tips and segments. Stem tips were those with only terminal buds. Figure 1 In section A), the segment is a stem segment with only one pair of axillary buds. Figure 1 (B) Rinse the explants with running water for 30 min, then clean the surface of the explants with a soft brush to remove the hairs on the surface of the stem segments. Soak the cleaned explants in 0.1 g / L ascorbic acid solution for 1 h.

[0051] 2.2 Detoxification of explants

[0052] Detoxification of pretreated explants (including shoot tips and segments) was performed in a clean bench. The explants were placed in glass culture flasks and first immersed in 75% (v / v) ethanol for 0, 15, 30, and 60 seconds, respectively. Then, they were detoxified by immersion in 0.1%, 0.2%, and 0.3% (w / v) mercuric chloride solutions for four time gradients: 3 min, 6 min, 9 min, and 12 min. Afterward, they were immersed in a solution containing 100 mg / L penicillin and 100 mg / L streptomycin for 5 minutes to remove endophytic bacteria. Finally, the surface moisture of the explants was blotted dry with sterile filter paper.

[0053] 2.3 Germination and growth of explants

[0054] Virus-free explants were inoculated into MS basal medium for shoot germination. After shoot elongation, the new shoots (segments) were cut off and used as new explants in shoot proliferation medium. In the shoot proliferation medium, IAA was used in combination with different concentrations of 6-BA, 2-iP, KT, and inositol, and 0.5 mg / L silver nitrate (AgNO3) and 0.2 g / L water-based casein were added to reduce the browning of explants. The IAA concentration was 0.5 mg / L, the 6-BA concentrations were 0.1, 0.3, 0.5, and 1.0 mg / L, the 2-iP concentrations were 0.2 and 0.5 mg / L, the KT concentrations were 0.2 and 0.5 mg / L, and the inositol concentrations were 30, 50, 80, and 100 mg / L. One explant was inoculated per bottle of medium, with 10 bottles constituting one replicate, and the replicates were performed three times. The cultivation conditions were 25±2℃, 2500 lux light intensity, 16h light per day, and the bud growth was observed after 25 days.

[0055] 2.4 Rooting of tissue culture seedlings

[0056] After 3-4 subcultures, tissue culture seedlings can be cultured for rooting. When the subcultured seedlings grow to 3 cm, the swollen callus tissue at the base is removed and the seedlings are inoculated into rooting medium. In the rooting medium, 6-BA is used in combination with different concentration gradients of NAA, IBA, activated charcoal (AC), and biotin. The concentrations are: 6-BA 0.5 mg / L, NAA 0.5, 1.0, and 1.5 mg / L, IBA 0.5, 1.0, and 1.5 mg / L, AC 0.1, 0.25, 0.3, 0.5, and 1.0 g / L, and biotin 0.25, 0.5, and 0.75 mg / L. Six seedlings are inoculated per bottle of medium, with 10 bottles constituting one replicate, repeated three times. The culture conditions are 25±2℃, 2500 lux light intensity, 16 h light per day, and rooting status is observed after 30 days.

[0057] 2.5 Seedling hardening and transplanting

[0058] After obtaining rooted seedlings, allow them to grow to approximately 5 cm in length and 3 cm in root length before hardening them off. First, loosen the cap of the tissue culture bottle and place it in a greenhouse for 3-5 days. Carefully remove the seedlings from the bottle and gently wash away any remaining culture medium residue from the roots with running water, taking care not to damage the roots or young leaves. Soak the cleaned seedling roots in a 2000-fold diluted carbendazim solution for 2 minutes to sterilize. Then, transplant the regenerated seedlings into flowerpots. The flowerpots and substrate should be sterilized under high temperature and pressure beforehand. The substrate should be a mixture of loess, peat moss, and vermiculite in a 3:3:1 volume ratio. Place the transplanted seedlings in a nursery for further cultivation.

[0059] 2.6 Experimental Results

[0060] (1) Effect of alcohol on detoxification of stem segments of teak clone 7029

[0061] Table 1 shows that the detoxification time with 75% alcohol significantly affected the contamination rate and mortality rate of teak clone 7029 stem segments. As the detoxification time increased from 0 s to 60 s, the explant contamination rate decreased from 100% to 33.33%, indicating that the detoxification effect of using 75% alcohol in combination with 0.1% mercuric chloride was better than using 0.1% mercuric chloride alone, and that 75% alcohol played a major role in reducing the contamination rate. However, with the extension of the detoxification time, the explant mortality rate gradually increased. When the disinfection time was extended to 60 s, the explant mortality rate reached as high as 66.67%, indicating that excessively long alcohol disinfection time can cause penetrating damage to the explant epidermal cells that is irreversible, ultimately leading to browning and death of the explants. Although the contamination rate was relatively high when 75% alcohol disinfection was performed for 15 s, two buds emerged from the surviving explants, indicating a significantly better detoxification effect than other treatment combinations. Therefore, a detoxification time of 15 s with 75% alcohol is optimal (Table 1).

