An in vitro regeneration culture method of begonia fimbristipula

Abstract

This invention provides a method for in vitro regeneration culture of Begonia clavatum, comprising the following steps: S1: Explant selection and treatment: Selecting leaves, leaf bases, or stem segments from aseptic seedlings of Begonia clavatum, and obtaining explants after cutting and treatment; S2: Bud induction culture: Inoculating the explants obtained in step S1 into a bud induction medium for bud induction culture to induce adventitious buds; S3: Bud proliferation culture: Transferring the adventitious buds induced in step S2 into a bud proliferation medium for bud proliferation culture for 30 days. Using the method of this invention, with leaf bases as explants, the regeneration cycle can be shortened from the traditional 130 days to 45 days, a reduction of approximately 65%. This invention effectively solves the problems of slow adventitious bud differentiation, low proliferation efficiency, and excessively long genetic transformation verification cycle in existing Begonia clavatum in vitro regeneration systems, providing effective technical support for the genetic transformation and molecular breeding of Begonia clavatum.

Description

Technical Field

[0001] This invention relates to an in vitro regeneration culture method, and more particularly to an in vitro regeneration culture method for Begonia spp. Background Technology

[0002] Begonia spp. are highly valued for their colorful leaves and diverse flower shapes, playing an important role in horticultural breeding and indoor and outdoor landscaping. Begonia masoniana, also known as the Iron Cross Begonia, is a perennial herbaceous plant belonging to the genus Begonia in the family Begoniaceae. Its unique and stably inherited leaf markings and pubescent structure make it an ideal material for studying plant morphology and development, and it has significant applications in functional genomics and molecular breeding research.

[0003] Genetic transformation relies heavily on efficient in vitro regeneration systems, and significant progress has been made in the in vitro culture of Begonia species. Studies have shown that leaves, leaf bases, and stem segments of Begonia species can all serve as explants for regeneration, and the combination of 6-BA and NAA is a classic hormone system for in vitro regeneration of Begonia species. For example, tuberous Begonias exhibited a 100% callus induction rate and 7.2 adventitious buds induced on MS medium supplemented with 1.5 mg / L 6-BA and 0.2 mg / L NAA. Leaf base explants of Begonia species also demonstrated strong callus induction and differentiation capabilities.

[0004] However, the existing in vitro regeneration system of Begonia truncatum still has prominent problems in practical applications: First, the differentiation of adventitious buds is slow, taking about 70 days; second, the proliferation efficiency is low, making it difficult to meet the demand for the number of regenerated seedlings for genetic transformation; third, the regeneration speed of different explants varies greatly, and there is currently a lack of systematic comparison and targeted optimization. The regeneration after transformation takes a long time, and the preliminary verification cycle is too long, which seriously affects the progress of gene function verification. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an in vitro regeneration culture method for Begonia spp., which can solve the problems of slow adventitious bud differentiation, low proliferation efficiency, and excessively long genetic transformation verification cycle.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] A method for in vitro regeneration culture of Begonia spp. includes the following steps:

[0008] S1: Explant selection and treatment:

[0009] Leaves, leaf bases, or stem segments from healthy, pollution-free, sterile seedlings of Begonia truncatum were selected, cut, and processed to obtain explants.

[0010] S2: Bud induction culture:

[0011] The explants obtained in step S1 were inoculated into a bud induction medium for bud induction culture to induce adventitious buds.

[0012] S3: Bud proliferation culture:

[0013] The adventitious buds induced in step S2 were transferred into bud proliferation medium for bud proliferation culture for 30 days.

[0014] Furthermore, in step S1 of the present invention, during the cutting process, the leaves are cut into small pieces of 0.8cm×0.8cm by a sterile scalpel, the leaf base is cut into small pieces of 0.8cm×0.8cm by a sterile scalpel while retaining the leaf base tissue, and the stem segments are cut into small segments of 0.5-1.0cm in length by a sterile scalpel.

[0015] Further, in step S2 of the present invention, MS + (0.05-0.1) mg / L TDZ + (0.5-1.0) mg / L 6-BA + (0.1-0.2) mg / L NAA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8-6.2.

[0016] Furthermore, in step S2 of this invention, the conditions for bud induction culture are a temperature of 22-26℃, a light intensity of 1000 lx, and a photoperiod of 16h light / 8h darkness. When leaf bases are used as explants, the bud induction time is 15 days, with an induction rate of 100%; when stem segments are used as explants, the bud induction time is 25 days, with an induction rate of 100%; and when leaves are used as explants, the bud induction time is greater than 30 days.

