A culture medium and culture method for balsa wood callus culture

Abstract

This invention provides a culture medium and method for culturing balsa wood callus, belonging to the field of plant tissue culture technology. The culture medium, through a scientific combination of seed germination medium and callus induction medium, achieves highly efficient induction of balsa wood callus. Using 1 / 2 MS medium as the seed germination medium effectively improves the seed germination rate; the callus induction medium significantly enhances the callus induction rate while ensuring that the induced callus is loose in texture, grows vigorously, and exhibits no obvious browning. This solves the technical problems of easy browning, low induction efficiency, and poor stability in the callus induction process of balsa wood as a woody plant. The culture medium and method provided by this invention provide key technical support for the establishment of a balsa wood regeneration system, thereby helping to overcome the shortage of balsa wood raw materials in my country and providing important guarantees for the sustainable and healthy development of the wind power industry and related fields.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a culture medium and culture method for culturing balsa wood callus. Background Technology

[0002] Balsa wood (scientific name: Ochroma lagopus Balsa wood (Swiss.) is an evergreen tree belonging to the genus Balsa in the family Malvaceae. Native to tropical America, it is also known as balsa wood or basil wood. Balsa wood thrives in warm, humid conditions and grows well in deep, fertile, well-drained alluvial or loamy soils. It is typically propagated by seed. Its dry density is only 0.12 g / cm³. 3 Wood is the lightest wood in the world and has a short growth cycle, making it one of the fastest-growing tree species. With its excellent strength-to-weight ratio, good elasticity, and impact resistance, it has wide applications in aerospace, shipbuilding, and high-end model making. Balsa wood, as a key core material for wind turbine blades, can effectively reduce the blade's rotational inertia, improve wind energy conversion efficiency, and enhance the blade's structural stability and fatigue resistance, making it an indispensable core material for large-scale wind turbine blades. However, balsa wood has demanding requirements for growing climate conditions, is not very cold-resistant, and is easily affected by frost damage. The success rate of large-scale cultivation is low, and commercial production capacity has not yet been formed.

[0003] Therefore, overcoming the supply difficulties of balsa wood raw materials and establishing a localized breeding system are crucial to ensuring the development of the wind power industry. Traditional seed propagation methods have significant limitations: balsa wood seeds have a germination rate of only 30% to 50%, resulting in low propagation efficiency, and seedlings are prone to trait segregation, making it difficult to meet the demand for high-quality seedlings in large-scale production. Tissue culture technology, as an efficient asexual reproduction method, can quickly achieve the mass propagation of superior germplasm, and callus induction is the core first step in establishing a balsa wood regeneration system. However, tissue culture of woody plants generally faces technical bottlenecks such as severe browning, low regeneration rate, and strong genotype dependence, and research on callus induction in balsa wood is particularly weak. Existing studies mostly focus on shoot proliferation and rooting culture, lacking systematic exploration of key technical parameters for callus induction. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide a culture medium and culture method for culturing balsa wood callus, which can achieve efficient induction of balsa wood callus.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a culture medium for balsa wood callus culture, comprising a seed germination medium and a callus induction medium; The seed germination medium is based on 1 / 2 MS medium and also includes 30 g / L sucrose and 7 g / L agar powder; the pH value of the seed germination medium is 5.8~5.9. The callus induction medium is based on MS medium and further includes: 6-BA 0.05~0.2 mg / L, 2,4-D 0.1~0.2 mg / L, sucrose 30 g / L and agar powder 7 g / L; or, based on MS medium and further includes: 2,4-D 0.1~0.2 mg / L, KT 0.1 mg / L, sucrose 30 g / L and agar 7 g / L; the pH of the callus induction medium is 5.8~5.9.

[0006] Preferably, the culture medium further includes a callus proliferation culture medium; the callus proliferation culture medium is based on MS medium and further includes: 6-BA 0.05~0.2 mg / L, 2,4-D 0.1~0.2 mg / L, sucrose 30 g / L and agar 7 g / L; or, based on MS medium, it further includes: 2,4-D 0.1~0.2 mg / L, KT 0.1 mg / L, sucrose 30 g / L and agar 7 g / L; the pH value of the callus proliferation culture medium is 5.8~5.9.

[0007] This invention provides the application of the culture medium described in the above technical solution in the culture of balsa wood callus.

[0008] This invention provides a method for improving the germination rate of balsa seeds, comprising: After soaking the balsa seeds in water, they were subjected to a water bath treatment to obtain water bath-treated seeds. Disinfect the seeds after water bath treatment; The sterilized seeds are inoculated into the seed germination medium described in the above technical solution for germination culture.

[0009] Preferably, the water bath temperature is 55°C and the water bath time is 50 minutes.

[0010] Preferably, the soaking temperature is 18~25℃; the soaking time is 8~14h.

[0011] Preferably, the disinfection process includes a first disinfection with an ethanol solution followed by a second disinfection with a NaClO solution.

