Induction culture medium for embryogenic callus of rubber tree variety heat reclamation 628, method for inducing somatic embryogenesis by induction culture medium and application of induction culture medium

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to an induction culture medium for embryogenic calluses of rubber tree variety thermal reclamation 628, a method for inducing somatic embryogenesis by using the induction culture medium and application of the induction culture medium. According to the induction culture medium, an improved MS culture medium is used as a basic culture medium, and the induction culture medium further comprises 0.1 g / L to 1 g / L of proline, 0.1 g / L to 1 g / L of arginine, 0.1 g / L to 1 g / L of glutamine, 10 mg / L to 70 mg / L of adenine sulfate, 1 mg / L to 10 mg / L of silver nitrate, 0.2 mg / L to 2 mg / L of 2, 4-D, 0.2 mg / L to 2 mg / L of Zetin and 0.2 mg / L to 2 mg / L of Picloram. According to the method, the somatic embryo occurrence rate can be remarkably increased by optimizing the induction culture medium of the embryogenic callus of the heat reclamation 628, the anther somatic embryo occurrence rate of the heat reclamation 628 of the rubber tree variety can be increased to 50% or above by utilizing the induction culture medium, and the cotyledon embryo occurrence rate can be increased to 20% or above.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to an induction culture medium for embryogenic callus of the rubber tree variety Reken 628, and its method and application for inducing somatic embryogenesis. Background Technology

[0002] Reken 628 is an excellent new rubber tree variety, characterized by high yield, rapid growth, and strong resistance to adverse conditions. It is one of the fastest-growing varieties and also one of the most promising new rubber tree varieties for widespread promotion. Due to its narrow crown and sparse plant type, it is particularly suitable for a wide-narrow row planting pattern: a 4-meter row spacing and a 2-meter plant spacing in the narrow rows, and a 20-meter row spacing in the wide rows. This planting pattern not only improves land resource utilization but also effectively improves light and ventilation conditions for the rubber trees, promoting healthy growth and increasing latex yield and quality. Reken 628 is one of the few rubber tree varieties currently best suited for this planting pattern and is a popular variety widely promoted in rubber-growing areas.

[0003] Somatic embryogenesis seedlings of rubber trees are the third generation of planting materials for rubber trees. They are seedlings cultivated using somatic embryogenesis technology with anthers of superior varieties as explants. Compared with budded seedlings (the second generation of planting materials for rubber trees), they have advantages such as complete inheritance of the characteristics of the mother plant, faster growth rate, and better resistance. The large-scale production of somatic embryogenesis seedlings of rubber trees was first achieved using the rubber tree variety Reyan 73397. However, large-scale production of somatic embryogenesis seedlings for other rubber tree varieties still faces bottlenecks, especially for Reken 628, which has a low anther embryogenic callus induction rate and an even lower cotyledon somatic embryogenesis rate, limiting the application of somatic embryogenesis seedlings of this variety. Summary of the Invention

[0004] The purpose of this invention is to provide an induction culture medium for embryogenic callus of the rubber tree variety Reken 628, as well as a method and application for inducing somatic embryogenesis, wherein the induction culture medium can significantly improve the somatic embryogenesis rate of Reken 628.

[0005] This invention provides an induction culture medium for embryogenic callus of the rubber tree variety Reken 628. The induction culture medium is based on modified MS medium and further includes: proline 0.1-1 g / L, arginine 0.1-1 g / L, glutamine 0.1-1 g / L, adenine sulfate 10-70 mg / L, silver nitrate 1-10 mg / L, 2,4-D 0.2-2 mg / L, zeatin 0.2-2 mg / L, and picloram 0.2-2 mg / L.

[0006] As a preferred embodiment, the modified MS medium uses MS medium as the base medium, with the concentration of ammonium nitrate adjusted to 330–1800 mg / L, the concentration of copper sulfate pentahydrate to 0.1–0.3 mg / L, the concentration of nicotinic acid to 2.5–7.5 mg / L, and the concentration of VB1 (vitamin B1) to 0.25–0.75 mg / L; it also includes: biotin 0.01–0.1 mg / L, folic acid 0.1–1 mg / L, asparagine 100–600 mg / L, hydrolyzed casein 100–600 mg / L, sucrose 50–90 g / L, gellan gum 2–3 g / L, and coconut water 40–90 mL / L.