[0062] Table 1. Explant contamination rate and germination rate at different alcohol detoxification times

[0063]

[0064] (2) Effects of mercuric chloride on detoxification of teak clone stem segments

[0065] Table 2 shows that both mercuric chloride concentration and detoxification time significantly affect the contamination rate and mortality rate of explants. Under the same mercuric chloride concentration, the contamination rate and mortality rate gradually decrease and increase, respectively, with the extension of detoxification time. Excessive disinfection time makes it difficult for explants to withstand the penetrating damage of mercuric chloride, easily leading to browning and death; insufficient disinfection time fails to completely kill bacteria on the explant surface, ultimately resulting in explant contamination. Increasing the mercuric chloride concentration can effectively reduce the contamination rate. For example, when the disinfection time is 9 min, the contamination rate decreases from 63.33% to 0 with increasing mercuric chloride concentration. When the mercuric chloride concentration is 0.3% and the detoxification time is extended from 3 min to 12 min, the explant mortality rate increases from 70% to 100%, indicating that at this point, the mercuric chloride concentration has caused significant damage to the explants, almost reaching the threshold that the explants can tolerate. Both excessively low and excessively high mercuric chloride concentrations are detrimental to explant detoxification. At low concentrations, some tolerant bacteria can survive, and some endophytic bacteria cannot be effectively killed, potentially leading to widespread contamination during subsequent culture. Excessively high mercuric chloride concentrations not only kill the surface cells of the explant but may even penetrate the interior, causing explant death. Therefore, finding a balance between minimizing contamination and reducing toxicity to explants is crucial. Results showed that when 75% alcohol was disinfected for 15 seconds and 0.2% mercuric chloride for 6 minutes, both the contamination rate and explant mortality rate were at moderate levels, and six explants successfully germinated, indicating that this method achieved the best detoxification effect (Table 2).

[0066] Table 2. Effects of mercuric chloride concentration and detoxification time on explants

[0067]

[0068] (3) Differences in germination among different explant types

[0069] Explants were divided into two types: shoot tips (including shoot tips) and segments (including axillary buds). Under optimal virus-free conditions, virus-free treatment was performed according to step 2.2. The virus-free explants were then inoculated into MS basal medium for bud germination. Results showed that most shoot tips did not bud after inoculation into MS basal medium and gradually withered and died with prolonged culture time. A very small number of terminal buds showed two true leaves gradually unfolding at the top after approximately 20 days of culture. Figure 2 (A) The shoot tip budding rate was 7.78% (Table 3); after the segments were inoculated into MS basal medium, the cut surface easily turned brown, but the browning range did not expand to the axillary buds. After about 20 days of culture, the axillary buds began to germinate and grow tender leaves. Each stem segment produced two axillary buds, and usually both axillary buds would germinate at the same time. Figure 2 (B in the table), the germination rate of axillary buds was 57.78% (Table 3). Compared with terminal buds, axillary buds are more numerous and easier to germinate, therefore segments are more suitable as explants for rapid propagation of teak.

[0070] Table 3. Effects of explant type on bud break

[0071]

[0072] (4) Effect of 6-BA on bud proliferation

[0073] Segments of newly grown shoots from explants were used as explants for shoot proliferation experiments to determine the optimal culture medium for shoot proliferation. The effect of 6-BA on shoot proliferation was investigated by combining 0.5 mg / L IAA with different concentrations of 6-BA. The results showed that adding 6-BA to the culture medium significantly promoted shoot proliferation. Furthermore, as the concentration of 6-BA increased, the shoot proliferation rate first increased and then decreased. The optimal concentration of 6-BA was 0.5 mg / L, at which the shoot proliferation rate was the highest (33.33%), and the shoots in this medium were robust, with green, spreading leaves and rapid growth. When the concentration of 6-BA was too low, shoot proliferation was difficult or the proliferating shoots grew very slowly; while when the concentration of 6-BA was too high, multiple shoots were produced simultaneously from the explants. Due to competition for nutrients in the culture medium, each shoot failed to grow robustly (Table 4).