[0017] Further, in step S2 of the present invention, MS + (0.5-1.5) mg / L 6-BA + (0.1-0.3) mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH is 5.8-6.2.

[0018] Furthermore, in step S3 of this invention, the conditions for bud proliferation culture are a temperature of 22-26℃, a light intensity of 1000 lx, and a photoperiod of 16h light / 8h darkness. After 30 days of bud proliferation culture, the proliferation ratio reaches 8.61 times. When using the leaf base as an explant, the regeneration cycle is 45 days, which is 65% shorter than the traditional method using leaves as explants.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. This invention is the first to systematically compare the regeneration rates of three explants (leaf, leaf base, and stem segment) of Begonia 'Imperata', confirming that the leaf base is the optimal explant type. Experimental results show that under the same optimized culture medium conditions, leaf base explants sprout 15 days after inoculation, stem segments require 25 days, while leaves require more than 30 days. This is likely because the leaf base tissue is loose, allowing cells with strong meristematic ability around the vascular bundles to easily sense hormone signals and directly initiate division, saving the time cost of the callus stage.

[0021] 2. This invention optimizes the hormone ratio and selects the optimal bud induction culture medium. In this medium, TDZ and 6-BA work synergistically, allowing explants to directly form adventitious buds. This is a direct organogenesis pathway, avoiding the time delay caused by the indirect regeneration pathway of "root first, then bud." Among the three explants, the induction rate of leaf base and stem segment both reached 100%, while the induction rate of leaf was relatively low but still has application value.

[0022] 3. This invention optimizes the bud proliferation medium and selects the optimal bud proliferation medium. In this medium, a high concentration of cytokinin and a suitable concentration of auxin are used in combination, and the proliferation multiple can reach 8.61 times (based on fresh weight). The buds grow vigorously, the number of leaves increases significantly, the leaves are dark green, and the number of subcultures is significantly reduced.

[0023] 4. Using the optimized method of this invention, when using the leaf base as the explant, the regeneration cycle is shortened from 130 days in the traditional method (using the leaf as the explant) to 45 days, with an overall reduction of 65%. Stem segment explants can also achieve rapid regeneration (100% induction rate, budding in 25 days), providing a variety of explant selection options for different experimental needs.

[0024] 5. In the method of this invention, the induction rate of leaf base and stem segment both reach 100%, and the 8.61-fold multiplication factor ensures a sufficient number of screenable explants, laying a good foundation for genetic transformation. At the same time, the significant reduction in culture time effectively reduces the risk of explant browning and somatic cell mutation, making the transformation system more stable and reliable.

[0025] 6. The method of the present invention is not only applicable to Begonia spp., but can also provide a reference for the optimization of in vitro regeneration systems of other Begonia species, and has good prospects for promotion and application. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0027] Figure 1 This is a diagram showing the induction process one month after inoculation when the explants were stem segments and the bud induction medium was BY3 in Experiment Example 1 of this invention.

[0028] Figure 2 This is a diagram showing the induction process one month after inoculation when the explants were leaf bases and the bud induction medium was BY3 in Experiment Example 1 of this invention.

[0029] Figure 3 This is a diagram showing the induction process one month after inoculation when the explants were leaves and the bud induction medium was BY3 in Experiment Example 1 of this invention.

[0030] Figure 4 This is a diagram showing the induction process of the explants in Experiment Example 1 of this invention, after bud induction and bud proliferation culture medium of CY4, one month after inoculation. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0032] Example

[0033] Perform in vitro regeneration culture of Begonia spp. following these steps:

[0034] S1: Explant Selection and Treatment

[0035] Leaves, leaf bases, or stem segments from aseptic seedlings of Begonia truncatum were selected and cut to obtain explants. The leaves were cut into 0.8cm×0.8cm pieces with a sterile scalpel, the leaf bases were cut into 0.8cm×0.8cm pieces with a sterile scalpel while retaining the leaf base tissue, and the stem segments were cut into 0.5-1.0cm long segments with a sterile scalpel.

[0036] S2: Bud induction culture

[0037] The explants obtained in step S1 were inoculated into the shoot induction medium for shoot induction culture to induce adventitious shoots. The conditions for shoot induction culture were: temperature 22-26℃, light intensity 1000 lx, and photoperiod of 16h light / 8h dark.

