A method for in-vitro culture of rhododendron anthopogon using flower stalk as explant

Abstract

The application discloses a kind of flower stalk as explant's in vitro culture method of Rhododendron simsii, belongs to plant tissue culture technical field.The method includes the following steps: after the flower stalk of Rhododendron simsii is treated with disinfectant, inoculation is carried out to induction medium, differentiation produces adventitious bud, and then inoculation is carried out to proliferation culture medium and rooting culture medium in sequence to obtain well-grown single seedling.The method can induce differentiation of the flower stalk of Rhododendron simsii to produce adventitious bud, avoid the inevitable damage to Rhododendron simsii bulb caused by cutting bulb, provide new material for in vitro rapid propagation of Rhododendron simsii, and have important significance for protection and effective utilization of Rhododendron simsii germplasm resource.The application solves the key technical bottleneck of explant selection and disinfection in Rhododendron simsii tissue culture, greatly reduces the production cost of Rhododendron simsii factory production and new variety breeding and propagation.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for in vitro culture of amaryllis using pedicels as explants. Background Technology

[0002] Amaryllis (Hippeastrum vittatum), also known as the solitary hibiscus, is a perennial bulbous herbaceous plant belonging to the genus Hippeastrum in the family Amaryllidaceae. It originates from tropical regions of the Americas, including Peru and Brazil. With its bright green, long leaves, upright flower stalks, large flowers, and vibrant colors, it is highly valued for its ornamental qualities. It is suitable for flower borders, potted plants, and cut flowers, and is very popular among gardening enthusiasts and consumers. In recent years, amaryllis has been widely introduced and promoted as a trendy flower, and has a broad market prospect in China.

[0003] Traditional propagation methods for amaryllis typically include seed sowing, natural bulb division, and scale cuttings. However, these conventional methods are inefficient, slow, require large quantities of bulbs, and are costly, making it difficult to meet market demand. Tissue culture, on the other hand, offers a high propagation coefficient and produces seedlings with consistent traits, facilitating commercial and industrial production. Currently, amaryllis tissue culture primarily uses bulb cuttings as explants. However, imported amaryllis bulbs carry significantly higher levels of foreign pathogenic fungi in their roots than in other parts of the plant. Furthermore, using bulb cuttings as explants causes irreversible damage to the amaryllis bulbs, severely hindering the industrial development of amaryllis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for in vitro culture of amaryllis using flower stalks as explants, in order to overcome the shortcomings of existing amaryllis tissue culture methods, such as high contamination rate, high cost, and serious restriction on the industrialized production of amaryllis tissue culture by cutting bulbs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for in vitro culture of amaryllis using pedicels as explants includes the following steps:

[0007] 1) Selection of explants: Select healthy, disease-free amaryllis with unopened flower stalks, cut the flower stalks at the base of the flowers as explants, and then disinfect the flower stalks.

[0008] 2) Induction of differentiation culture: The sterilized flower stalks were inoculated into the induction of differentiation culture medium. The culture temperature was controlled at 22-25℃, the light intensity was 2500-3000 lx, the photoperiod was 9.5-10.5 h / d, and adventitious shoots were obtained after 30-40 days of culture.

[0009] 3) Subculture and proliferation: Adventitious shoots are inoculated into proliferation medium, and the culture temperature is controlled at 22-25℃, the light intensity is 2500-3000 lx, the photoperiod is 9.5-10.5 h / d, and adventitious shoots that have grown to 2-3 cm are obtained after 30-40 days of culture.

[0010] 4) Rooting culture: Transfer the adventitious buds that have grown to 2-3cm to the rooting medium. The culture temperature is controlled at 22-25℃, the light intensity is 2500-3000lx, the photoperiod is 9.5-10.5h / d, and the culture is carried out for 25-50 days.

[0011] Further, the disinfection method in step 1) is as follows: cut the flower stalk into 5±0.2cm lengths, soak it in a 1-1.1% detergent solution for 10-15 minutes, rinse it with running water for 60-70 minutes, transfer it to a clean bench, cut the flower stalk at the base of the flower as the explant, soak it in 75% ethanol for 30-35 seconds, then disinfect it with 0.1% HgCl2 for 20 minutes, and finally rinse it repeatedly with sterile water 4-6 times. After the disinfected explant is cut into small flower stalk segments of 1±0.1cm length, it is laid flat in the induction medium.

[0012] Further, the method for preparing the differentiation induction medium in step 2) is as follows: add 1.0 mg·L⁻¹ to MS medium. -1 6-BA and 0.1 mg·L -1 NAA.

