In-vitro culture method of hippeastrum rutilum by taking flower stalks as explants

Abstract

The invention discloses a hippeastrum striatum in-vitro culture method with flower stalks as explants, and belongs to the technical field of plant tissue culture. The method comprises the following steps: disinfecting flower stalks of hippeastrum rutilum, inoculating the flower stalks to an induction culture medium, differentiating to generate adventitious buds, and sequentially inoculating the adventitious buds to a proliferation culture medium and a rooting culture medium for culturing to obtain single seedlings with good growth vigor. According to the method, the flower stalks of the hippeastrum hippeastrum can be induced and differentiated to generate adventitious buds, inevitable damage to the hippeastrum hippeastrum bulbs due to bulb cutting is avoided, a new material is provided for in-vitro rapid propagation of the hippeastrum hippeastrum, and the method has important significance on germplasm resource protection and effective utilization of the hippeastrum hippeastrum. According to the method, the key technical bottleneck of explant selection and disinfection in hippeastrum hippeastrum tissue culture is solved, and the production cost of industrialized production of hippeastrum hippeastrum and new variety breeding and propagation is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to an in vitro culture method of amaryllis with pedicels as explants. Background Art

[0002] Hippeastrum vittatum, also known as amaryllis, Huazhoulan, and Junzihong, is a perennial bulbous herbaceous plant of the genus Hippeastrum in the Amaryllidaceae family, native to tropical regions of the Americas such as Peru and Brazil. Its leaves are bright green and slender, its flower stems are upright, its flowers are large, and its colors are colorful. It has great ornamental value and can be used for flower landscaping, potted plants, and fresh cut flowers. It is deeply favored by gardening enthusiasts and consumers. In recent years, Hippeastrum has been introduced and promoted in large quantities as a trendy flower, and has broad market prospects in China.

[0003] The traditional propagation methods of Amaryllis are usually seed sowing, natural bulb division, scale cutting, etc., but the conventional propagation methods are inefficient, slow, require a large number of bulbs, and have high production costs, which are difficult to meet market demand; while tissue culture has a high coefficient of rapid propagation and consistent seedling characteristics, which is more convenient for commercialization and industrial production. At present, the tissue culture of Amaryllis mainly cuts the bulbs of Amaryllis bulbs as explants, but the foreign pathogenic fungi carried in the roots of imported Amaryllis bulbs are significantly higher than other parts, and cutting the bulbs as explants will cause irreversible damage to Amaryllis bulbs, which seriously restricts the industrial development of Amaryllis. Summary of the invention

[0004] The purpose of the present invention is to provide an in vitro culture method of Amaryllis with pedicels as explants, so as to overcome the shortcomings of the existing Amaryllis tissue culture, such as high contamination rate and high cost of cutting bulbs, which seriously restrict the factory production of Amaryllis tissue culture.

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

[0006] An in vitro culture method of Amaryllis with pedicels as explants comprises the following steps:

[0007] 1) Selection of explants: Select healthy, pest-free, and unopened Amaryllis plants, cut the pedicels at the base of the flowers as explants, and then disinfect the pedicels;

[0008] 2) Induced differentiation culture: The sterilized pedicels were inoculated into the induction differentiation medium, the culture temperature was controlled at 22-25°C, the light intensity was 2500-3000 lx, the photoperiod was 9.5-10.5 h / d, and the adventitious buds were obtained after 30-40 days of culture;

[0009] 3) Subculture proliferation culture: inoculate the adventitious buds into the proliferation culture medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 30-40 days to obtain adventitious buds that grow to 2-3 cm;

[0010] 4) Rooting culture: transfer the adventitious buds that have grown to 2-3 cm to the rooting medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the photoperiod at 9.5-10.5 h / d, and culture for 25-50 days.

[0011] Furthermore, the disinfection method of step 1) is: cutting the flower arrow into a length of 5±0.2 cm, soaking it in a 1-1.1% laundry detergent solution for 10-15 minutes, rinsing it with running water for 60-70 minutes, transferring it to a clean bench, cutting the pedicel at the base of the flower as an explant, soaking it in 75% ethanol for 30-35 seconds, and then disinfecting it with 0.1% HgCl2 for 20 minutes, and finally rinsing it repeatedly with sterile water for 4-6 times, cutting the disinfected explant into small pedicels of 1±0.1 cm in length, and then spreading it flat on the induction culture medium.

[0012] Furthermore, the preparation method of the differentiation induction medium in step 2) is: adding 1.0 mg·L -1 6-BA and 0.1mg·L -1 NAA.

[0013] Furthermore, the preparation method of the proliferation medium in step 3) is: adding 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.6mg·L -1 NAA and 30g·L -1 sucrose.