[0038] The bud induction culture medium is shown in Table 1:

[0039] BY1 MS + 0.1 mg / L TDZ + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY2 MS + 0.05 mg / L TDZ + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY3 MS + 0.1 mg / L TDZ + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY4 MS + 0.05 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY5 MS + 0.1 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY6 MS + 0.1 mg / L TDZ + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY7 MS + 0.05 mg / L TDZ + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY8 MS + 0.15 mg / L TDZ + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY9 MS + 0.1 mg / L TDZ + 0.1 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY10 MS + 0.1 mg / L TDZ + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar BY11 MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar

[0040] Table 1

[0041] Note: BY3 is the optimal formula.

[0042] The results of leaf base culture for each bud induction medium formulation are shown in Table 2:

[0043] BY1 10 10 10 80 >20 The new shoots are small and grow relatively slowly. BY2 10 10 7 100 25 The new shoots are small and grow relatively slowly. BY3 10 10 11 100 15 The new shoots are robust and grow relatively quickly. BY4 10 10 6 100 25 The new shoots are small, grow relatively slowly, and root directly. BY5 10 10 9 100 17 The new shoots are robust, grow quickly, and root directly. BY6 10 10 9 100 >20 The new shoots are robust, grow quickly, and root directly. BY7 10 10 6 100 >20 The new shoots are small and grow relatively slowly. BY8 10 10 8 100 >20 The new shoots are robust and grow relatively quickly. BY9 10 10 9 100 >20 The new shoots are robust and grow relatively quickly. BY10 10 8 5 80 >20 The new shoots are small and grow relatively slowly. BY11 10 8 4 80 >20 The new shoots are small and grow relatively slowly.

[0044] Table 2

[0045] The stem segment culture results for each bud induction medium formulation are shown in Table 3:

[0046] BY1 10 2 8 25 >20 The new shoots are small and grow relatively slowly. BY2 10 1 3 10 >20 The new shoots are small and grow relatively slowly. BY3 10 3 19 30 25 The new shoots are robust and grow relatively quickly. BY4 10 0 0 0 >30 The new shoots are small and grow relatively slowly. BY5 10 3 15 30 17 The new shoots are robust, grow quickly, and root directly. BY6 10 0 0 0 >30 The new shoots are robust and grow relatively quickly. BY7 10 2 6 20 >20 The new shoots are small and grow relatively slowly. BY8 10 3 11 30 >20 The new shoots are robust and grow relatively quickly. BY9 10 2 14 20 >20 The new shoots are robust and grow relatively quickly. BY10 10 3 12 30 >20 The new shoots are small and grow relatively slowly. BY11 10 0 0 0 >30 The new shoots are small and grow relatively slowly.

[0047] Table 3

[0048] As can be seen from Tables 2 and 3:

[0049] (1) The optimal bud induction medium formula for direct budding is BY3.

[0050] BY3: Leaf base explants sprouted 15 days after inoculation, with an induction rate of 100% and a total of 11 sprouts. The new sprouts were robust and grew rapidly. Stem segment explants sprouted 25 days after inoculation, with an induction rate of 30% and a total of 19 sprouts. The new sprouts were robust and grew rapidly. This formula is the optimal sprout induction medium of this invention.

[0051] (2) The formula for the shoot induction medium for root-to-bud induction is BY4, BY5, and BY6.

[0052] BY4: The explant first forms adventitious roots, and then differentiates into buds, with the bud differentiation time exceeding 20 days.

[0053] BY5: The explant first forms adventitious roots, and then differentiates into buds. The bud differentiation time is about 17 days. The new buds are robust and grow relatively quickly.

[0054] BY6: The explant first forms adventitious roots, and then differentiates into buds. The bud differentiation time exceeds 20 days, and the new buds are robust and grow relatively quickly.

[0055] (3) The formulation containing a combination of low concentration TDZ and 6-BA is BY7.

[0056] BY7: Leaf base induction rate 100%, total number of buds 6, new buds are small and grow relatively slowly, budding time exceeds 20 days; stem segment induction rate 20%, total number of buds 6. The concentration of TDZ in this formula is low (0.05mg / L), although it is combined with 0.5mg / L 6-BA, the overall budding effect is not as good as BY3, showing fewer buds and slower growth rate.