[0013] Further, the preparation method of the proliferation medium in step 3) is as follows: add 2.0 mg·L⁻¹ to MS medium. -1 6-BA, 1.0 mg·L -1 KT, 0.6 mg·L -1 NAA and 30g·L -1 sucrose.

[0014] Furthermore, the rooting medium in step 4) is prepared by adding 15 g·L⁻¹ to 1 / 2 MS medium. -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA.

[0015] By adopting the above technical solution, the beneficial effects of the present invention are as follows: The method of the present invention can induce the differentiation of amaryllis flower stalks to produce adventitious buds, avoid irreversible damage to amaryllis bulbs caused by cutting the bulbs, provide new materials for the rapid in vitro propagation of amaryllis, solve the key technical bottlenecks of explant selection and disinfection in amaryllis tissue culture, greatly reduce the production cost of amaryllis industrial production and new variety breeding and propagation, and have important significance for the protection and effective utilization of amaryllis germplasm resources. Detailed Implementation

[0016] The present invention will be described in detail below with reference to specific embodiments.

[0017] The raw materials used in this invention are described below:

[0018] The amaryllis plant is a healthy, disease-free new amaryllis variety, 'Snowy Dawn', cultivated in the greenhouse of the Fujian Provincial Forestry Science and Technology Experiment Center.

[0019] MS medium is currently the most widely used plant tissue culture medium. 6-BA: 6-benzylaminopurine; NAA: naphthaleneacetic acid; KT: chlorpyrifos; IBA: indolebutyric acid.

[0020] Example 1

[0021] Selection of HgCl2 treatment time during explant disinfection

[0022] Select healthy, disease-free, and unopened amaryllis flower stalks. Use the flower stalks at the base of the flowers as explants. Disinfect the explants as follows: cut the flower stalks to approximately 5cm lengths, soak them in a 1% detergent solution for 10 minutes, rinse with running water for 60 minutes, transfer to a clean bench, cut the flower stalks at the base of the flowers, soak them in 75% ethanol for 30 seconds, then disinfect with 0.1% HgCl2 (for 10, 15, 20, and 25 minutes), and finally rinse repeatedly with sterile water 5 times. Cut the disinfected explants into 1cm segments of flower stalks and lay them flat on the induction medium.

[0023] The pollution rate and survival rate were statistically analyzed, and the results are shown in Table 1.

[0024] Contamination rate / % = Number of contaminated explants / Total number of inoculated explants × 100

[0025] Survival rate = 100% - pollution rate - mortality rate.

[0026] Table 1. Disinfection effect of HgCl2 at different treatment times

[0027] 10 30.0a 17.0d 53.0c 15 20.0b 24.3c 55.7b 20 6.7d 26.6b 66.7a 25 13.3c 32.4a 54.3bc

[0028] *: Different lowercase letters after the values ​​in the same column indicate significant differences (P<0.05), the same applies below.

[0029] Table 1 shows that different treatment times with HgCl2 have a significant impact on the disinfection effect on the petioles of Amaryllis. The contamination rate gradually decreases with increasing disinfection time. The best effect is achieved when the disinfection time is 20 minutes, with a contamination rate of 6.7%, a mortality rate of 26.6%, and a survival rate of 66.7%.

[0030] Example 2

[0031] Screening of differentiation induction media

[0032] Sterilized flower stalks were inoculated into an induction differentiation medium to obtain adventitious shoots. The culture temperature was controlled at 22-25℃, the light intensity was 2500-3000 lx, and the photoperiod was 10 h / d.

[0033] Screening method for induction differentiation medium: MS + 0.1 mg·L⁻¹ -1 NAA was used as the basal medium, with different mass concentrations of 6-BA (0, 1, 2, 3 mg·L⁻¹) added. -1 Four different culture media (four treatments) were used, with 30 flower stalks per treatment group and three replicates.

[0034] Table 2. Effects of different concentrations of 6-BA on the induction of pedicel in Amaryllis.

[0035]

[0036]

[0037] After disinfection, amaryllis flower stalks were inoculated onto induction medium. After 30 days of culture, the germination and growth of adventitious buds were observed and recorded daily. After 40 days of culture, adventitious buds from the flower stalks of all treatment groups germinated. Table 2 shows that within a certain concentration range, the induction rate increased with increasing 6-BA concentration. When the concentration reached 1.0 mg·L⁻¹, the induction rate increased further. -1 At that time, the induction rate of flower stalks reached 46.7%.

