Tissue culture and rapid propagation method of new variety of Dendrobium nobile

Abstract

The invention relates to the technical field of tissue culture, and particularly discloses a tissue culture and rapid propagation method of a new variety of Dendrobium nobile, which comprises the following steps: S1, selecting current-year new lateral buds, removing leaf sheaths and retaining bud points, and sequentially washing with running water, soaking with 75% alcohol, disinfecting with 0.1% mercury bichloride and washing with sterile water; s2, the disinfected stem segments with buds are inoculated into an induction culture medium, the induction culture medium is a 1 / 2 MS culture medium, and 3.0 mg / L of 6-BA, 0.5 mg / L of NAA and 60 g / L of potato powder are added; s3, adventitious buds generated through induction are cut off and inoculated into a proliferation culture medium, the proliferation culture medium is a 1 / 2 MS culture medium, and 3.0 mg / L of 6-BA, 0.5 mg / L of NAA and 80 g / L of potato powder are added; s4, the proliferated adventitious buds are inoculated into a rooting culture medium, the rooting culture medium is a 1 / 2 MS culture medium, and 0.5 mg / L of NAA or 0.5 mg / L of IBA is added; according to the method, the culture medium formula and the culture process are optimized, so that the tissue culture efficiency of the Dendrobium nobile 'Hooke Chaoya' is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture and rapid propagation method for a new variety of Dendrobium officinale. Background Art

[0002] Dendrobium cultivar 'CATAS Morning Glow' is a new commercial variety with excellent ornamental traits and strong adaptability, which was bred through artificial hybridization with Dendrobium 'Nopporn Pink' as the female parent and Dendrobium 'Swirl' as the male parent. This variety has both the bright flower colors of the female parent and the full flower shape of the male parent, and has broad market prospects in tropical and subtropical regions. As an important member of the orchid family, Dendrobium cultivar 'CATAS Morning Glow' occupies an important position in the cut flower and potted plant markets because of its elegant flowers, rich colors, long flowering period and strong adaptability, and has extremely high ornamental and economic value.

[0003] In recent years, advances in flower breeding technology have led to the emergence of new varieties of autumn dendrobium with superior ornamental qualities, greater stress resistance, or unique flower colors. However, the successful selection of new varieties is only the first step; their commercial value depends on efficient, stable, and large-scale propagation techniques that maintain the variety's excellent characteristics.

[0004] Traditional propagation methods for Dendrobium nobile, such as division, can maintain varietal characteristics, but their reproduction coefficient is extremely low, making it difficult to meet the needs of rapidly introducing new varieties to the market and large-scale production, severely restricting its industrialization process. While seed propagation can theoretically produce a large number of seedlings, the process is lengthy. Furthermore, because orchid seeds are small, lack endosperm, and have difficulty germinating naturally, they require symbiotic fungi. More importantly, seedlings are prone to genetic variation, resulting in separation in key ornamental traits such as flower color, flower shape, and plant form in subsequent generations. This makes it impossible to stably maintain the excellent characteristics of the new variety and cannot be used for the faithful propagation of commercial varieties. Furthermore, different varieties require different types and concentrations of exogenous hormones for induction, proliferation, and rooting of explants, resulting in significant variations in the optimal formula. Summary of the Invention

[0005] The object of the present invention is to provide a method for rapid tissue culture propagation of a new variety of Dendrobium officinale, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A tissue culture and rapid propagation method for a new variety of Dendrobium officinale comprises the following steps:

[0008] S1. Select new lateral buds of the current year, remove the leaf sheaths and retain the buds, and sequentially rinse with running water, soak in 75% alcohol, disinfect with 0.1% mercuric chloride, and rinse with sterile water;

[0009] S2, inoculating the sterilized stem segments with buds into an induction medium, wherein the induction medium is 1 / 2 MS medium supplemented with 3.0 mg / L 6-BA, 0.5 mg / L NAA, and 60 g / L potato flour;

[0010] S3, cutting the induced adventitious buds and inoculating them into a proliferation medium, wherein the proliferation medium is 1 / 2 MS medium supplemented with 3.0 mg / L 6-BA, 0.5 mg / L NAA, and 80 g / L potato flour;

[0011] S4. The proliferated adventitious buds were inoculated into a rooting medium, wherein the rooting medium was 1 / 2 MS medium supplemented with 0.5 mg / L NAA or 0.5 mg / L IBA.