[0012] This invention provides a method for culturing balsa wood callus, using the culture medium described in the above-mentioned technical solution as the culture medium, comprising the following steps: Cotyledons with petioles were taken from balsa seedlings and inoculated into callus induction medium for induction culture to obtain balsa callus tissue; The balsa wood callus was inoculated into a callus proliferation medium for proliferation culture.

[0013] Preferably, the induction culture temperature is 28℃; the induction culture time is 20~30 days; the induction culture is a dark culture; the proliferation culture temperature is 28℃; the proliferation culture is subcultured every 30 days; and the proliferation culture is a dark culture.

[0014] Preferably, the petiolate cotyledons include petiolate cotyledons with the half-cotyledon removed from the end of the petiole and / or cotyledons cut in half.

[0015] The beneficial effects of this invention are: This invention provides a culture medium for balsa wood callus culture, comprising a seed germination medium and a callus induction medium. The seed germination medium uses 1 / 2 MS medium as its base, further comprising 30 g / L sucrose and 7 g / L agar powder; the pH of the seed germination medium is 5.8–5.9. The callus induction medium uses MS medium as its base, further comprising: 0.05–0.2 mg / L 6-BA, 0.1–0.2 mg / L 2,4-D, 30 g / L sucrose, and 7 g / L agar; or, using MS medium as its base, further comprising: 0.1–0.2 mg / L 2,4-D, 0.1 mg / L KT, 30 g / L sucrose, and 7 g / L agar; the pH of the callus induction medium is 5.8–5.9. This culture medium, through the scientific combination of seed germination medium and callus induction medium, achieves highly efficient induction of balsa wood callus. The 1 / 2 MS medium used as the seed germination medium provides suitable nutrition and a suitable growth environment for seeds, effectively improving the germination rate and promoting robust growth of balsa seedlings, thus laying a good material foundation for subsequent callus induction. The callus induction medium, based on MS medium, employs a combination of two hormones in specific ratios. By regulating the concentration ratio of auxin and cytokinin, it effectively stimulates the callus differentiation potential of balsa seedling explants with petioles and cotyledons, significantly improving the callus induction rate. It also ensures that the induced callus is loose in texture, grows vigorously, and exhibits no obvious browning, solving the technical problems of easy browning, low induction efficiency, and poor stability in the callus induction process of balsa as a woody plant. The culture medium and cultivation method provided by this invention provide key technical support for the establishment of a balsa regeneration system, thereby helping to overcome the shortage of balsa raw material supply and providing important guarantees for the sustainable and healthy development of the wind power industry and related fields. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 This is a diagram showing the callus induction process in Example 2; Figure 2 This is a diagram showing the results of callus proliferation culture in Example 2; Figure 3 This is a diagram showing the callus induction process in Example 3; Figure 4 This is a diagram showing the callus induction process in Example 4; Figure 5 This is a diagram showing the callus induction process in Example 5; Figure 6 This is a diagram showing the callus induction process in Example 6; Figure 7 This is a diagram showing the callus induction process in Example 7; Figure 8 This is a diagram showing the callus induction process in Example 8; Figure 9 This is a diagram showing the callus induction process in Example 9; Figure 10 This is a diagram showing the callus induction process in Example 11; Figure 11 This is a diagram showing the callus induction in Example 12 when the hormone concentrations were 0.2 mg / L 2,4-D and 0.5 mg / L 6-BA in MS medium. Figure 12 This is a diagram showing the callus induction in Example 12 when the hormones were 0.2 mg / L 2,4-D and 1.0 mg / L 6-BA in MS medium. Figure 13 This is a diagram showing the callus induction process in Example 14; Figure 14 This is a diagram of callus subculture and proliferation in Example 14. Detailed Implementation

[0018] This invention provides a culture medium for balsa wood callus culture, comprising a seed germination medium and a callus induction medium; The seed germination medium is based on 1 / 2 MS medium and also includes 30 g / L sucrose and 7 g / L agar powder; the pH value of the seed germination medium is 5.8~5.9. The callus induction medium is based on MS medium and further includes: 6-BA 0.05~0.2 mg / L, 2,4-D 0.1~0.2 mg / L, sucrose 30 g / L and agar 7 g / L; or, based on MS medium and further includes: 2,4-D 0.1~0.2 mg / L, KT 0.1 mg / L, sucrose 30 g / L and agar 7 g / L; the pH of the callus induction medium is 5.8~5.9.

[0019] In this invention, the callus induction medium is based on MS medium, and further supplemented with: 0.05~0.2 mg / L 6-BA, 0.1~0.2 mg / L 2,4-D, 30 g / L sucrose, and 7 g / L agar. As an optional embodiment of this invention, the concentration of 6-BA in the callus induction medium can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg / L; the concentration of 2,4-D in the callus induction medium can be 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg / L.