[0038] Therefore, the present invention selects the following induction differentiation medium: MS medium supplemented with 1.0 mg·L⁻¹ -1 6-BA and 0.1 mg·L -1 NAA.

[0039] Example 3

[0040] Screening of proliferation culture media

[0041] Adventitious shoots generated by induced differentiation were inoculated into proliferation medium for cultivation to obtain well-growing adventitious shoots. The cultivation temperature was controlled at 22–25℃, the light intensity at 2500–3000 lx, and the photoperiod at 10 h / d. The proliferation medium was selected using MS medium supplemented with 30 g·L⁻¹. -1 Sucrose as the basal medium, supplemented with 6-BA, KT, and NAA, using L9(3) 4Orthogonal experimental design was used. Induced adventitious shoots were inoculated into different proliferation media, with 30 shoots inoculated into each medium, replicated three times. After 30 days of culture, growth was observed, and the proliferation coefficient of the adventitious shoots was calculated and analyzed (proliferation coefficient = total number of adventitious shoots / initial number of inoculated adventitious shoots). The effects of different combinations of exogenous plant growth regulators on the state and proliferation coefficient of adventitious shoots were compared to screen for the optimal proliferation medium.

[0042] Table 3 Results of the orthogonal experiment for the proliferation culture of *Hippeastrum*

[0043]

[0044]

[0045] Table 4. Results of Variance Analysis of Orthogonal Experiments for Induction Culture

[0046] A(6-BA) 34.15 2 17.07 85.99** B(KT) 3.00 2 1.50 7.55** C(NAA) 8.94 2 4.47 22.51** error 3.09 2 1.55 sum 50.07

[0047] * indicates that the difference is significant at the p=0.01 level.

[0048] Tables 3 and 4 show that different combinations of plant growth regulators significantly affected the proliferation coefficient of adventitious shoots in *Amaryllis*. Adding appropriate concentrations of 6-BA, KT, and NAA was beneficial for adventitious shoot proliferation. Among the nine treatments, treatment 8 had the highest average proliferation coefficient, reaching 5.66. Orthogonal analysis indicated that the optimal combination of medium for *Amaryllis* proliferation was A3B2C3, i.e., MS + 2.0 mg·L⁻¹. -1 6-BA + 1.0 mg·L -1 KT+0.6mg·L -1 NAA+30g·L -1 sucrose.

[0049] The three factors, A (6-BA), B (KT), and C (NAA), had different degrees of influence on the proliferation of adventitious shoots in *Amaryllis rubra*, with their influence ranking as A > C > B. That is, 6-BA had the most significant effect, followed by NAA, and then KT. The analysis of variance results (Table 4) showed that all three factors had highly significant effects on the proliferation coefficient of *Amaryllis rubra* (P < 0.01).

[0050] Therefore, the proliferation medium selected in this invention is: MS medium supplemented with 2.0 mg·L⁻¹ -1 6-BA, 1.0 mg·L - 1 KT, 0.6 mg·L -1 NAA and 30g·L -1 sucrose.

[0051] Example 4

[0052] Screening of rooting culture media

[0053] Adventitious shoots that have grown to 2–3 cm were transferred to rooting medium. The culture temperature was controlled at 22–25℃, the light intensity at 2500–3000 lx, and the photoperiod at 10 h / d. The selection method for the rooting medium was as follows: adventitious shoots that grew to 2–3 cm were transferred to rooting medium. The medium was prepared using 1 / 2 MS + 15 g·L⁻¹. -1 Sucrose + 0.2 g·L -1 Activated carbon was used as the basic culture medium, with different concentration gradients (0.2, 0.4, 0.6 mg·L⁻¹) added. -1 Six rooting media combinations were prepared, including NAA and IBA. Thirty bottles of each media were used, with five adventitious buds per bottle, and the media were replicated three times. After 45 days of culture, rooting progress was observed and recorded daily to select the optimal rooting medium.

[0054] Table 5. Effects of different concentrations of NAA and IBA on rooting of Amaryllis.

[0055]

[0056] Observations revealed that white root points began to appear approximately 25 days after rooting culture. After 45 days of rooting culture, the growth status of the rooted seedlings was observed and recorded daily. At 50 days, the average number of roots, average root length, and average rooting rate of the rooted seedlings were calculated. Table 5 shows that both NAA and IBA effectively promoted root formation, with a rooting rate of 100%. Compared to NAA, IBA had a more significant effect on the rooting of amaryllis. The effective rate of IBA was 0.4 mg / L. -1 At that time, the average number of roots was as high as 4.3, and the average root length was 4.88cm.