[0074] Table 4. Effects of different concentrations of 6-BA on shoot proliferation

[0075]

[0076] (5) Effects of plant growth regulator combinations on bud proliferation

[0077] (5.1) Effect of combined use of 6-BA and 2-iP on bud proliferation

[0078] The results showed that after explants were inoculated into the shoot proliferation medium, the basal callus swelled, and the terminal or axillary buds of the explants elongated; typically, only one of the two axillary buds could grow. The combined use of 6-BA and 2-iP was beneficial in improving the shoot proliferation rate. During shoot proliferation, 6-BA played a dominant role, and its concentration was crucial; both excessively low and high concentrations were detrimental to shoot proliferation, and even the addition of 2-iP did not effectively improve the shoot proliferation rate. Furthermore, compared to low concentrations of 2-iP, the combined use of high concentrations of 2-iP and 6-BA significantly improved the shoot proliferation rate. Specifically, the shoot proliferation rate reached 63.33% when 0.5 mg / L 6-BA and 0.5 mg / L 2-iP were added to the medium, followed by 46.67% when 0.5 mg / L 6-BA and 0.2 mg / L 2-iP were added (Table 5).

[0079] Table 5. Effects of combined use of 6-BA and 2-iP on bud proliferation.

[0080]

[0081] (5.2) Effect of the combined use of 6-BA and KT on bud proliferation

[0082] The results showed that, compared with 6-BA alone, the combined use of 6-BA and KT promoted shoot proliferation, but the effect was not significant. Shoot proliferation rates were generally higher under low KT concentrations than under high KT concentrations. The highest shoot proliferation rate (40.00%) was observed when 0.5 mg / L 6-BA and 0.2 mg / L KT were added to the culture medium; followed by a rate of 36.67% when 0.3 mg / L 6-BA and 0.2 mg / L KT were added (Table 6).

[0083] Table 6. Effects of the combined use of 6-BA and KT on bud proliferation.

[0084]

[0085] (5.3) Effects of combined use of 6-BA, 2-ip and inositol on bud proliferation

[0086] The results showed that the combined use of 6-BA, 2-ip, and inositol significantly improved the bud proliferation rate. Inositol was used at concentrations of 30, 50, 80, and 100 mg / L. The highest bud proliferation effect was achieved when the inositol concentration was 80 mg / L and the 2-iP concentration was 0.5 mg / L, with a bud proliferation rate of 86.67% (Table 7).

[0087] Table 7. Effects of combined use of 6-BA, 2-ip and inositol on bud proliferation.

[0088]

[0089] In conclusion, the combined use of 6-BA, 2-ip, and inositol is superior to that of 6-BA with 2-iP or 6-BA with KT; the optimal bud proliferation medium is MS + 0.5 mg / L IAA + 0.5 mg / L 6-BA + 0.5 mg / L 2-iP + 80 mg / L inositol + 0.5 mg / L AgNO3 + 0.2 g / L water + casein + 30 g / L sucrose + 7 g / L agar.

[0090] (6) Effects of plant growth regulators on rooting of tissue culture seedlings

[0091] (6.1) Effects of NAA, IBA and AC on rooting of tissue culture seedlings

[0092] After inoculating the tissue culture seedlings onto the rooting medium, the basal callus tissue begins to swell after about 15 days of culture. As the culture time extends, the callus tissue gradually swells into a hard mass, with single or multiple root growth points appearing on its surface, which is white. After 25 days of culture, small roots grow from the root growth points on the surface of the callus tissue and gradually elongate, eventually forming a complete plant with roots, stems, and leaves. Figure 3 ).

[0093] By combining 6-BA with plant growth regulators such as NAA, IBA, and activated carbon, different rooting culture medium formulations were established to investigate the effects of plant growth regulators on rooting. The results showed that when the culture medium contained only 6-BA, teak tissue culture seedlings could not root, with a rooting rate of 0%. The combination of 6-BA and IBA had the best effect on inducing rooting in teak tissue culture seedlings, with rooting rates all above 70%. The highest rooting rate, reaching 86.67%, was achieved when the IBA concentration was 1.0 mg / L. NAA had no significant effect on promoting rooting in teak tissue culture seedlings; the number of roots in the NAA-added medium was low, with rooting rates all below 20%. AC also promoted rooting in teak tissue culture seedlings; adding 0.25 g / L AC to the culture medium significantly increased the rooting rate to 41.11%, but the rooting rate did not increase with increasing AC concentration (Table 8).