[0020] In this invention, the callus induction medium is based on MS medium, and further supplemented with: 0.1-0.2 mg / L 2,4-D, 0.1 mg / L KT, 30 g / L sucrose, and 7 g / L agar. As an optional embodiment of this invention, the concentration of 2,4-D in the callus induction medium can be 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg / L.

[0021] The seed germination medium provided by this invention provides suitable nutrition and a suitable growth environment for seeds, effectively improving seed germination rate, promoting robust growth of balsa seedlings, and laying a good material foundation for subsequent callus induction. In the callus induction medium provided by this invention, 2,4-D can induce explant dedifferentiation, promote rapid cell division and proliferation, inhibit premature bud differentiation, and thus facilitate callus formation. The 6-BA and 2,4-D work synergistically to promote cell division, significantly enhancing cell division activity, resulting in faster callus growth, a denser and more uniform structure, delayed cell senescence, regulation of dedifferentiation direction, and improved callus quality. KT promotes cell division and increases the induction success rate. The callus induction medium provided by this invention is based on MS medium and uses a combination of two hormones in a specific ratio. By regulating the concentration ratio of auxin and cytokinin, it can effectively stimulate the callus differentiation potential of balsa seedling explants with petioles and cotyledons, significantly improve the callus induction rate, and ensure that the induced callus is loose in texture, grows vigorously, and has no obvious browning phenomenon. This solves the technical problems of easy browning, low induction efficiency, and poor stability of balsa as a woody plant callus induction process.

[0022] As an optional embodiment of the present invention, the culture medium further includes a callus proliferation culture medium; the callus proliferation culture medium is based on MS medium and further includes: 6-BA 0.05~0.2 mg / L, 2,4-D 0.1~0.2 mg / L, sucrose 30 g / L and agar 7 g / L; or, based on MS medium, it further includes: 2,4-D 0.1~0.2 mg / L, KT 0.1 mg / L, sucrose 30 g / L and agar 7 g / L; the pH value of the callus proliferation culture medium is 5.8~5.9.

[0023] In this invention, the callus proliferation medium is based on MS medium, and further supplemented with: 0.05~0.2 mg / L 6-BA, 0.1~0.2 mg / L 2,4-D, 30 g / L sucrose, and 7 g / L agar. As an optional embodiment of this invention, the concentration of 6-BA in the callus proliferation medium can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg / L; the concentration of 2,4-D in the callus proliferation medium can be 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg / L.

[0024] In this invention, the callus proliferation medium is based on MS medium, and further supplemented with: 0.1-0.2 mg / L 2,4-D, 0.1 mg / L KT, 30 g / L sucrose, and 7 g / L agar. As an optional embodiment of this invention, the concentration of 2,4-D in the callus proliferation medium can be 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20 mg / L.

[0025] This invention provides the application of the culture medium described in the above-mentioned technical solution in the culture of balsa wood callus. The culture medium provided by this invention, when used with appropriate culture methods, can significantly improve the callus induction rate while ensuring that the induced callus is loose in texture, grows vigorously, and shows no obvious browning.

[0026] This invention provides a method for improving the germination rate of balsa seeds, comprising: After soaking the balsa seeds in water, they were subjected to a water bath treatment to obtain water bath-treated seeds. Disinfect the seeds after water bath treatment; The sterilized seeds were inoculated into the seed germination medium described in the above technical solution and cultured.

[0027] This invention involves soaking balsa seeds in water followed by a water bath treatment to obtain water-bath treated seeds. Preferably, the balsa seeds are plump, intact, with clean seed coats, and free from mold, insect infestation, and damage. The balsa seeds can be screened using a submersion method. After obtaining the balsa seeds, they are soaked in water to obtain a balsa seed-water mixture; the water can be distilled water; the soaking temperature can be 18-25℃, or 18, 19, 20, 21, 22, 23, 24, or 25℃; the soaking time can be 8-14 hours, or 8, 9, 10, 11, 12, 13, or 14 hours, or overnight. After soaking, the balsa seed-water mixture is subjected to a water bath treatment to obtain water-bath treated seeds. The water bath temperature can be 55℃; the water bath time can be 50 minutes. After the water bath treatment, water-bath treated seeds are obtained.

[0028] After obtaining the water bath-treated seeds, this invention further disinfects them. The disinfection process includes a first disinfection with an ethanol solution followed by a second disinfection with a NaClO solution. The ethanol solution can be a 75% v / v aqueous ethanol solution; the first disinfection time can be 30-60 seconds, or 30, 35, 40, 45, 50, 55, or 60 seconds. After the first disinfection, the ethanol solution is discarded, and the seeds are rinsed with sterile water to remove residual ethanol; preferably, sterile water is used for rinsing. After rinsing, the seeds are then disinfected a second time with a NaClO solution. The NaClO solution can be a 10%-20% NaClO solution; the second disinfection time can be 30 minutes. During the second disinfection process, the disinfection system is preferably shaken to ensure full contact between the seeds and the disinfectant. After the second disinfection, the NaClO solution is discarded, and the seeds are rinsed with sterile water to remove residual NaClO solution. After rinsing, the pretreated seeds are obtained.