[0057] Therefore, the rooting medium selected in this invention is: 15 g·L⁻¹ added to 1 / 2 MS medium. -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA.

[0058] Example 5

[0059] A method for in vitro culture of amaryllis using pedicels as explants includes the following steps:

[0060] 1) Selection of explants

[0061] Select healthy, disease-free, and unopened amaryllis flower stalks. Cut the flower stalks at the base of the flowers as explants. Then, disinfect the flower stalks as follows: cut the flower stalks into 5±0.2cm lengths, soak them in a 1% detergent solution for 10 minutes, rinse them under running water for 60 minutes, transfer them to a clean bench, cut the flower stalks at the base of the flowers as explants, soak them in 75% ethanol for 30 seconds, then disinfect them with 0.1% HgCl2 for 20 minutes, and finally rinse them repeatedly with sterile water 5 times. After disinfecting, cut the flower stalks into small segments of 1±0.1cm length and lay them flat on the induction medium.

[0062] 2) Induced differentiation culture

[0063] Add 1.0 mg·L⁻¹ to MS medium. -1 6-BA and 0.1 mg·L -1 NAA was used to obtain the induction differentiation medium. The sterilized flower stalks were inoculated into the induction differentiation medium, and the culture temperature was controlled at 22-25℃, the light intensity was 2500-3000 lx, the photoperiod was 9.5-10.5 h / d, and adventitious shoots were obtained after 30 days of culture.

[0064] 3) Subculture and proliferation

[0065] To obtain a proliferation medium, 2.0 mg·L⁻¹ 6-BA, 1.0 mg·L⁻¹ KT, 0.6 mg·L⁻¹ NAA, and 30 g·L⁻¹ sucrose were added to MS medium. Adventitious shoots were inoculated into the proliferation medium and cultured at a temperature of 22–25 °C, a light intensity of 2500–3000 lx, and a photoperiod of 9.5–10.5 h / d. After 30 days of culture, adventitious shoots that had grown to 2–3 cm were obtained.

[0066] 4) Rooting culture

[0067] Add 15 g·L to 1 / 2 MS medium -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA was used to obtain the rooting medium. Adventitious buds that had grown to 2-3 cm were transferred to the rooting medium and cultured at a temperature of 22-25℃, a light intensity of 2500-3000 lx, a photoperiod of 9.5-10.5 h / d, for 45 days.

[0068] Example 6

[0069] A method for in vitro culture of amaryllis using pedicels as explants includes the following steps:

[0070] 1) Selection of explants

[0071] Select healthy, disease-free, and unopened amaryllis flower stalks. Cut the flower stalks at the base of the flowers as explants. Then, disinfect the flower stalks as follows: cut the flower stalks into 5±0.2cm lengths, soak them in a 1% detergent solution for 15 minutes, rinse them under running water for 70 minutes, transfer them to a clean bench, cut the flower stalks at the base of the flowers as explants, soak them in 75% ethanol for 35 seconds, then disinfect them with 0.1% HgCl2 for 20 minutes, and finally rinse them repeatedly with sterile water 6 times. After disinfecting, cut the flower stalks into small segments of 1±0.1cm length and lay them flat on the induction medium.

[0072] 2) Induced differentiation culture

[0073] Add 1.0 mg·L⁻¹ to MS medium. -1 6-BA and 0.1 mg·L -1 NAA was used to obtain the induction differentiation medium. The sterilized flower stalks were inoculated into the induction differentiation medium, and the culture temperature was controlled at 22-25℃, the light intensity was 2500-3000 lx, the photoperiod was 9.5-10.5 h / d, and adventitious shoots were obtained after 35 days of culture.

[0074] 3) Subculture and proliferation

[0075] To obtain a proliferation medium, 2.0 mg·L⁻¹ 6-BA, 1.0 mg·L⁻¹ KT, 0.6 mg·L⁻¹ NAA, and 30 g·L⁻¹ sucrose were added to MS medium. Adventitious shoots were inoculated into the proliferation medium and cultured at a temperature of 22–25 °C, a light intensity of 2500–3000 lx, and a photoperiod of 9.5–10.5 h / d for 35 days to obtain adventitious shoots that had grown to 2–3 cm.

[0076] 4) Rooting culture

[0077] Add 15 g·L to 1 / 2 MS medium -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA was used to obtain the rooting medium. Adventitious buds that had grown to 2-3 cm were transferred to the rooting medium and cultured at a temperature of 22-25℃, a light intensity of 2500-3000 lx, a photoperiod of 9.5-10.5 h / d, for 50 days.