[0007] The present invention provides a method for inducing somatic embryogenesis of Reken 628, comprising the following steps: inoculating the stamens of Reken 628 into the above-mentioned induction medium, and inducing callus tissue to form after induction culture; inoculating the formed callus tissue into the differentiation medium, and inducing somatic embryogenesis after differentiation culture.

[0008] As a preferred embodiment, the stamens are derived from immature male flower buds.

[0009] As a preferred embodiment, the induction culture temperature is 25–29°C and the humidity is 50%–75%.

[0010] As a preferred embodiment, the induction culture includes: culturing in the dark for 35–50 days.

[0011] As a preferred embodiment, the differentiation culture temperature is 24–28°C and the humidity is 50%–75%.

[0012] As a preferred embodiment, the differentiation culture includes: culturing in the dark for 45–55 days.

[0013] As a preferred embodiment, the stamens are further disinfected before inoculation; the disinfection time is 0.5 to 16 minutes.

[0014] This invention provides the application of the above-mentioned induction culture medium or the above-mentioned method in the large-scale production of rubber tree seedlings.

[0015] Beneficial Effects: This invention provides an induction medium for embryogenic callus of the rubber tree variety Reken 628. The induction medium is based on a modified MS medium and further includes: proline 0.1–1 g / L, arginine 0.1–1 g / L, glutamine 0.1–1 g / L, adenine sulfate 10–70 mg / L, silver nitrate 1–10 mg / L, 2,4-D 0.2–2 mg / L, Zeatin 0.2–2 mg / L, and Picloram 0.2–2 mg / L. By optimizing the induction medium for embryogenic callus of Reken 628, this invention can significantly improve the somatic embryogenesis rate of Reken 628. Results from the examples show that, using the induction medium described in this invention, the anther somatic embryogenesis rate of the rubber tree variety Reken 628 can be increased to over 50%, and the cotyledonary embryogenesis rate can be increased to over 20%.

[0016] This invention provides a method for inducing somatic embryogenesis in *Hot Ken 628*, comprising the following steps: inoculating the stamens of *Hot Ken 628* into the aforementioned induction medium, and inducing callus formation through induction culture; inoculating the formed callus into a differentiation medium, and inducing somatic embryogenesis through differentiation culture. This invention, by optimizing the composition of the induction medium and improving culture conditions, can significantly increase the somatic embryogenesis rate of *Hot Ken 628*.

[0017] This invention provides the application of the above-described induction medium or method in the large-scale production of rubber tree seedlings. Using the induction medium and method provided by this invention, the embryogenesis rate of Reken 628 is significantly improved, enabling the large-scale production of rubber tree seedlings. Attached Figure Description

[0018] Figure 1 This is a photograph of the callus tissue cultured for 42 days after inoculation with stamens in Example 5. The scale bar is 1 cm.

[0019] Figure 2 The image shows the callus tissue of Comparative Example 2 after 42 days of culture following inoculation with stamens. The scale bar is 1 cm.

[0020] Figure 3 This is a photograph of a differentiated somatic embryo cultured for 49 days after callus inoculation in Example 5.

[0021] Figure 4 This is a photograph of the differentiated embryonic tissue cultured for 49 days after callus inoculation, as shown in Comparative Example 2. Detailed Implementation

[0022] This invention provides an induction culture medium for embryogenic callus of the rubber tree variety Reken 628. The induction culture medium is based on a modified MS medium and further includes: proline 0.1-1 g / L, arginine 0.1-1 g / L, glutamine 0.1-1 g / L, adenine sulfate 10-70 mg / L, silver nitrate 1-10 mg / L, 2,4-D 0.2-2 mg / L, zeatin 0.2-2 mg / L, and picloram 0.2-2 mg / L.