[0029] After obtaining pretreated seeds, the present invention inoculates the pretreated seeds into a seed germination medium for cultivation to obtain balsa seedlings. The present invention does not specifically limit the inoculation method; any conventional inoculation method in the art can be used. As an optional embodiment of the present invention, the inoculation method may involve embedding approximately one-third of the seed tip into the culture medium. In the present invention, the cultivation temperature can be 28°C; the photoperiod can be 16 hours of light and 8 hours of darkness per day; the cultivation time can be 3–10 days, or 3, 4, 5, 6, 7, 8, 9, or 10 days. After cultivation, the present invention obtains balsa seedlings. The cultivation method of the present invention can significantly improve the germination rate of balsa seeds.

[0030] This invention provides a method for culturing balsa wood callus, using the culture medium described in the above-mentioned technical solution as the culture medium, comprising the following steps: Cotyledons with petioles were taken from balsa seedlings and inoculated into callus induction medium for induction culture to obtain balsa callus tissue; The balsa wood callus was inoculated into a callus proliferation medium for proliferation culture.

[0031] This invention preferably employs the method described above for improving the germination rate of balsa seeds to cultivate balsa seedlings. After obtaining balsa seedlings, the cotyledons with petioles of the balsa seedlings are inoculated into a callus induction medium for induction culture to obtain balsa callus. As an optional embodiment of this invention, the cotyledons with petioles include those with the half-cotyledon removed from the end furthest from the petiole. This invention does not have a specific limitation on the inoculation method; any conventional inoculation method in the art can be used. As an optional embodiment of this invention, the inoculation method can be to lay the cotyledons with petioles facing down on the callus induction medium, ensuring sufficient contact between them and the medium. In this invention, the induction culture temperature can be 28℃; the induction culture time can be 20-30 days, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days; the induction culture is a dark culture. This invention can rapidly obtain callus through the induction culture, and the obtained callus exhibits good growth.

[0032] After obtaining balsa wood callus, this invention inoculates the balsa wood callus into a callus proliferation culture medium for proliferation culture. This invention does not have a specific limitation on the inoculation method; any conventional inoculation method in the art can be used. As an optional embodiment of this invention, the inoculation method can be to use forceps to pick up a piece of highly active outer tissue, about the size of a soybean, and place it into the callus proliferation culture medium. In this invention, the proliferation culture temperature can be 28℃; the proliferation culture can be subcultured every 30 days; and the proliferation culture is conducted in the dark. This invention, through the proliferation culture, can cause a large proliferation of callus clusters with good growth.

[0033] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] The balsa seeds were screened using the water immersion method. The balsa seeds with plump kernels, intact shape, clean seed coat, and no mold, insect infestation, or damage were selected for subsequent experiments.

[0035] KT stands for Kinetin; 6-BA stands for 6-benzylaminopurine; and 2,4-D stands for 2,4-dichlorophenoxyacetic acid.

[0036] Example 1 Seed treatment method screening 1. Take 300 balsa seeds and divide them into 3 experimental groups, with 100 seeds in each group.

[0037] Experiment 1: Balsa seeds were soaked in distilled water at room temperature overnight. After soaking, the seed-distilled water mixture was subjected to a water bath treatment at 55°C for 50 minutes. After the water bath treatment, the distilled water was discarded.

[0038] Experiment 2: Prepare a 10% PEG-6000 solution and soak the seeds for 12 hours. Air dry naturally to their original weight.

[0039] Experimental Group 3: Seeds were soaked in a 100 mg / L GA3 solution for 12 hours and then air-dried to their original weight.

[0040] The seeds treated in each of the above treatment groups were disinfected and inoculated, and the germination rate was observed.

[0041] 2. Seed disinfection: In a clean bench, the surface was disinfected with 75% ethanol solution for 1 min. After discarding the ethanol, the seeds were rinsed with sterile water to remove any residual ethanol. Then, the seeds were disinfected with 10% NaClO solution for 30 min, shaking several times during the process to ensure that the seeds were in full contact with the disinfectant. The disinfectant was then discarded, and the seeds were rinsed again with sterile water to remove any residual disinfectant.