[0094] Table 8 Effects of plant growth regulators on rooting of teak tissue culture seedlings

[0095]

[0096] (6.2) Effects of combined use of IBA, AC and biotin on rooting of tissue culture seedlings

[0097] The results showed that, compared with IBA alone, the combined use of IBA, AC and biotin significantly promoted the rooting of teak tissue culture seedlings. The highest rooting rate, reaching 92.22%, was achieved in the culture medium MS + 0.5 mg / L 6-BA + 1.0 mg / L IBA + 0.1 g / L AC + 0.5 mg / L biotin + 30 g / L sucrose + 7 g / L agar (Table 9).

[0098] Table 9. Effects of combined use of IBA, AC, and biotin on rooting of tissue culture seedlings.

[0099]

[0100] In conclusion, the optimal culture medium for rooting teak tissue culture seedlings is MS + 0.5 mg / L 6-BA + 1.0 mg / L IBA + 0.1 g / L AC + 0.5 mg / L biotin + 30 g / L sucrose + 7 g / L agar.

[0101] (7) Transplanting of teak rooted tissue culture seedlings

[0102] Ninety healthy tissue culture seedlings, approximately 5 cm in height and 3 cm in root length, were selected for hardening-off and transplanting. The caps of the tissue culture bottles were loosened, and the seedlings were hardened off for 3 days under natural light at room temperature. Afterward, the sterilized seedlings were transplanted into the prepared substrate, and a transparent cap was placed over them to maintain humidity. The transparent cap was removed 10 days after transplanting, and the survival rate was measured after 30 days of growth, reaching 94.44%.

[0103] Comparative Example 1:

[0104] The explants used for disinfection were segments of the teak clone 7029; the explants used for bud proliferation were segments of new buds grown from the explants in Example 1, which were then used for bud proliferation experiments. Explant detoxification was performed according to the disinfection method described in the literature published by Tiwari SK et al. (An improved micropropagation protocol for teak. 2002, Plant Cell, Tissue and Organ Culture 71.1: 1-6.). The specific disinfection method was as follows: the surface was wiped with gauze soaked in 50% alcohol (volume concentration). Stem segments were cut into 3-4 cm long segments with nodes, washed in 1% Teepol detergent solution for 30 minutes, then treated with 80% ethanol for 30 seconds, then disinfected with a 0.1% mercuric chloride solution containing a few drops of Tween-20 for 5 minutes, and finally rinsed 5 times with sterile distilled water. The optimal bud proliferation medium (MS + 6-BA 5.0 mg / L + IAA 0.1 mg / L + 30 g / L sucrose + 8 g / L agar) was used for proliferation culture. The rooting method was to take out the tissue culture seedlings, dip them in IBA rooting powder, and then transplant them into the soil for rooting.

[0105] Comparative Example 2:

[0106] The explants used for shoot proliferation were segments of new shoots grown from the explants in Example 1. Shoot proliferation experiments were conducted using these explants. Propagation culture was performed according to the optimal shoot proliferation medium (MS + 6-BA 2.0 mg / L + 30 g / L sucrose + 8 g / L agar) published by Stephane Gbehe et al. (Effect of Cytokinins on Micropropagation of Teak (Tectona grandis L.) Grown in Cote D'ivoire. 2023, Journal of Advances in Biology & Biotechnology, 26.10: 1-11.). Rooting culture was performed using the optimal rooting medium (1 / 2 WPM + AIB 4.0 mg / L + 60 g / L sucrose + 8 g / L agar).

[0107] Comparative Example 3:

[0108] The explants used for disinfection were segments of the teak clone 7029; the explants used for bud proliferation were segments of new buds grown from the explants in Example 1, which were then used as subsequent explants for bud proliferation experiments. Explants were detoxified according to the disinfection method described by Chen Xiaoming et al. in "Rapid Propagation Technology of Teak Stem Segment Tissue Culture" [J]. Guangxi Forestry Science, 2013, 42(04):319-323. The specific disinfection method was as follows: the explants were soaked in 0.1% HgCl2 for 4 min, rinsed twice with sterile water, and then soaked in 0.1% HgCl2 for 4 min. The optimal bud proliferation medium (MS + 6-BA 1.0 mg / L + 30 g / L sucrose + 3 g / L agar) was used for proliferation culture, and the optimal rooting culture (1 / 2MS + NAA 1.0 mg / L + IBA 0.5 mg / L + 30 g / L sucrose + 3 g / L agar) was used for rooting culture.