[0012] A culture medium for inducing adventitious buds of a new variety of Dendrobium officinale, comprising 1 / 2MS basal culture medium, 3.0 mg / L 6-BA, 0.5 mg / L NAA, and 60 g / L potato flour.

[0013] A culture medium for the proliferation of adventitious buds of a new variety of Dendrobium officinale, comprising 1 / 2MS basal culture medium, 3.0 mg / L 6-BA, 0.5 mg / L NAA, and 80 g / L potato flour.

[0014] A culture medium for rooting adventitious buds of a new variety of Dendrobium officinale, comprising 1 / 2MS basal culture medium and 0.5 mg / L NAA or 0.5 mg / L IBA.

[0015] According to any of the above culture media, 20 g / L sucrose and 7.5 g / L carrageenan are added to the culture media at each stage, the pH is 5.8-6.0, the culture temperature is 24±2°C, the light intensity is 1000-1200 lx, and the light intensity is 14 hours per day.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention is based on the characteristics of the new variety of Dendrobium officinale. Through the efficient adventitious bud induction and rapid proliferation mechanism, a large number of high-quality tissue culture seedlings can be obtained in a unit time, which effectively solves the problem of low efficiency of traditional division reproduction and difficulty in meeting the needs of large-scale production and market promotion of new varieties. The excellent genetic characteristics of the new variety are retained. The obtained tissue culture seedlings have highly consistent and stable characteristics. After transplanting, the plants are strong, the root system is well-developed, the flowering performance is uniform, and there is no genetic variation.

[0018] (2) The present invention optimizes the culture medium formula and culture process, significantly improving the success rate and efficiency of each stage. The operation process is simplified, standardized and reproducible, greatly reducing the contamination risk and batch differences in the production process. At the same time, the addition of potato flour not only provides a carbon source and natural growth-promoting substances, but also enhances the physical support of the culture medium, thereby realizing the large-scale breeding and industrial promotion of new varieties of seedlings. Potato flour, as a natural organic additive, is rich in carbohydrates, vitamins and growth factors, which can significantly promote the division and differentiation of explant cells and shorten the culture cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the effect of different potato flour contents on adventitious bud proliferation in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Example 1:

[0022] See also Figure 1 As shown, a tissue culture and rapid propagation method for a new variety of Dendrobium officinale comprises:

[0023] S1. Explant disinfection

[0024] The new lateral buds of the current year of Dendrobium officinale 'Reke Zhaoxia' provided by the Institute of Tropical Crops Variety Resources of the Chinese Academy of Tropical Agricultural Sciences were selected. They were strong and about 10 cm long.

[0025] Remove the leaf sheaths, retain the buds on the nodes, rinse with running water for 20 minutes to remove surface impurities, soak in 75% alcohol for 30 seconds, rinse once with sterile water, soak in 0.1% mercuric chloride solution for 8-10 minutes, gently shake 2-3 times, rinse with sterile water 4-5 times, drain and set aside;

[0026] S2. Adventitious bud induction

[0027] Use 1 / 2MS as the basal medium, add 3.0 mg / L 6-BA, 0.5 mg / L NAA, 60 g / L potato flour, 20 g / L sucrose, and 7.5 g / L carrageenan, adjust the pH to 5.8-6.0, sterilize with high temperature and high pressure, and dispense into culture bottles. Cut the sterilized explants into 1.5 cm stem segments with buds and inoculate them into the induction medium. The culture conditions are set at a temperature of 24±2°C, a light intensity of 1000-1200 lx, 14 hours of light per day, and 10 hours of dark culture.

[0028] S3, adventitious bud proliferation

[0029] Use 1 / 2MS as the basal medium, add 3.0 mg / L 6-BA, 0.5 mg / L NAA, 80 g / L potato flour, 20 g / L sucrose and 7.5 g / L carrageenan, adjust the pH to 5.8-6.0, cut the induced 1-2 cm adventitious buds, and inoculate them into the proliferation medium. The culture conditions are the same as the previous stage.

[0030] S4. Rooting culture

[0031] Use 1 / 2MS as the basal medium, add 0.5mg / L NAA or 0.5mg / L IBA, 20g / L sucrose and 7.5g / L carrageenan, adjust the pH to 5.8-6.0, select the proliferation seedlings of about 3cm, inoculate them into the rooting medium, and culture conditions are the same as the previous stage;

[0032] S5. Transplanting and acclimatization

[0033] Remove the rooted seedlings from the culture bottle, wash the root culture medium, place them in a greenhouse with shade for 3 days, use sterilized sphagnum moss, maintain humidity at 60% to 80%, temperature at 25-30°C, and gradually increase the light intensity until the greenhouse is shaded by about 60%.