[0042] 3. Vaccination: After sterilization, blot the seeds dry on sterile filter paper. Under aseptic conditions, use tweezers to inoculate the seeds into disposable sterile petri dishes. The culture medium is 1 / 2 MS solid medium (components: 2.17 g / L 1 / 2 MS powder, 30 g / L sucrose, 7 g / L agar powder, pH 5.8–5.9). When inoculating, ensure that approximately one-third of the seed tip is embedded in the medium. Place the petri dishes in a light incubator at 28°C and a light intensity of 180 μmol / m². 2 / s, with a photoperiod of 16 h light / 8 h darkness. Seeds entered a concentrated germination period after 3-7 days. Germination was recorded for different seed treatments, and the germination rate was calculated. Seeds that showed signs of sprouting and were uncontaminated were transferred to tissue culture bottles containing 1 / 2 MS medium for further cultivation. After about 10 days, when the seedlings had grown to the stage where the two cotyledons were fully expanded but the true leaves had not yet developed, healthy seedlings with consistent growth (i.e., balsa seedlings) were selected as materials for subsequent experiments.

[0043] 4. Results The germination rates of seeds treated in Experiments 1 through 3 are shown in Table 1.

[0044] Table 1 Seed germination rate

[0045] The results in Table 1 show that the highest germination rate was achieved by treating the food in a water bath at 55℃ for 50 min.

[0046] Example 2 1. Vaccination The balsamina seedlings cultured in Example 1 were removed from the tissue culture flasks. Under aseptic conditions, cotyledons with petioles were cut using a sterile scalpel as material for callus induction. Subsequently, the half-cotyledon furthest from the petiole was removed, and the remaining explants (including the half-cotyledon with petiole) were laid face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium was MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), and different plant hormone gradients were set up. The concentrations of different plant hormones are detailed in Table 2. At least 5 explants were inoculated into each culture dish, and 5 replicates were set up for each treatment. After aliquoting, the pH of all media was adjusted to 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0047] Table 2. Different plant hormone concentration settings

[0048] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0049] 3. Callus induction status Callus induction was observed during the culture process. After 30 days of culture, the callus induction rate was calculated. The callus induction status of treatment groups 1-5 after 30 days of culture is shown below. Figure 1 As shown.

[0050] Callus induction rate = (number of explants that form callus / number of inoculated explants × 100%).

[0051] Statistical requirements: The induced callus should form distinct clumps, be white or pale yellow in color, and not brown over time.

[0052] During callus induction, significant deformation occurred at the cotyledon cut site after 5 days of induction culture, indicating the start of callus induction. After 20 days, the experimental group treated with 0.2 mg / L 2,4-D + 0.1 mg / L 6-BA formed visible callus clusters, which were pale yellow in color. With increasing 2,4-D concentration, increasingly severe vitrification was observed in the leaves, and no obvious callus clusters appeared. Furthermore, when the 2,4-D concentration increased to 2 mg / L, browning and death of the cotyledons occurred.

[0053] The statistical results of callus induction rate after 30 days of culture are shown in Table 3.

[0054] Table 3. Statistical results of callus induction rate

[0055] Based on the induction statistics and callus formation status, significant differences were observed among groups 1, 2, 3, 4, and 5. The optimal induction hormone concentration was 0.2 mg / L 2,4-D + 0.1 mg / L 6-BA. With increasing 2,4-D concentration, the callus induction rate decreased and the callus condition worsened; therefore, high concentrations of 2,4-D were the main factor affecting the differences in induction rates.

[0056] 6. Callus proliferation culture Callus induced by a combination of 0.2 mg / L 2,4-D and 0.1 mg / L 6-BA was collected by forceps, and the outermost, highly viable tissue, approximately the size of a soybean, was placed on a fresh culture medium containing the same hormones. The callus was cultured in the dark at 28°C under light. The callus proliferated significantly and grew well, and was subcultured every 30 days. The resulting callus was shown in the image. Figure 2 As shown.

[0057] Example 3 Investigating the effects of different explants on callus induction 1. Vaccination The balsam seedlings cultured in Example 1 were removed from the tissue culture flasks, and hypocotyls were excised under aseptic conditions using a sterile scalpel as material for callus induction. The hypocotyls were placed horizontally on the callus induction medium to ensure adequate contact with the medium. The callus induction medium was MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), with different plant hormone gradients. Details of the different plant hormone concentrations are shown in Table 2. At least five explants were inoculated into each culture dish, and five replicates were set up for each treatment. After aliquoting, all media were adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0058] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0059] 3. Callus induction status Three days later, the hypocotyl showed obvious deformation and swelling at the incision site, indicating the start of callus induction. Around 20 days later, callus clusters accumulated at both ends of the hypocotyl, appearing pale yellow in color. With increasing 2,4-D concentration, the swelling at both ends of the hypocotyl ceased, and vitrification and browning occurred. The callus induction status of treatment groups 1-5 at 30 days of culture is shown below. Figure 3 As shown.

[0060] 4. After culturing for 30 days, calculate the callus induction rate. See Table 4 for details of the callus induction rate.

[0061] Table 4 Callus induction rate

[0062] The results showed that the above hormone ratios were all poor for callus induction of hypocotyls, with low induction rates and numerous vitrification and browning phenomena.