[0109] Comparative Example 4:

[0110] The explants used for disinfection were segments of the teak clone 7029; the explants used for bud proliferation were segments of new buds grown from the explants in Example 1, which were then used as subsequent explants for bud proliferation experiments. The explants were detoxified according to the disinfection method published by Li Xuedong et al., “The Effects of NAA and 6-BA on Tissue Culture of Black Teak” [J]. Subtropical Plant Science, 2017, 46(4):379-382. The specific disinfection method was as follows: first, surface disinfection with 70% ethanol for 1 min, then soaking in potassium permanganate solution for 20 min, soaking in 75% ethanol for 30 s, then soaking in 0.2% mercuric chloride (2-3 drops of Tween) solution for 3 min, and finally rinsing with sterile water 4-5 times. The optimal bud proliferation medium (MS + 0.5 mg / L NAA + 30 g / L sucrose + 12 g / L agar) was used for proliferation culture, and the optimal rooting medium (MS + 0.8 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 12 g / L agar) was used for rooting culture.

[0111] Comparative Example 5:

[0112] The explants used for disinfection were segments of the teak clone 7029; the explants used for bud proliferation were segments of new buds grown from the explants in Example 1, which were then used as subsequent explants for bud proliferation experiments. Explant detoxification was performed according to the disinfection method published by Aguilar ME et al. (Simple protocol for the micropropagation of teak (Tectona grandis linn.) in semi-solid and liquid media in RITA® bioreactors and ex vitro rooting[J]. American Journal of Plant Sciences, 2019, 10(7): 1121-1141.). Specifically, the stem tip material was immersed in a solution containing 3 g / L of mancozeb, benomyl, and agricultural streptomycin sulfate for 30 minutes, followed by rinsing three times with sterile distilled water. Subsequently, the stem tips were disinfected a second time with 10% and 8% calcium hypochlorite solutions, soaking for 20 minutes and 15 minutes respectively, followed by rinsing three times with sterile distilled water. The optimal bud proliferation medium (MS + 0.5 mg / L 6-BA + 0.5 mg / L KT + 30 g / L sucrose + 7 g / L agar) was used for proliferation culture. The rooting method was to take out the tissue culture seedlings, dip them in 0.3% IBA rooting powder, and then transplant them into the soil for in vitro rooting.

[0113] Comparative Example 6:

[0114] The explants used for disinfection were segments of the teak clone 7029; the explants used for bud proliferation were segments of new buds grown from the explants in Example 1, which were then used as subsequent explants for bud proliferation experiments. Explant detoxification was performed according to the disinfection method published by Kozgar et al. (Animproved protocol for micropropagation of teak tree (Tectona grandis L.)[J].Rendiconti Lincei, 2012, 23(2): 195-202.). The specific disinfection method was as follows: first, rinse with double-distilled water 4-5 times, then treat with 5% Bavastin bactericide, and then soak in Teepol detergent solution for 5 minutes. Under a clean bench, rinse with sterile distilled water 3-4 times, then soak in 70% alcohol for 30 seconds, disinfect with 1% (w / v) mercuric chloride solution for 3 minutes, and finally rinse thoroughly with sterile distilled water. The optimal bud proliferation medium (MS + 0.6 mg / LTDZ + 0.5 mg / L KT + 30 g / L sucrose + 8 g / L agar) was used for proliferation culture, and the optimal rooting medium (MS + 0.5 mg / L NAA + 30 g / L sucrose + 8 g / L agar) was used for rooting culture.

[0115] Table 10 Comparison Results

[0116]

[0117] Note: The number of explants used for disinfection was 30, the number of explants used for bud proliferation was 60, the number of tissue culture seedlings used for rooting was 180, and the number of tissue culture seedlings used for transplanting was 90.

[0118] As shown in Table 10, in Example 1, the teak clone 7029 had the highest bud proliferation rate and rooting rate, at 86.67% and 92.22%, respectively, and the rooted seedlings were robust with a transplant survival rate as high as 94.44%. In Comparative Examples 1-6, the bud proliferation rate of the teak clone 7029 was less than 30%, and the rooting rate was less than 45%; among them, no proliferating buds were obtained in Comparative Example 4, so subsequent rooting experiments could not be conducted.