[0023] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0024] The induction medium described in this invention is based on modified MS medium. The amount of proline added per 1L of medium can be any value within the range of 0.1–1 g / L, for example, 0.1, 0.25, 0.5, 0.75, or 1 g / L. Proline mainly enhances the stress resistance and regeneration efficiency of isolated tissue cells through osmotic regulation, stabilizing the structure of biological macromolecules, participating in metabolism, supplying energy, and antioxidation. The amount of arginine added per 1L of medium can be any value within the range of 0.1–1 g / L, for example, 0.1, 0.25, 0.5, 0.75, or 1 g / L. Arginine mainly promotes the proliferation, differentiation, and regeneration capacity of isolated tissue cells by regulating metabolic balance, signal transduction, and antioxidant defense. Based on 1L of culture medium, the amount of glutamine added can be any value within the range of 0.1 to 1 g / L, for example, 0.1, 0.25, 0.5, 0.75, or 1 g / L. Glutamine significantly affects the proliferation, differentiation, and organ regeneration of isolated cells mainly through mechanisms such as nitrogen metabolism regulation, energy supply, and cell protection. Based on 1L of culture medium, the amount of adenine sulfate added can be any value within the range of 10 to 70 mg / L, for example, 10, 30, 50, or 70 mg / L. Adenine sulfate significantly affects the proliferation and differentiation of isolated tissues mainly through mechanisms such as regulating cell metabolism, promoting division, and synergistic hormone effects. Based on 1L of culture medium, the amount of silver nitrate added can be any value within the range of 1 to 10 mg / L, for example, 1, 2.5, 5, 7.5, or 10 mg / L. Silver nitrate plays a key role mainly through mechanisms such as regulating ethylene metabolism, promoting organ regeneration, and improving morphogenesis. Based on 1 L of culture medium, the amount of 2,4-D added can be any value within the range of 0.2–2 mg / L, for example, 0.2, 0.5, 1, 1.5, or 2 mg / L; the amount of Zeatin added can be any value within the range of 0.2–2 mg / L, for example, 0.2, 0.5, 1, 1.5, or 2 mg / L; the amount of Picloram added can be any value within the range of 0.2–2 mg / L, for example, 0.2, 0.5, 1, 1.5, or 2 mg / L. Specific hormone combinations can promote callus growth, induce embryogenic callus formation, and significantly improve the induction rate of embryogenic callus. In a preferred embodiment, the induction medium of the present invention is based on modified MS medium, with the addition of proline 0.1 g / L, arginine 0.5 g / L, glutamine 0.25 g / L, adenine sulfate 30 mg / L, silver nitrate 10 mg / L, 2,4-D 1 mg / L, Zeatin 1 mg / L and Picloram 1 mg / L.

[0025] The modified MS medium of this invention can be based on MS medium, with adjustments made to the concentrations of ammonium nitrate, copper sulfate pentahydrate, nicotinic acid, and vitamin B1. Based on 1L of modified MS medium, the concentration of ammonium nitrate can be adjusted to any value within the range of 330–1800 mg / L, for example, 330, 660, 900, 1200, 1500, or 1800 mg / L; ammonium nitrate provides nitrogen nutrition to plant cells and acts as a buffer to regulate the pH of the medium. Based on 1L of modified MS medium, the concentration of copper sulfate pentahydrate can be adjusted to any value within the range of 0.1–0.3 mg / L, for example, 0.1, 0.15, 0.2, 0.25, or 0.3 mg / L; copper sulfate pentahydrate is an essential micronutrient for plants, participating in the redox reactions of various enzymes. Based on 1L of modified MS medium, the concentration of nicotinic acid can be any value within the range of 2.5–7.5 mg / L, for example, 2.5, 5, or 7.5 mg / L; nicotinic acid is an essential intermediate and metabolic catalyst for plant cell growth. Based on 1L of modified MS medium, the concentration of VB1 can be adjusted to any value within the range of 0.25–0.75 mg / L, for example, 0.25, 0.5, or 0.75 mg / L; VB1 is an essential intermediate and metabolic catalyst for plant cell growth. This invention allows the addition of biotin, folic acid, asparagine, hydrolyzed casein, sucrose, Gellangum, and coconut water to the adjusted MS medium. Based on 1L of modified MS medium, the amount of biotin added can be any value within the range of 0.01–0.1 mg / L, for example, 0.01, 0.03, 0.05, 0.07, or 0.1 mg / L. Based on 1L of modified MS medium, the amount of folic acid added can be any value within the range of 0.1–1 mg / L, for example, 0.1, 0.3, 0.5, 0.7, or 1 mg / L. Based on 1L of modified MS medium, the amount of asparagine added can be any value within the range of 100–600 mg / L, for example, 100, 200, 300, 400, 500, or 600 mg / L; asparagine provides amino acids for plant cells. Based on 1L of modified MS medium, the amount of hydrolyzed casein added can be any value within the range of 100–600 mg / L, for example, 100, 200, 300, 400, 500, or 600 mg / L; hydrolyzed casein provides amino acids for plant cells. Based on 1L of modified MS medium, the amount of sucrose added can be any value within the range of 50–90 g / L, for example, 50, 60, 70, 80, or 90 g / L; sucrose provides a carbon source for plant cells and acts as an osmotic agent to adjust the osmotic potential of the medium. Based on 1L of modified MS medium, the amount of gellan gum added can be any value in the range of 2 to 3 g / L, such as 2, 2.2, 2.5, 2.8 or 3 g / L; gellan gum can semi-solidify the medium as a coagulant to support the cultured plant material.Based on 1L of modified MS medium, the amount of coconut water added can be any value within the range of 40 to 90 mL / L, such as 40, 50, 60, 70, 80 or 90 mL / L; coconut water provides plant cells with a variety of nutrients such as plant hormones, amino acids, and vitamins.