[0063] 5. Callus proliferation The induced callus clusters were used to pick off the outermost, highly active tissue, about the size of a soybean, and placed on a new culture medium with the same hormones. The callus clusters were found to proliferate rapidly and grow well, requiring subculturing every 30 days.

[0064] Example 4 Investigating the effect of sterile water immersion on callus induction in balsa seedlings 1. Vaccination The balsa seedlings cultured in Example 1 were opened in a clean bench and filled with sterile water to submerge them for 1 hour. The seedlings were then removed, the cotyledons were cut off, and the half of the cotyledon furthest from the petiole was removed. The surface water was then blotted dry with sterile filter paper, and the seedlings were placed face down on callus induction medium (MS medium as the basal medium) containing 0.2 mg / L 2,4-D + 0.1 mg / L 6-BA. At least 5 explants were inoculated into each culture dish, and 5 replicates were set up for each treatment.

[0065] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0066] 3. Callus induction status Callus clusters appeared on the cotyledonary leaves after about 20 days. However, waterlogging increased vitrification of the leaves, affecting callus induction. The callus induction status after 30 days of culture is as follows: Figure 4 As shown.

[0067] After 30 days of culture, the callus induction rate was 62.2% (number of explants that formed callus / number of inoculated explants × 100%).

[0068] 4. Callus proliferation The induced callus clusters were used to pick off the outermost, highly active tissue, about the size of a soybean, and placed on a new culture medium with the same hormones. The callus clusters were found to proliferate rapidly and grow well, requiring subculturing every 30 days.

[0069] Example 5 Investigating the effects of different cytokinin KT relative to 6-BA on callus induction 1. Vaccination The cultured balsamic seedlings were removed from the tissue culture flasks, and under aseptic conditions, cotyledons with petioles were excised using a sterile scalpel as material for callus induction. The cotyledons were then cut in half, and the explants were laid face down on the callus induction medium to ensure adequate contact with the medium. The callus induction medium was MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), with the addition of 0.2 mg / L 2,4-D and 0.1 mg / L KT. At least five explants were inoculated into each culture dish, and five replicates were set up for each treatment. After aliquoting, all media were adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0070] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0071] 3. Callus induction status Five days later, significant deformation occurred at the cotyledon incision site, indicating the start of callus induction. Callus tissue clusters formed at the incision site after 20 days of culture. The callus induction status at 30 days of culture is as follows... Figure 5 As shown.

[0072] After 30 days of culture, the callus induction rate was 90.4% (number of explants that formed callus / number of inoculated explants × 100%).

[0073] Example 6 Investigating the effect of NAA on callus induction 1. Remove the cultured balsam seedlings from the tissue culture flasks. Under aseptic conditions, use a sterile scalpel to cut off the cotyledons with petioles as material for callus induction. Then, remove the half of the cotyledon furthest from the petiole, and lay the remaining explant (including the half of the cotyledon with petiole) face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium is MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), supplemented with 0.2 mg / L NAA + 0.1 mg / L 6-BA. At least 5 explants are inoculated into each culture dish, and 5 replicates are set for each treatment. After aliquoting, all media are adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0074] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0075] 3. Callus induction status Twenty days later, no obvious callus tissue had formed at the cotyledon incision site, such as Figure 6 As shown.

[0076] Example 7 Investigating the effect of reducing 2,4-D concentration on callus induction 1. Remove the cultured balsam seedlings from the tissue culture flasks. Under aseptic conditions, use a sterile scalpel to cut off the cotyledons with petioles as material for callus induction. Then, remove the half of the cotyledon furthest from the petiole, and lay the remaining explant (including the half of the cotyledon with the petiole) face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium is MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), supplemented with 0.1 mg / L 2,4-D + 0.1 mg / L 6-BA. At least 5 explants are inoculated into each culture dish, and 5 replicates are set for each treatment. After aliquoting, all media are adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0077] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0078] 3. Callus induction status Five days later, obvious deformation occurred at the cotyledon incision site, indicating the start of callus induction. Twenty days later, visible callus clusters formed, pale yellow in color. Callus induction progressed as follows after 30 days of culture. Figure 7 As shown.

[0079] After 30 days of culture, the callus induction rate was 94.2% (number of explants that formed callus / number of inoculated explants × 100%).

[0080] Example 8 Investigating the effect of reducing 2,4-D concentration on callus tissue 1. Remove the cultured balsam seedlings from the tissue culture flasks. Under aseptic conditions, use a sterile scalpel to cut off the cotyledons with petioles as material for callus induction. Then, remove the half-cotyledon furthest from the petiole end, and lay the remaining explant (including the half-cotyledon with petiole) face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium is MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), supplemented with 0.05 mg / L 2,4-D + 0.1 mg / L 6-BA. At least 5 explants are inoculated into each culture dish, and 5 replicates are set for each treatment. After aliquoting, all media are adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0081] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0082] 3. Callus induction status Under conditions of 0.05 mg / L 2,4-D, callus induction became difficult, and no obvious callus clusters were produced after 30 days of culture. The results were as follows: Figure 8 As shown.