[0034] Experimental Example 1: Effect of 6-BA and NAA combination on adventitious bud induction

[0035] The sterilized explants were cut into 1.5 cm long stem segments with buds, and inoculated into induction medium supplemented with different mass concentrations of 6-BA and NAA. The concentrations of 6-BA were 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / L, and the concentrations of NAA were 0, 0.1, 0.5, and 1.0 mg / L, respectively. There were 24 treatment combinations in total, with 20-30 explants each time. The experiment was repeated 3 times. After 40 days, the number of buds was counted and the induction rate was calculated, as shown in Table 1:

[0036] Table 1 Effects of different concentrations of 6-BA and NAA combinations on adventitious bud induction

[0037]

[0038]

[0039] As can be seen from the above, the sterilized stem segments with buds were inoculated into the induction medium with different mass concentrations of 6-BA and NAA. After culturing for about 2 weeks, it was found that the buds germinated to form small protrusions and continued to expand. After culturing for about 5-6 weeks, the induction of adventitious buds was statistically analyzed. The results showed that when no 6-BA was added, the induction rate was about 10%. When the 6-BA concentration reached 2.0 mg / L, the induction rate increased sharply, reaching more than 82.78%, but the number of adventitious buds induced at this time was only 1-2.

[0040] When the 6-BA concentration reaches 3.0-5.0 mg / L, the induction rate reaches about 90%, and the number of induced adventitious buds is more than 3. A significant variance analysis shows that when the 6-BA concentration is 3.0 mg / L and the NAA concentration is 0.5-1.5 mg / L, the induction rate is as high as more than 98%, and the number of induced adventitious buds is more than 4. Among them, when the NAA concentration is 0.5 mg / L, the induction effect is the best, the induction rate reaches 99.44%, and the number of buds is 4.99. At this time, the buds grow vigorously. Therefore, the most suitable culture medium for adventitious bud induction is 3.0 mg / L 6-BA + 0.5 mg / L NAA.

[0041] Experimental Example 2: Effect of 6-BA and NAA combination on adventitious bud induction

[0042] The induced adventitious buds of about 2 cm in length were cut off and inoculated into induction medium supplemented with different mass concentrations of 6-BA and NAA. The concentrations of 6-BA were 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / L, and the concentrations of NAA were 0, 0.1, 0.5, and 1.0 mg / L, respectively. There were 24 treatment combinations in total, with 20-30 explants each time. The experiment was repeated 3 times. After 60 days, the number of buds was counted and the proliferation coefficient was calculated, as shown in Table 2:

[0043] Table 2 Effects of different concentrations of 6-BA and NAA combinations on adventitious bud proliferation

[0044]

[0045]

[0046] As can be seen from the above, adventitious buds of approximately 2 cm in diameter were inoculated into induction medium containing different mass concentrations of 6-BA and NAA. After about 8 weeks of culture, the proliferation of the adventitious buds was statistically analyzed. The results showed that when the 6-BA concentration was 3.0 mg / L and the NAA concentration was 0.5-1.0 mg / L, the adventitious bud proliferation coefficient reached approximately 5, significantly higher than that of other combinations. Among them, when the NAA concentration was 0.5 mg / L, the average proliferation coefficient reached a maximum of 5.17. Therefore, the optimal medium for adventitious bud proliferation was determined to be 3.0 mg / L 6-BA + 0.5 mg / L NAA.

[0047] Experimental Example 3: Effect of potato flour on adventitious bud induction

[0048] Different concentrations of potato flour (0, 20, 40, 60, 80, 100, 120 g / L) were added to the basal induction medium (1 / 2MS + 3.0 mg / L 6-BA + 0.5 mg / L NAA). 20-30 explants were used each time. The experiment was repeated 3 times. After 40 days, the number of buds was counted and the induction rate was calculated, as shown in Table 3:

[0049] Table 3 Effects of different potato flour contents on adventitious bud induction

[0050]

[0051] As can be seen from the above, adding the potato flour of different contents, the induction rate does not have a significant effect, all about 98% (Table 3), but there is a significant difference in the number of sprouts of induction. When the potato flour content is 60-80g / L, the number of sprouts of induction is all about 6, significantly higher than other, while ensuring high induction rate, realizing the optimal number of sprouts, and cost controllable, therefore, after considering actual production cost, germination rate and number of sprouts, determining that the optimum content of adding potato is 60g / L.