[0119] In Example 1 and Comparative Examples 1-6, initial explants (segments) were used for virus elimination treatment, and the initial explants were collected from the nursery. Since no virus elimination method was provided in Comparative Example 2, relevant data could not be collected. In Comparative Examples 1, 3, and 4, no buds sprouted after virus elimination treatment of the initial explants (segments); in Comparative Examples 5 and 6, only one bud sprouted after virus elimination treatment of the initial explants (segments). Because the number of new buds produced in the comparative examples was too small to support subsequent experiments, the new buds produced from their respective explants were not used as explants in subsequent bud proliferation experiments. Instead, the new buds obtained in Example 1 were used as explants. Therefore, the explants used in Example 1 and Comparative Examples 1-6 were in the same state in the bud proliferation and rooting experiments, ensuring the reliability of the experimental data.

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A tissue culture rapid propagation method of Tectona grandis clone 7029, characterized by, Includes the following steps: (1) Pretreatment of explants: Take stem segments with only one pair of axillary buds as explants, rinse the explants with running water, clean the surface hairs, and then soak them in ascorbic acid solution to obtain pretreated explants; (2) Detoxification of explants: The pretreated explants were disinfected sequentially with 75% alcohol, mercuric chloride, and penicillin and streptomycin solutions to obtain detoxified explants; the alcohol disinfection time was 15±1 s; the mercuric chloride disinfection conditions were 0.2% mercuric chloride disinfection for 6 min; the penicillin and streptomycin solutions were solutions containing 90-100 mg / L penicillin and 90-100 mg / L streptomycin; the sterilization time was 5-7 min. (3) Sprouting and bud proliferation culture of explants: The virus-free explants were inoculated into MS basic medium for bud germination. After the buds elongated, the new buds were cut off and used as new explants for inoculation into bud proliferation medium for culture. The formula of the MS basic medium was MS + 30±0.5 g / L sucrose and 7±0.5 g / L agar, pH 5.8~6.

0. The formula of the bud proliferation medium was MS + 0.5 mg / L IAA + 0.5 mg / L 6-BA + 0.5 mg / L 2-iP + 80 mg / L inositol + 0.5±0.02 mg / L AgNO3 + 0.2±0.02 g / L water casein + 30±0.5 g / L sucrose + 7±0.5 g / L agar, pH 5.8~6.

0. (4) Rooting culture: After subculture, the tissue culture seedlings are inoculated into the rooting medium for rooting culture; the formula of the rooting medium is MS + 0.5 mg / L 6-BA + 1.0 mg / L IBA + 0.1 g / L activated carbon + 0.5 mg / L biotin + 30±0.5 g / L sucrose + 7±0.5 g / L agar, pH 5.8~6.

0.

2. The method for rapid propagation of teak clonal line 7029 by tissue culture according to claim 1, characterized in that, Also includes: (5) Hardening and transplanting: Harden and sterilize the rooted seedlings before transplanting.

3. The method for rapid propagation of teak clone 7029 via tissue culture according to claim 1 or 2, characterized in that: In step (3), the culture time is 25-30 days; In step (4), the rooting culture time is 30 to 35 days.

4. The method for rapid propagation of teak clone 7029 via tissue culture according to claim 1 or 2, characterized in that: In step (3), the culture conditions are 25±2℃, 2400~2500 lux light intensity, and 16 h of light per day; In step (4), the conditions for rooting culture are 25±2℃, 2400~2500 lux light intensity, and 16h light per day.

5. The method for rapid propagation of teak clone 7029 via tissue culture according to claim 1 or 2, characterized in that: In step (1), the rinsing time with running water is 30 to 50 minutes; the concentration of the ascorbic acid solution is 0.1 to 0.2 g / L; and the soaking time is 45 to 60 minutes.

6. The method for rapid propagation of teak clone 7029 by tissue culture according to claim 1 or 2, characterized in that: In step (4), the succession is 3-4 generations.

7. The method for rapid propagation of teak clone 7029 via tissue culture according to claim 2, characterized in that: In step (5), the seedling hardening time is 3 to 5 days; The sterilization process involves immersing the roots in a carbendazim solution diluted 1800–2000 times for 1–2 minutes. The substrate used for transplanting is a mixture of loess, peat moss, and vermiculite in a volume ratio of 3:3:

1.

8. The application of the rapid propagation method of teak clone 7029 by tissue culture according to any one of claims 1-7 in the rapid propagation of teak clone 7029 by tissue culture.

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