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

[0015] By adopting the above technical scheme, the beneficial effects of the present invention are as follows: the method of the present invention can induce differentiation of the pedicels of Amaryllis to produce adventitious buds, avoid irreversible damage to the Amaryllis bulbs due to cutting the bulbs, provide new materials for the in vitro rapid propagation of Amaryllis, solve the key technical bottlenecks of explant selection and disinfection in Amaryllis tissue culture, greatly reduce the production cost of Amaryllis factory production and new variety breeding and expansion, and is of great significance to the protection and effective utilization of Amaryllis germplasm resources. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with specific implementation modes.

[0017] The raw materials used in the present invention are described as follows:

[0018] The amaryllis plants are the new amaryllis variety 'Xue Ran Chu Xia', which are grown in the greenhouse of Fujian Forestry Science and Technology Experimental Center and are healthy and free of diseases and insect pests.

[0019] MS medium is the most commonly used plant tissue culture medium. 6-BA: 6-benzylaminoadenine; NAA: naphthylacetic acid; KT: chlorfenapyr; IBA: indolebutyric acid.

[0020] Example 1

[0021] Selection of HgCl2 Treatment Time in Explant Disinfection Process

[0022] Select healthy, pest-free, and unopened amaryllis, cut the pedicel at the base of the flower as an explant, and then disinfect the explant. The disinfection method is: cut the flower arrow into a length of about 5 cm, soak it in a 1% washing powder solution for 10 minutes, rinse it with running water for 60 minutes, transfer it to a clean bench, cut the pedicel at the base of the flower, soak it in 75% ethanol for 30 seconds, and then disinfect it with 0.1% HgCl2 (10, 15, 20, 25 minutes), and finally rinse it repeatedly with sterile water for 5 times. Cut the disinfected explant into small pedicels of 1 cm in length and spread it flat in the induction medium.

[0023] The contamination 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% - contamination rate - mortality rate.

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

[0027] Disinfection time / min Pollution rate % mortality rate% Survival rate% 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 below.

[0029] As can be seen from Table 1, different HgCl2 treatment times have a significant effect on the disinfection effect of Amaryllis pedicels. As the disinfection time increases, the contamination rate gradually decreases. When the disinfection time is 20 minutes, the effect is the best, 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 medium

[0032] The sterilized pedicels are inoculated into an induction differentiation medium for cultivation to obtain adventitious buds. The culture temperature is controlled at 22-25°C, the light intensity is 2500-3000lx, and the light cycle is 10h / d.

[0033] Screening method of inducing differentiation medium: MS + 0.1mg·L -1 NAA was used as the basic culture medium, and different concentrations of 6-BA (0, 1, 2, 3 mg·L -1 ), a total of 4 culture media (4 treatments), 30 flower stalks in each treatment, repeated 3 times.

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

[0035]

[0036]

[0037] After disinfection, the pedicels of Amaryllis were inoculated into the induction medium. After 30 days of culture, the germination and growth of adventitious buds were observed and recorded every day. After 40 days of culture, the adventitious buds of the pedicels of all treatment groups germinated. As shown in Table 2, within a certain concentration range, as the mass concentration of 6-BA increased, the induction rate increased. When the mass concentration reached 1.0 mg·L -1 When , the induction rate of pedicel reached 46.7%.

[0038] Therefore, the induction differentiation medium selected in the present invention is: add 1.0 mg·L -1 6-BA and 0.1mg·L -1 NAA.

[0039] Example 3

[0040] Screening of proliferation medium

[0041] The adventitious buds produced by induced differentiation are inoculated into the proliferation medium for cultivation to obtain adventitious buds with good growth. The culture temperature is controlled at 22-25°C, the light intensity is 2500-3000lx, and the light cycle is 10h / d. Screening method of proliferation medium: MS+30g·L -1 Sucrose was used as the basic medium, 6-BA, KT and NAA were added, and L9 (3 4) Orthogonal experimental design. The induced adventitious buds were inoculated into different proliferation culture media, 30 adventitious buds were inoculated into each culture medium, and repeated 3 times. After 30 days of culture, the growth status was observed, and the proliferation coefficient of the adventitious buds was calculated and analyzed (proliferation coefficient = total number of adventitious buds / initial number of adventitious buds inoculated). The effects of different combinations of exogenous plant growth regulators on the state of adventitious buds, proliferation coefficient, etc. were compared to screen out the most suitable proliferation culture medium.

[0042] Table 3 Results of orthogonal test for the proliferation and cultivation of Amaryllis

[0043]

[0044]

[0045] Table 4 Results of variance analysis of orthogonal test for induction culture

[0046] Sources of variation sum of squares Degrees of Freedom variance F-number 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 significant difference at p=0.01 level.