[0078] Example 7

[0079] A method for in vitro culture of amaryllis using pedicels as explants includes the following steps:

[0080] 1) Selection of explants

[0081] Select healthy, disease-free, and unopened amaryllis flower stalks. Cut the flower stalks at the base of the flowers as explants. Then, disinfect the flower stalks as follows: cut the flower stalks into 5±0.2cm lengths, soak them in a 1% detergent solution for 12 minutes, rinse them under running water for 65 minutes, transfer them to a clean bench, cut the flower stalks at the base of the flowers as explants, soak them in 75% ethanol for 33 seconds, then disinfect them with 0.1% HgCl2 for 20 minutes, and finally rinse them repeatedly with sterile water 5 times. After disinfecting, cut the flower stalks into small segments of 1±0.1cm length and lay them flat on the induction medium.

[0082] 2) Induced differentiation culture

[0083] Add 1.0 mg·L⁻¹ to MS medium. -1 6-BA and 0.1 mg·L -1 NAA was used to obtain the induction differentiation medium. The sterilized flower stalks were inoculated into the induction differentiation medium, and the culture temperature was controlled at 22-25℃, the light intensity was 2500-3000 lx, the photoperiod was 9.5-10.5 h / d, and adventitious shoots were obtained after 40 days of culture.

[0084] 3) Subculture and proliferation

[0085] To obtain a proliferation medium, 2.0 mg·L⁻¹ 6-BA, 1.0 mg·L⁻¹ KT, 0.6 mg·L⁻¹ NAA, and 30 g·L⁻¹ sucrose were added to MS medium. Adventitious shoots were inoculated into the proliferation medium and cultured at a temperature of 22–25 °C, a light intensity of 2500–3000 lx, and a photoperiod of 9.5–10.5 h / d for 40 days to obtain adventitious shoots that had grown to 2–3 cm.

[0086] 4) Rooting culture

[0087] Add 15 g·L to 1 / 2 MS medium -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA was used to obtain the rooting medium. Adventitious buds that had grown to 2-3 cm were transferred to the rooting medium and cultured at a temperature of 22-25℃, a light intensity of 2500-3000 lx, a photoperiod of 9.5-10.5 h / d, for 50 days.

[0088] The above are merely specific embodiments of the present invention, but the design concept of the present invention is not limited thereto. Any non-substantial modifications made to the present invention using this concept shall be considered as infringing upon the protection scope of the present invention.

Claims

1. A method for in vitro culture of Amaryllis using pedicels as explants, characterized in that, Includes the following steps: 1) Selection of explants: Select healthy, disease-free, and unopened flower stalks of amaryllis. Cut the flower stalks into 5±0.2 cm lengths, soak them in 1-1.1% detergent solution for 10-15 minutes, rinse them under running water for 60-70 minutes, transfer them to a clean bench, cut the flower stalks at the base of the flowers as explants, soak them in 75% ethanol for 30-35 seconds, then disinfect them with 0.1% HgCl2 for 20 minutes, and finally rinse them repeatedly with sterile water 4-6 times. After disinfecting, cut the explants into small flower stalk segments of 1±0.1 cm length and lay them flat on the induction medium. 2) Induction of differentiation culture: The sterilized flower stalks are inoculated into the induction of differentiation culture medium. The culture temperature is controlled at 22~25℃, the light intensity is 2500~3000lx, the photoperiod is 9.5~10.5 h / d, and the culture is carried out for 30~40 days to obtain adventitious buds. The method for preparing the induction differentiation medium is as follows: add 1.0 mg·L⁻¹ to MS medium. -1 6-BA and 0.1 mg·L -1 NAA; 3) Subculture and proliferation: Adventitious shoots are inoculated into proliferation medium, and the culture temperature is controlled at 22~25℃, the light intensity is 2500~3000lx, the photoperiod is 9.5~10.5 h / d, and the culture is carried out for 30~40 days to obtain adventitious shoots that have grown to 2~3 cm. The method for preparing the proliferation medium is as follows: add 2.0 mg·L⁻¹ to MS medium. -1 6-BA, 1.0 mg·L -1 KT, 0.6 mg·L -1 NAA and 30 g·L -1 sucrose; 4) Rooting culture: Transfer adventitious buds that have grown to 2-3 cm to rooting medium, control the culture temperature at 22-25℃, light intensity at 2500-3000 lx, photoperiod at 9.5-10.5 h / d, and culture for 25-50 days; The method for preparing the rooting medium is as follows: add 15 g·L to 1 / 2 MS medium. -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA.

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