[0026] The present invention provides a method for inducing somatic embryogenesis of Reken 628, comprising the following steps: inoculating the stamens of Reken 628 into the above-mentioned induction medium, and inducing callus tissue to form after induction culture; inoculating the formed callus tissue into the differentiation medium, and inducing somatic embryogenesis after differentiation culture.

[0027] This invention allows for the initial harvesting of immature male flower buds from the Reken 628 variety. These immature buds can be harvested using conventional methods. The immature male flower buds can be fully unopened, pale yellowish-green. In this invention, the immature male flower buds of Reken 628 can be disinfected first. The disinfection time can be any value within the range of 0.5 to 16 minutes, for example, 0.5, 1, 5, 10, 11, 15, or 16 minutes. The disinfection reagents can include: a 70%–75% (v / v) alcohol aqueous solution and / or a 0.1% (w / w) mercuric chloride aqueous solution. As a preferred embodiment, the immature male flower buds are surface-disinfected with a 70% or 75% (v / v) alcohol aqueous solution for 0.5–1 minute; after surface disinfection with the alcohol aqueous solution, they are disinfected with a 0.1% (w / w) mercuric chloride aqueous solution for 10–15 minutes; after disinfection with the mercuric chloride aqueous solution, they are rinsed with sterile water 3–6 times, each time for 2–3 minutes.

[0028] After sterilization, the immature male flower buds of the *Hot Ken 628* variety described in this invention can be sterilized, and the stamens can be extracted under aseptic conditions. The stamens are then inoculated into the aforementioned induction medium for induction culture. The induction culture temperature can be any value within the range of 25–29°C, for example, 25, 26, 27, 28, or 29°C; the humidity can be any value within the range of 50%–75%, for example, 50%, 55%, 60%, 65%, 70%, or 75%. This invention allows for induction culture under dark conditions for any value within the range of 35–50 days, for example, 35, 38, 42, 47, or 50 days, to form callus tissue.