[0048] Tables 3 and 4 show that different combinations of plant growth regulators have a significant effect on the proliferation coefficient of Amaryllis adventitious buds. Adding appropriate concentrations of 6-BA, KT and NAA is conducive to the proliferation of adventitious buds. Among the 9 treatments, the average proliferation coefficient of treatment 8 was the highest, reaching 5.66. From the results of orthogonal analysis, the best combination of Amaryllis proliferation culture medium is A3B2C3, that is, MS+2.0mg·L -1 6-BA+1.0mg·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 effects on the proliferation of adventitious buds of Amaryllis, and their influence was ranked as A>C>B, that is, 6-BA had the most significant effect, followed by NAA, and KT the least. The results of variance analysis (Table 4) showed that the effects of the three factors on the proliferation coefficient of Amaryllis were extremely significant (P<0.01).

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

[0051] Example 4

[0052] Screening of rooting medium

[0053] The adventitious buds that grow to 2-3 cm are transferred to the rooting medium. The culture temperature is controlled at 22-25°C, the light intensity is 2500-3000 lx, and the photoperiod is 10h / d. The screening method of the rooting medium is: when the adventitious buds grow to 2-3 cm, they are transferred to the rooting medium. 1 / 2MS+15g·L -1 Sucrose + 0.2 g·L -1 Activated carbon was used as the basic culture medium, and different concentration gradients (0.2, 0.4, 0.6 mg·L -1 )NAA and IBA, a total of 6 combinations of rooting medium. 30 bottles of each medium, 5 adventitious buds per bottle, repeated 3 times. After 45 days of culture, the rooting situation was observed and counted every day to screen out the most suitable rooting medium.

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

[0055]

[0056] Through observation, it was found that white root spots began to appear after about 25 days of rooting culture; after 45 days of rooting culture, the growth status of the rooted seedlings was observed and recorded every day, and the average number of roots, average root length, and average rooting rate of the rooted seedlings were calculated after 50 days of culture. As shown in Table 5, NAA and IBA can effectively promote root formation, and the rooting rate is 100%. Compared with NAA, IBA has a more significant effect on the rooting of Amaryllis. IBA is 0.4 mg·L -1 The average number of roots was 4.3 and the average root length was 4.88 cm.

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

[0058] Example 5

[0059] An in vitro culture method of Amaryllis with pedicels as explants comprises the following steps:

[0060] 1) Selection of explants

[0061] Select healthy, disease-free and insect-free Amaryllis with flower arrows that have not yet opened, cut the pedicels at the base of the flower as explants, and then disinfect the pedicels, specifically: cut the flower arrows into 5±0.2 cm in length, soak them in 1% laundry detergent solution for 10 minutes, rinse them with running water for 60 minutes, transfer them to a clean bench, cut the pedicels at the base of the flower 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 for 5 times, cut the disinfected explants into small pedicels of 1±0.1 cm in length, and then spread them flat on the induction medium.

[0062] 2) Induced differentiation culture

[0063] Add 1.0 mg·L -1 6-BA and 0.1mg·L -1 NAA, obtain the induction differentiation medium. Inoculate the disinfected pedicels into the induction differentiation medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000lx, the light cycle at 9.5-10.5h / d, and culture for 30 days to obtain adventitious buds.

[0064] 3) Subculture proliferation

[0065] Add 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 to the MS medium to obtain a proliferation medium. Inoculate the adventitious buds into the proliferation medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 30 days to obtain adventitious buds that grow to 2-3 cm.

[0066] 4) Rooting culture

[0067] Add 15 g·L -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA, obtain rooting medium. Transfer the adventitious buds that grow to 2-3 cm to the rooting medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 45 days.

[0068] Example 6

[0069] An in vitro culture method of Amaryllis with pedicels as explants comprises the following steps:

[0070] 1) Selection of explants

[0071] Select healthy, pest-free, and unopened amaryllis, cut the pedicel at the base of the flower as an explant, and then disinfect the pedicel, specifically: cut the flower arrow into 5±0.2 cm in length, soak it in a 1% laundry detergent solution for 15 minutes, rinse it with running water for 70 minutes, transfer it to a clean bench, cut the pedicel at the base of the flower as an explant, soak it in 75% ethanol for 35 seconds, then disinfect it with 0.1% HgCl2 for 20 minutes, and finally rinse it repeatedly with sterile water for 6 times, cut the disinfected explant into small pedicels of 1±0.1 cm in length, and then spread it flat on the induction medium.

[0072] 2) Induced differentiation culture

[0073] Add 1.0 mg·L -1 6-BA and 0.1mg·L -1 NAA, obtain the induction differentiation medium. Inoculate the disinfected pedicels into the induction differentiation medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000lx, the light cycle at 9.5-10.5h / d, and culture for 35 days to obtain adventitious buds.