[0052] Experimental Example 4: Effect of potato flour on adventitious bud proliferation

[0053] Different concentrations of potato flour (0, 20, 40, 60, 80, 100, and 120 g / L) were added to the basal induction medium (1 / 2MS + 3.0 mg / L 6-BA + 0.5 mg / L NAA). 20-30 explants were used each time, and the experiment was repeated three times. After 60 days, the number of buds was counted and the proliferation coefficient was calculated.

[0054] like Figure 1 As shown, adding different levels of potato flour resulted in significant differences in the proliferation coefficient of adventitious sprouts. When the potato flour content was between 60-120 g / L, the proliferation coefficient reached approximately 6. When the potato flour content reached 80 g / L, the average proliferation coefficient reached 6.3. Therefore, the optimal concentration of potato flour was determined to be 80 g / L.

[0055] Experimental Example 5: Effects of different concentrations of NAA and I BA on rooting of adventitious buds

[0056] The uniformly grown seedlings of about 3 cm in diameter were inoculated into rooting medium containing NAA or IBA at concentrations of 0, 0.1, 0.5, and 1.0 mg / L, respectively. 20-30 explants were used each time. The experiment was repeated three times. The rooting status was statistically analyzed after 60 days, as shown in Table 4:

[0057] Table 4: Effects of different concentrations of NAA and I BA on rooting of adventitious buds

[0058]

[0059]

[0060] As shown above, when adventitious buds reached approximately 3 cm in length, they were inoculated into rooting media supplemented with various concentrations of NAA or IBA. The results showed that the rooting rate, plant height, and root length in media supplemented with NAA or IBA were significantly higher than in media without growth regulators, while no significant differences were observed in stem diameter or number of roots. When NAA or IBA concentrations ranged from 0.1 to 1.0 mg / L, the average rooting rate reached over 92%, with no significant differences. However, when NAA or IBA concentrations were 0.5 or 1.0 mg / L, the average plant height reached over 10.24 cm, significantly higher than at other concentrations. At these concentrations, the average rooting rate reached a maximum of 98%, the average number of leaves reached 6.8, the average stem diameter exceeded 5.37 mm, the average number of roots reached over 6.40, and the average root length reached approximately 1.82 cm. Therefore, the optimal medium for rooting was determined to be 1 / 2 MS supplemented with 0.5 mg / L NAA or 1 / 2 MS supplemented with 0.5 mg / L IBA.

[0061] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid propagation of a new variety of Dendrobium officinale, characterized in that: The following steps are involved: S1. Select new lateral buds of the current year, remove the leaf sheaths and retain the buds, and sequentially rinse with running water, soak in 75% alcohol, disinfect with 0.1% mercuric chloride, and rinse with sterile water; S2, inoculating the sterilized stem segments with buds into an induction medium, wherein the induction medium is 1 / 2 MS medium supplemented with 3.0 mg / L 6-BA, 0.5 mg / L NAA, and 60 g / L potato flour; S3, cutting the induced adventitious buds and inoculating them into a proliferation medium, wherein the proliferation medium is 1 / 2 MS medium supplemented with 3.0 mg / L 6-BA, 0.5 mg / L NAA, and 80 g / L potato flour; S4. The proliferated adventitious buds were inoculated into a rooting medium, wherein the rooting medium was 1 / 2 MS medium supplemented with 0.5 mg / L NAA or 0.5 mg / L IBA.

2. A culture medium for inducing adventitious buds of new varieties of Dendrobium officinale, characterized by: The culture medium comprises 1 / 2 MS basal medium, 3.0 mg / L 6-BA, 0.5 mg / L NAA and 60 g / L potato flour.

3. A culture medium for the proliferation of adventitious buds of new varieties of Dendrobium officinale, characterized by: The culture medium comprises 1 / 2 MS basal medium, 3.0 mg / L 6-BA, 0.5 mg / L NAA and 80 g / L potato flour.

4. A culture medium for rooting adventitious buds of a new variety of Dendrobium officinale, characterized by: The culture medium contains 1 / 2 MS basal medium and 0.5 mg / L NAA or 0.5 mg / L IBA.

5. The culture medium according to any one of claims 2 to 4, characterized in that: In each stage, 20 g / L sucrose and 7.5 g / L carrageenan were added to the culture medium, the pH was 5.8-6.0, the culture temperature was 24±2° C., the light intensity was 1000-1200 lx, and the light intensity was 14 hours per day.

Images