[0057] (4) The formulation containing a high concentration of TDZ and 6-BA is BY8.

[0058] BY8: Leaf base induction rate 100%, total number of buds 8, new buds are robust and grow relatively quickly, but budding time exceeds 20 days; Stem segment induction rate 30%, total number of buds 11, new buds are robust and grow relatively quickly. In this formula, the TDZ concentration is increased to 0.15 mg / L. Although the bud quality is good, the budding speed is not as good as BY3, and the higher TDZ concentration may increase the risk of somatic cell mutation.

[0059] (5) The formulation containing a low concentration of 6-BA and TDZ is BY9.

[0060] BY9: Leaf base induction rate 100%, total number of buds 9, new buds are robust and grow relatively quickly, budding time exceeds 20 days; stem segment induction rate 20%, total number of buds 14, new buds are robust and grow relatively quickly. This formula has a low 6-BA concentration (0.1 mg / L), although the bud quality is good, the budding speed is not as good as BY3, and the stem segment induction rate is also lower.

[0061] (6) The formulation containing a high concentration of 6-BA and TDZ is BY10.

[0062] BY10: The leaf base induction rate was only 80%, with a total of 5 buds. The new buds were small and grew relatively slowly, with a budding time exceeding 20 days. The stem segment induction rate was 30%, with a total of 12 buds. The new buds were also small and grew relatively slowly. The concentration of 6-BA in this formula was too high (1.0 mg / L). Although the TDZ was 0.1 mg / L, the excessively high concentration of 6-BA actually inhibited the quality and induction rate of the buds.

[0063] (7) Formulas with slow germination or ineffective germination are BY1, BY2, and BY11.

[0064] BY1: Higher 6-BA concentration significantly delays budding time, with a leaf base induction rate of 80%, resulting in smaller new shoots and relatively slow growth.

[0065] BY2: Higher 6-BA concentration significantly delays germination time (25 days), resulting in smaller new shoots and relatively slow growth.

[0066] BY11: Without TDZ, the leaf base induction rate is only 80%, the total number of buds is the fewest (4), the new buds are smaller and grow relatively slowly; the stem segment induction rate is 0%. This formula proves that TDZ is the key hormone for bud induction in Begonia spp.

[0067] Usage recommendations:

[0068] For rapid acquisition of adventitious buds (direct organogenesis), the BY3 formula is recommended. Leaf base explants can sprout in 15 days with an induction rate of 100%.

[0069] If you need to obtain regenerated seedlings through the root-before-budding method, you can choose BY4, BY5 or BY6 formulas, among which BY5 germinates the fastest (17 days).

[0070] If you need to propagate a large number of plants but have plenty of time, you can choose the BY1 formula.

[0071] While BY7, BY8, and BY9 formulations have high induction rates, their germination speed and bud quality are inferior to BY3, and therefore they are not recommended as first-line choices. BY10 is not recommended because its high 6-BA concentration leads to a decrease in induction rate and bud quality. BY11, lacking TDZ, has the worst effect and is not recommended.

[0072] S3: Bud proliferation culture

[0073] The adventitious buds induced in step S2 were transferred into bud proliferation medium for bud proliferation culture for 30 days. The conditions for bud proliferation culture were: temperature 22-26℃, light intensity 1000 lx, and photoperiod of 16h light / 8h darkness.

[0074] The bud proliferation culture medium is shown in Table 4:

[0075] CY1 MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar CY2 MS + 1.0 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar CY3 MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar CY4 MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar CY5 MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 7 g / L agar CY6 MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7 g / L agar

[0076] Table 4

[0077] The effects of various bud proliferation culture medium formulations on bud proliferation are shown in Table 5:

[0078] CY1 0.5 0.2 1.0 8.75 7.75 It has many buds, roots, and grows well. CY2 1.0 0.05 1.0 8.32 7.32 It has many buds, roots, and is growing well. CY3 1.0 0.1 1.0 7.47 6.48 Many buds, small leaves, and good growth CY4 1.0 0.2 1.0 9.61 8.61 Many buds, many leaves, vigorous growth CY5 1.0 0.3 1.0 9.58 8.58 Many buds, many leaves, vigorous growth CY6 1.5 0.2 1.0 8.11 7.11 Many buds, small leaves, and good growth

[0079] Table 5

[0080] As can be seen from Table 5:

[0081] (1) The optimal bud proliferation medium formula is CY4.