[0074] 3) Subculture proliferation

[0075] Add 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 to MS medium to obtain a proliferation medium. Inoculate the adventitious buds into the proliferation medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 35 days to obtain adventitious buds growing to 2-3 cm.

[0076] 4) Rooting culture

[0077] Add 15 g·L -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA, obtain rooting medium. Transfer the adventitious buds that grow to 2-3 cm to the rooting medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 50 days.

[0078] Example 7

[0079] An in vitro culture method of Amaryllis with pedicels as explants comprises the following steps:

[0080] 1) Selection of explants

[0081] Select healthy, pest-free, and unopened amaryllis, cut the pedicel at the base of the flower as an explant, and then disinfect the pedicel, specifically: cut the flower arrow into 5±0.2 cm in length, soak it in 1% laundry detergent solution for 12 minutes, rinse it with running water for 65 minutes, transfer it to a clean bench, cut the pedicel at the base of the flower as an explant, soak it in 75% ethanol for 33 seconds, then disinfect it with 0.1% HgCl2 for 20 minutes, and finally rinse it repeatedly with sterile water for 5 times, cut the disinfected explant into small pedicels of 1±0.1 cm in length, and then spread it flat on the induction medium.

[0082] 2) Induced differentiation culture

[0083] Add 1.0 mg·L -1 6-BA and 0.1mg·L -1 NAA, obtain the induction differentiation medium. Inoculate the disinfected pedicels into the induction differentiation medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000lx, the light cycle at 9.5-10.5h / d, and culture for 40 days to obtain adventitious buds.

[0084] 3) Subculture proliferation

[0085] Add 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 to MS medium to obtain a proliferation medium. Inoculate the adventitious buds into the proliferation medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 40 days to obtain adventitious buds growing to 2-3 cm.

[0086] 4) Rooting culture

[0087] Add 15 g·L -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA, obtain rooting medium. Transfer the adventitious buds that grow to 2-3 cm to the rooting medium, control the culture temperature at 22-25°C, the light intensity at 2500-3000 lx, the light cycle at 9.5-10.5 h / d, and culture for 50 days.

[0088] The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited thereto. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. A method for in vitro culture of Amaryllis using pedicels as explants, characterized in that: The following steps are involved: 1) Selection of explants: Select healthy, pest-free, and unopened amaryllis plants, cut the pedicels at the base of the flowers as explants, and then disinfect the pedicels; 2) Induced differentiation culture: Inoculate the sterilized pedicels into the induction differentiation medium, control the culture temperature at 22-25°C, light intensity at 2500-3000 lx, light cycle at 9.5-10.5 h / d, culture for 30-40 days to obtain adventitious buds; 3) Subculture proliferation culture: inoculate the adventitious buds into the proliferation culture medium, control the culture temperature at 22-25°C, light intensity at 2500-3000 lx, light cycle at 9.5-10.5 h / d, culture for 30-40 days, and obtain adventitious buds that grow to 2-3 cm; 4) Rooting culture: Transfer the adventitious buds that have grown to 2-3 cm to the rooting medium. The culture temperature is controlled at 22-25°C, the light intensity is 2500-3000 lx, the photoperiod is 9.5-10.5 h / d, and the culture is carried out for 25-50 days.

2. The method for in vitro culture of Amaryllis using pedicels as explants according to claim 1, characterized in that: The disinfection treatment method of step 1) is as follows: cutting the flower arrow into 5±0.2 cm in length, soaking it in a 1~1.1% laundry detergent solution for 10~15 minutes, rinsing it with running water for 60~70 minutes, transferring it to a clean bench, cutting the pedicel at the base of the flower as an explant, soaking it in 75% ethanol for 30~35 seconds, disinfecting it with 0.1% HgCl2 for 20 minutes, and finally rinsing it repeatedly with sterile water for 4~6 times, cutting the disinfected explant into small pedicels of 1±0.1 cm in length, and then spreading it flat on the induction medium.

3. The method for in vitro culture of Amaryllis using pedicels as explants according to claim 1, characterized in that: The preparation method of the differentiation induction medium in step 2) is as follows: 1.0 mg·L -1 6-BA and 0.1mg·L -1 NAA.

4. The method for in vitro culture of Amaryllis using pedicels as explants according to claim 1, characterized in that: The preparation method of the proliferation medium in step 3) is as follows: add 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.6 mg·L -1 NAA and 30 g·L -1 sucrose.

5. The method for in vitro culture of Amaryllis using pedicels as explants according to claim 1, characterized in that: The preparation method of the rooting medium in step 4) is as follows: 15 g·L -1 Sucrose, 0.2 g·L -1 Activated carbon and 0.4 mg·L -1 IBA.

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