[0083] Example 9 Investigating the effect of adding only 6-BA on callus induction 1. Remove the cultured *Clerodendrum trichotomum* seedlings from the tissue culture flasks. Under aseptic conditions, use a sterile scalpel to cut off the cotyledons with petioles as material for callus induction. Then, remove the half-cotyledon furthest from the petiole end, and lay the remaining explant (including the half-cotyledon with petiole) face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium is MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), with 0.1 mg / L 6-BA added. At least 5 explants are inoculated into each culture dish, and 5 replicates are set for each treatment. After aliquoting, all media are adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0084] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0085] 3. Callus induction status Under conditions of 0.1 mg / L 6-BA, callus induction became difficult, and no obvious callus tissue clusters were produced. The callus induction status after 30 days of culture was as follows: Figure 9 As shown.

[0086] Example 10 Investigating the effect of adding only 6-BA on callus induction 1. Remove the cultured balsam seedlings from the tissue culture flasks. Under aseptic conditions, use a sterile scalpel to cut off the cotyledons with petioles as material for callus induction. Then, remove the half-cotyledon furthest from the petiole end, and lay the remaining explant (including the half-cotyledon with petiole) face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium is MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), with 0.5 mg / L 6-BA added. At least 5 explants are inoculated into each culture dish, and 5 replicates are set for each treatment. After aliquoting, all media are adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0087] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0088] 3. Callus induction status Under the condition of 0.1 mg / L 6-BA, callus induction becomes difficult and callus tissue cannot be generated.

[0089] Example 11 1. Remove the cultured balsamic seedlings from the tissue culture flasks. Under aseptic conditions, use a sterile scalpel to cut off the cotyledons with petioles as material for callus induction. Then, remove the half-cotyledon furthest from the petiole end, and lay the remaining explant (including the half-cotyledon with petiole) face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium is MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), with hormones listed in Table 5 added. At least 5 explants are inoculated into each culture dish, and 5 replicates are set for each treatment. After aliquoting, all media are adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0090] Table 5. Different plant hormone concentration settings

[0091] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0092] 3. Callus induction Five days later, obvious deformation occurred at the cotyledon incision site, indicating the start of callus induction. Twenty days later, visible callus clusters formed, pale yellow in color. Callus induction progressed as follows after 30 days of culture. Figure 10 As shown in the figure, from left to right, they correspond to processing groups 1, 2, 3, and 4.

[0093] The callus induction rate after 30 days is detailed in Table 6.

[0094] Table 6 Callus induction rate in different treatment groups

[0095] Example 12 Investigating the effect of high concentrations of 6-BA on callus induction 1. Vaccination The balsamina seedlings cultured above were removed from the tissue culture flasks. Under aseptic conditions, cotyledons with petioles were excised using a sterile scalpel as material for callus induction. Subsequently, the half of the cotyledon furthest from the petiole was removed, and the remaining explant (including the half of the cotyledon with the petiole) was laid face down on the callus induction medium to ensure sufficient contact with the medium. The callus induction medium was MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), with 0.2 mg / L 2,4-D + 0.5 mg / L 6-BA and 0.2 mg / L 2,4-D + 1.0 mg / L 6-BA added, respectively. At least 5 explants were inoculated into each culture dish, and 5 replicates were set for each treatment. After aliquoting, the pH of all media was adjusted to 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0096] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0097] 3. Callus induction The induced callus is hard, and the callus becomes harder with increasing 6-BA concentration.

[0098] The callus induction rate was 55.3% in MS medium with hormone concentrations of 0.2 mg / L 2,4-D and 0.5 mg / L 6-BA. For details on callus induction, please refer to [link to relevant documentation]. Figure 11 In MS medium with hormone concentrations of 0.2 mg / L 2,4-D and 1.0 mg / L 6-BA, the callus induction rate was 47.9%. For details on callus induction, please refer to [link to relevant documentation]. Figure 12 .

[0099] Example 13 The effect of temperature on callus condition 1. Vaccination The cultured balsamic seedlings were removed from the tissue culture flasks, and under aseptic conditions, cotyledons with petioles were excised using a sterile scalpel as material for callus induction. The cotyledons were then cut in half, and the explants were laid face down on the callus induction medium to ensure adequate contact with the medium. The callus induction medium was MS-based (MS powder 4.43 g / L, sucrose 30 g / L, agar powder 7 g / L), supplemented with 0.2 mg / L 2,4-D and 0.1 mg / L 6-BA. At least five explants were inoculated into each culture dish, and five replicates were set up for each treatment. After aliquoting, all media were adjusted to pH 5.80–5.90 with 1 mol / L NaOH solution and autoclaved at 121°C for 15 minutes.