[0029] This invention allows the formed callus tissue to be inoculated onto a differentiation medium for differentiation culture. The differentiation culture temperature can be any value within the range of 24–28°C, for example, 24, 25, 26, 27, or 28°C; the humidity can be any value within the range of 50%–75%, for example, 50%, 55%, 60%, 65%, 70%, or 75%. This invention allows differentiation culture under dark conditions for any value within the range of 45–55 days, for example, 45, 47, 49, 53, or 55 days, developing into somatic embryos. This invention does not have special requirements for the differentiation medium; conventional differentiation media in the art can be used. As a preferred embodiment, the differentiation medium of this invention can be based on modified MS medium, with its macroelements adjusted to 4 / 5 of the original, asparagine removed, and 6-BA (6-benzylaminopurine), KT, 2,4-D, GA3 (gibberellin 3), and activated charcoal added. Based on 1L of modified MS medium, the amount of 6-BA added can be any value within the range of 0.1 to 1 mg / L, for example, 0.1, 0.5, or 1 mg / L; the amount of KT added can be any value within the range of 2 to 4 mg / L, for example, 2, 3, or 4 mg / L; the amount of 2,4-D added can be any value within the range of 0.01 to 0.1 mg / L, for example, 0.01, 0.06, or 0.1 mg / L; the amount of GA3 added can be any value within the range of 0.1 to 1 mg / L, for example, 0.1, 0.5, or 1 mg / L; and the amount of activated carbon added can be any value within the range of 0.5 to 1.5 g / L, for example, 0.5, 1, or 1.5 g / L. As another preferred embodiment, the differentiation medium of the present invention can be based on modified MS medium, with its macroelements adjusted to 4 / 5 of the original, asparagine removed, and then 6-BA, KT, NAA, GA3, and ABA (abscisic acid) added. Based on 1L of modified MS medium, the amount of 6-BA added can be any value within the range of 0.1 to 1 mg / L, for example, 0.1, 0.5, or 1 mg / L; the amount of KT added can be any value within the range of 2 to 4 mg / L, for example, 2, 3, or 4 mg / L; the amount of NAA added can be any value within the range of 0.1 to 0.3 mg / L, for example, 0.1, 0.2, or 0.3 mg / L; the amount of GA3 added can be any value within the range of 0.1 to 1 mg / L, for example, 0.1, 0.5, or 1 mg / L; and the amount of ABA added can be any value within the range of 0.5 to 1.5 mg / L, for example, 0.5, 1, or 1.5 mg / L.

[0030] This invention provides the application of the above-described induction medium or method in the large-scale production of rubber tree seedlings. Using the induction medium and method provided by this invention, the embryogenesis rate of Reken 628 is significantly improved, enabling the large-scale production of rubber tree seedlings.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides an induction culture medium for embryogenic callus of the rubber tree variety Reken 628, as well as its method and application for inducing somatic embryogenesis. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] Induction medium: Modified MS medium was used as the base medium, with the following added ingredients: proline 0.1 g / L, arginine 0.5 g / L, glutamine 0.25 g / L, adenine sulfate 30 mg / L, silver nitrate 10 mg / L, 2,4-D 1 mg / L, Zeatin 1 mg / L and Picloram 1 mg / L, pH 5.8.

[0034] Modified MS medium: Using MS medium as the basal medium, the concentrations of ammonium nitrate (1500 mg / L), copper sulfate pentahydrate (0.2 mg / L), nicotinic acid (5 mg / L), vitamin B1 (0.5 mg / L) were adjusted, and biotin (0.05 mg / L), folic acid (0.5 mg / L), asparagine (300 mg / L), hydrolyzed casein (100 mg / L), sucrose (70 g / L), gellan gum (2.2 g / L), and coconut water (50 mL / L) were added.

[0035] Differentiation medium: Using the above-mentioned modified MS medium as the basic medium, the macro-elements were adjusted to 4 / 5 of the original, asparagine was removed, and 6-BA 0.5 mg / L, KT 3 mg / L, 2,4-D 0.06 mg / L, GA3 0.5 mg / L and activated carbon 1 g / L were added, pH 5.8.

[0036] Example 2

[0037] The procedure was carried out as described in Example 1, except that the induction medium was modified MS medium as the base medium, with the following added ingredients: proline 0.5 g / L, arginine 0.1 g / L, glutamine 0.5 g / L, adenine sulfate 10 mg / L, silver nitrate 2.5 mg / L, 2,4-D 1 mg / L, Zeatin 1 mg / L and Picloram 1 mg / L, pH 5.8.

[0038] Example 3

[0039] The procedure was carried out as described in Example 1, except that the induction medium was modified MS medium as the base medium, with the following added components: proline 1 g / L, arginine 0.25 g / L, glutamine 1 g / L, adenine sulfate 10 mg / L, silver nitrate 5 mg / L, 2,4-D 1 mg / L, Zeatin 1 mg / L and Picloram 1 mg / L, pH 5.8.

[0040] Example 4

[0041] The procedure was carried out as described in Example 1, except that the induction medium was modified MS medium as the base medium, with the following added components: proline 0.5 g / L, arginine 1 g / L, glutamine 0.5 g / L, adenine sulfate 10 mg / L, silver nitrate 10 mg / L, 2,4-D 1 mg / L, Zeatin 1 mg / L and Picloram 1 mg / L, pH 5.8.