[0082] The CY4 treatment showed the best proliferation effect, with a proliferation ratio of 8.61 times in 30 days and vigorous bud growth, making it the optimal bud proliferation culture medium of this invention.

[0083] (2) The alternative bud proliferation medium formula is CY5.

[0084] CY5: The multiplication rate reaches 8.58 times in 30 days, which is similar to CY4. It has vigorous bud growth and many leaves, and can be used as an alternative.

[0085] (3) Comparison of other formulas

[0086] CY1: The multiplication rate is 7.75 times, rooting is observed, and growth is good.

[0087] CY2: The multiplication rate is 7.32 times, with more buds and rooting phenomena.

[0088] CY3: The multiplication rate is 6.48 times, with many buds but small leaves.

[0089] CY6: The proliferation rate is 7.11 times. It produces many buds but has small leaves. Excessive 6-BA concentration may inhibit leaf growth.

[0090] Usage recommendations:

[0091] For the most efficient bud proliferation, we recommend the CY4 formula, which can multiply the buds by 8.61 times in 30 days, resulting in vigorous bud growth and many leaves.

[0092] To reduce rooting, the NAA concentration can be appropriately reduced (e.g., CY2, CY3), but the multiplication rate will decrease accordingly.

[0093] If rapid propagation is required and a small number of roots are acceptable, CY1, CY2, and CY5 can all be considered as alternatives.

[0094] CY6 is not recommended as the first choice because its high 6-BA concentration (1.5 mg / L) results in smaller leaves.

[0095] Regeneration cycle comparison

[0096] As shown in Table 6, using the optimized method of this invention, the total regeneration cycle is 45 days when using leaf base as explant (15 days for bud induction + 30 days for bud proliferation); approximately 55 days when using stem segment as explant (25 days for bud induction + 30 days for bud proliferation); and over 90 days when using leaf as explant (over 60 days for bud induction + 30 days for bud proliferation). Compared to the traditional method (using leaf as explant and conventional hormone ratios) of 130 days, the optimized method using leaf base as explant shortens the cycle by 85 days, a reduction of approximately 65%.

[0097] Bud induction 70 15 45 bud proliferation 60 30 30 total 130 45 85 (reduced by 65%)

[0098] Table 6 Comparison of regeneration cycle between the optimized method of this invention and the traditional method

[0099] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for in vitro regeneration culture of Begonia spp., characterized in that: Includes the following steps: S1: Explant selection and treatment: Leaves, leaf bases, or stem segments of sterile Begonia truncatum seedlings were selected, cut, and processed to obtain explants; S2: Bud induction culture: The explants obtained in step S1 were inoculated into a bud induction medium for bud induction culture to induce adventitious buds. S3: Bud proliferation culture: The adventitious buds induced in step S2 were transferred into bud proliferation medium for bud proliferation culture for 30 days.

2. The method for in vitro regeneration culture of Begonia spp. according to claim 1, characterized in that: In step S1, the leaves are cut into 0.8cm×0.8cm pieces with a sterile scalpel, the leaf base is cut into 0.8cm×0.8cm pieces with a sterile scalpel while retaining the leaf base tissue, and the stem segments are cut into 0.5-1.0cm long segments with a sterile scalpel.

3. The method for in vitro regeneration culture of Begonia spp. according to claim 1, characterized in that: In step S2, the bud induction medium is MS + (0.05-0.1) mg / L TDZ + (0.5-1.0) mg / L 6-BA + (0.1-0.2) mg / L NAA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8-6.

2.

4. The method for in vitro regeneration culture of Begonia spp. according to claim 1, characterized in that: In step S2, the conditions for bud induction culture are a temperature of 22-26℃, a light intensity of 1000 lx, and a photoperiod of 16h light / 8h darkness.

5. The method for in vitro regeneration culture of Begonia spp. according to claim 1, characterized in that: In step S2, the bud proliferation medium is MS + (0.5-1.5) mg / L 6-BA + (0.05-0.3) mg / L NAA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8-6.

2.

6. The method for in vitro regeneration culture of Begonia spp. according to claim 1, characterized in that: In step S3, the conditions for bud proliferation culture are a temperature of 22-26℃, a light intensity of 1000 lx, and a photoperiod of 16h light / 8h darkness.

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