[0100] 2. Explant culture The explants were placed in light incubators at 26℃ and 28℃ respectively, and cultured in the dark. Their status was recorded.

[0101] 3. Callus induction status After 30 days, it was found that the amount of callus tissue was greater under the condition of 28°C.

[0102] Table 7 shows the callus induction under optimal or corresponding conditions in Examples 2-12.

[0103] Table 7. Callus induction under optimal or corresponding conditions in Examples 2-12

[0104] Based on Example 2, subsequent experiments were compared with it. It was found that cotyledons are more suitable as explants for balsa wood callus induction than hypocotyls; sterile water submersion has a counterproductive effect on callus induction in balsa wood cotyledons; and cytokinin 6-BA and KT show little difference during the balsa wood cotyledon callus induction stage.

[0105] Example 14 1. Vaccination Two months after culture, healthy balsa seedlings were taken from the tissue culture bottle. Under aseptic conditions, the leaves were cut into 1cm×1cm pieces with a sterile scalpel and placed in the culture media listed in Table 8. The basal culture medium was MS.

[0106] Table 8. Culture medium composition

[0107] At least five explants were inoculated into each culture dish, and five replicates were set up for each treatment. After aliquoting, the pH of all culture media was adjusted to 5.80–5.90 with 1 mol / L NaOH solution and then autoclaved at 121°C for 15 minutes.

[0108] 2. Explant culture The explants were placed in a light incubator at 28°C and cultured in the dark, and their status was recorded.

[0109] 3. Callus induction status The callus induction was observed during the culture process. After 30 days of culture, the callus induction rate was calculated. At 30 days, treatments 2-6 failed to induce callus, while treatment 1 showed a callus induction rate of 60%. The callus growth was as follows. Figure 13 As shown.

[0110] 4. Callus proliferation culture Using tweezers, a small piece of highly viable outer tissue, about the size of a soybean, was taken from the callus mass obtained from treatment #1 and placed on a new culture medium containing the same hormone. The callus was then cultured in the dark at 28°C. Repeated browning was observed in the subcultured callus masses, even after the addition of 1 g / L activated charcoal. Figure 14 As shown.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for culturing balsa wood callus, characterized in that, Including seed germination medium and callus induction medium; The seed germination medium is based on 1 / 2 MS medium, and also includes 30 g / L sucrose and 7 g / L agar powder; The pH value of the seed germination medium is 5.8~5.9; The callus induction medium is based on MS medium and further includes: 6-BA 0.05~0.2 mg / L, 2,4-D 0.1~0.2 mg / L, sucrose 30 g / L and agar 7 g / L; or, based on MS medium and further includes: 2,4-D 0.1~0.2 mg / L, KT 0.1 mg / L, sucrose 30 g / L and agar 7 g / L; the pH of the callus induction medium is 5.8~5.

9.

2. The culture medium according to claim 1, characterized in that, The culture medium further includes a callus proliferation medium; the callus proliferation medium is based on MS medium and further includes: 6-BA 0.05~0.2 mg / L, 2,4-D 0.1~0.2 mg / L, sucrose 30 g / L and agar 7 g / L; or, based on MS medium, it further includes: 2,4-D 0.1~0.2 mg / L, KT 0.1 mg / L, sucrose 30 g / L and agar 7 g / L; the pH of the callus proliferation medium is 5.8~5.

9.

3. The application of the culture medium described in claim 1 or 2 in the culture of balsa wood callus.

4. A method for improving the germination rate of balsa seeds, characterized in that, include: After soaking the balsa seeds in water, they were subjected to a water bath treatment to obtain water bath-treated seeds. Disinfect the seeds after water bath treatment; The sterilized seeds are inoculated into the seed germination medium of claim 1 and cultured.

5. The method according to claim 4, characterized in that, The water bath treatment temperature is 55℃; the water bath treatment time is 50 minutes.

6. The method according to claim 4, characterized in that, The soaking temperature is 18~25℃; the soaking time is 8~14h.

7. The method according to claim 4, characterized in that, The disinfection process includes a first disinfection using an ethanol solution, followed by a second disinfection using a NaClO solution.

8. A method for culturing balsa wood callus, characterized in that, Using the culture medium described in claim 1 or 2 as the culture medium, the method includes the following steps: Cotyledons with petioles were taken from balsa seedlings and inoculated into callus induction medium for induction culture to obtain balsa callus tissue; The balsa wood callus was inoculated into a callus proliferation medium for proliferation culture.

9. The method according to claim 8, characterized in that, The induction culture temperature is 28℃; the induction culture time is 20~30 days; the induction culture is a dark culture; the proliferation culture temperature is 28℃; the proliferation culture is subcultured every 30 days; the proliferation culture is a dark culture.

10. The method according to claim 8, characterized in that, The petiolate cotyledons include petiolate cotyledons with the half-cotyledon removed from the end of the petiole and / or cotyledons cut in half.

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