[0042] Example 5

[0043] The procedure was carried out in accordance with Example 2, except that the differentiation medium was modified MS medium as the basic medium, the macroelements were adjusted to 4 / 5 of the original, asparagine was removed, and 6-BA 0.5 mg / L, KT 3 mg / L, NAA 0.2 mg / L, GA 30.5 mg / L and ABA 1 mg / L were added, with pH 5.8.

[0044] Comparative Example 1

[0045] The procedure was carried out as described in Example 1, except that the induction medium was based on modified MS medium with the addition of only 1 mg / L 2,4-D, 1 mg / L Zeatin and 1 mg / L Picloram, pH 5.8.

[0046] Example 6

[0047] Immature flower buds of the rubber tree variety Reken 628 were harvested and sterilized in a laminar flow hood. The surface was disinfected with a 75% ethanol aqueous solution for 60 seconds (1 minute), followed immediately by disinfection with a 0.1% mercuric chloride aqueous solution for 10 minutes. Finally, the surface was rinsed five times with sterile water for 3 minutes each time. After sterilization, the stamens were aseptically removed and inoculated into the induction medium of Example 1. The stamens were then placed in a dark culture room at 27°C and 65% humidity for 6 weeks (42 days) to induce callus formation (see [link to example]). Figure 1 The resulting callus tissue was inoculated into the differentiation medium of Example 1 and then cultured in a dark culture room at 25°C and 65% humidity for 7 weeks (49 days) to induce development into somatic embryos (see Example 1). Figure 3 ).

[0048] Example 7

[0049] The procedure was carried out in accordance with Example 6, except that the induction medium was replaced with the induction medium of Example 2.

[0050] Example 8

[0051] The procedure was carried out in accordance with Example 6, except that the induction medium was replaced with the induction medium of Example 3.

[0052] Example 9

[0053] The procedure was carried out in accordance with Example 6, except that the induction medium was replaced with the induction medium of Example 4.

[0054] Example 10

[0055] The procedure was carried out in accordance with Example 6, except that the differentiation medium was replaced with that of Example 5.

[0056] Comparative Example 2

[0057] The procedure was carried out as described in Example 6, except that the induction medium was replaced with the induction medium of Comparative Example 1. The resulting callus tissue is shown in [reference needed]. Figure 2 The developed somatic embryo is seen Figure 4 .

[0058] Test Example 1

[0059] The examples and comparative examples were conducted in parallel, repeated three times, with each combination treatment inoculated with 84 stamens each time.

[0060] After 6 weeks of induction culture, the condition of the formed callus was observed. The callus formed in Example 6 is shown in the figure. Figure 1 The callus tissue formed in Comparative Example 2 is shown Figure 2 The total number of induced callus tissues was counted, and the callus induction rate was calculated according to the following formula. The results are shown in Table 1.

[0061] Callus induction rate = Total number of induced callus / (Total number of inoculated stamens - Number of contaminated stamens) × 100%.

[0062] After 7 weeks of differentiation culture, the development of the somatic embryos was observed. The somatic embryos developed in Example 6 are shown in the figure. Figure 3 Comparative Example 2 shows the somatic embryo that developed into a somatic embryo. Figure 4 The total number of callus tissues induced to produce somatic embryos and the total number of callus tissues induced to produce mature cotyledon embryos were counted. The embryogenic callus induction rate and cotyledon embryo induction rate were calculated according to the following formulas. The results are shown in Tables 2 and 3 to verify the embryogenicity of the callus tissues.

[0063] Embryogenic callus induction rate = Total number of callus tissues induced to produce somatic embryos / (Total number of inoculated callus tissues - Number of contaminated callus tissues) × 100%.

[0064] Cotyledon embryo formation rate = Total number of callus tissues that induce mature cotyledon embryos / (Total number of inoculated callus tissues - Number of contaminated callus tissues) × 100%.

[0065] Table 1. Effects of different induction culture medium combinations on callus induction in Reken 628.

[0066] Group Total number of callus tissues (pieces) Callus induction rate (%) Significance Example 6 204 83.4±14.3 ab Example 7 210 88.2±14.2 a Example 8 206 85.9±18.1 ab Example 9 223 86.6±6.4 ab Example 10 200 83.5±13 ab Comparative Example 2 193 85.5±4.3 ab

[0067] Table 2. Effects of different induction culture medium combinations on embryogenic callus induction in Reken 628.

[0068] Group Total number of callus tissues induced to form somatic embryos (number of callus tissues) Embryogenic callus induction rate (%) Significance Example 6 102 50.3±2.2 a Example 7 90 45.8±19.7 a Example 8 90 45.5±5.4 a Example 9 86 41.4±11.8 ab Example 10 88 45.2±8.9 a Comparative Example 2 26 13.3±5 c

[0069] Table 3. Effects of different induction culture medium combinations on cotyledon embryo formation in Reken 628.

[0070] Group Total number of callus tissues induced to produce mature cotyledon embryos (number of callus tissues) Cotyledon embryo formation rate (%) Significance Example 6 45 20.7±15.7 a Example 7 24 11.1±6.2 abc Example 8 23 11.2±5.5 abc Example 9 35 15.1±11.2 ab Example 10 24 12±5.8 abc Comparative Example 2 4 1.9±2.3 c

[0071] Note: Means marked with the same letter indicate no significant difference between them, while means marked with different letters indicate a significant difference.

[0072] Depend on Figure 1 and Figure 2 It can be seen that the majority of callus tissue formed in Example 6 is bright yellow in color, irregularly nodular, with a compact structure and smooth surface, while the majority of callus tissue formed in Comparative Example 2 has an uneven surface and lacks luster. Figure 3 and Figure 4 It can be seen that the cotyledon embryos formed in Example 6 are numerous and mature, while the cotyledon embryos in Comparative Example 2 are few and cannot develop normally.

[0073] As can be seen from Tables 1 to 3, by optimizing the callus induction culture medium of rubber tree, the anther embryogenesis rate of rubber tree variety Reken 628 was increased from a maximum of 13.3% to over 50%, and the cotyledon embryogenesis rate was increased from 1.9% to over 20%.

[0074] Therefore, it can be seen that by optimizing the culture medium composition and improving the culture conditions, the embryogenesis rate of the Reken 628 callus can be significantly improved.

[0075] 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 inducing embryogenic callus in the rubber tree variety Reken 628, characterized in that, The induction medium is based on modified MS medium and also includes: proline 0.1-1 g / L, arginine 0.1-1 g / L, glutamine 0.1-1 g / L, adenine sulfate 10-70 mg / L, silver nitrate 1-10 mg / L, 2,4-D 0.2-2 mg / L, Zeatin 0.2-2 mg / L and Picloram 0.2-2 mg / L.

2. The induction culture medium according to claim 1, characterized in that, The modified MS medium is based on MS medium, with the concentrations of ammonium nitrate adjusted to 330–1800 mg / L, copper sulfate pentahydrate to 0.1–0.3 mg / L, nicotinic acid to 2.5–7.5 mg / L, and vitamin B1 to 0.25–0.75 mg / L. It also includes: biotin 0.01–0.1 mg / L, folic acid 0.1–1 mg / L, asparagine 100–600 mg / L, hydrolyzed casein 100–600 mg / L, sucrose 50–90 g / L, Gellangum 2–3 g / L, and coconut water 40–90 mL / L.

3. A method for inducing somatic embryogenesis in Reken 628 embryos, characterized in that, The process includes the following steps: inoculating the stamens of Reken 628 into the induction medium described in claim 1 or 2, and inducing callus formation through induction culture; inoculating the formed callus into the differentiation medium, and inducing it to develop into a somatic embryo through differentiation culture.

4. The method according to claim 3, characterized in that, The stamens are derived from immature male flower buds.

5. The method according to claim 3, characterized in that, The induction culture was conducted at a temperature of 25–29°C and a humidity of 50%–75%.

6. The method according to claim 3 or 5, characterized in that, The induction culture includes: culturing in the dark for 35–50 days.

7. The method according to claim 3, characterized in that, The differentiation culture was conducted at a temperature of 24–28°C and a humidity of 50%–75%.

8. The method according to claim 3 or 7, characterized in that, The differentiation culture includes: culturing in the dark for 45–55 days.

9. The method according to claim 3, characterized in that, The stamens are disinfected before inoculation; the disinfection time is 0.5 to 16 minutes.

10. The application of the induction culture medium according to claim 1 or 2 or the method according to any one of claims 3 to 9 in the large-scale production of rubber tree seedlings.

Images