A method for in vitro culture and rapid propagation of Zingiber officinale
By using closed-shelled ginger seeds as explants and adding 6-BA to MS+NAA 0.2 mg/L basal culture medium to induce germination, combined with the use of cytokinin and growth hormone, the problems of slow reproduction and difficult disinfection in the prior art were solved, and efficient reproduction and low-cost production were achieved.
Patent Information
- Application Number
- CN202310683598.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-07
- Publication Date
- 2025-03-07
- Estimated Expiration
- 2043-06-07
AI Technical Summary
In the prior art, the reproduction speed of red-shelled ginger is slow, the production cost is high, and the germplasm quality is prone to deterioration, which makes it difficult to expand the cultivation scale, and the disinfection and sterilization of stems and rhizomes as explants is difficult and the pollution rate is high.
Closed ginger seeds were used as explants. After disinfection, 6-BA was added to MS+NAA 0.2 mg/L basal culture medium to induce germination, and then cytokinin and growth hormone were used for indefinite bud proliferation and rooting and seedling cultivation.
It significantly improves the reproduction effect of closed-sheaven ginger, has a high germination rate and a fast reproduction rate, reduces production costs, expands the cultivation range, simplifies the disinfection process, and reduces the pollution rate.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of Zingiber officinale cultivation, and in particular to an in vitro culture and rapid propagation method of Zingiber officinale. Background Art
[0002] Hellenia speciosa is a perennial herbaceous plant of the genus Hellenia in the family Costaceae. Domestic wild resources are mainly distributed in tropical and subtropical areas such as Hainan, Guangdong, Guangxi, and Yunnan. It prefers a warm and humid environment. Its tender stems are like bamboo shoots and grow vigorously in the rainy season, so it is also called "thunder god shoots". As a wild vegetable, Hellenia speciosa is often used for pickling or fresh consumption. This resource is rich in steroids, terpenes, flavonoids and other compounds, and has hypoglycemic activity, anti-inflammatory and antipyretic, antibacterial and liver-protecting effects. In addition, Hellenia speciosa has a unique and elegant red leathery spike inflorescence, which can be used as fresh cut flowers and greening, and is a wild resource with great application value and development prospects.
[0003] Existing technology: The reproduction of red clostridium is mainly based on vegetative propagation by cutting rhizomes, which has a slow reproduction speed, high production cost, and easy degradation of germplasm quality, making it difficult to expand the scale of cultivation. In addition, the stems and rhizome pieces are used as explants, which is difficult to disinfect and sterilize, and has a high contamination rate.
[0004] In order to solve the above problems, the patent application number 2020106426747 discloses a method for callus induction and plant regeneration of red sheath ginger, comprising the following steps: 1) disinfection and callus induction of explants; 2) callus proliferation culture; 3) induction and proliferation culture of clustered buds; 4) rooting and strong seedling culture. The invention simplifies the conventional tissue culture disinfection procedure by selecting the florets in the immature inflorescence bracts of red sheath ginger as explants, selects the improved MS medium instead of the MS medium, and performs cluster bud induction and proliferation culture and rooting and strong seedling culture under continuous light, which significantly improves the callus induction rate of red sheath ginger. However, the above technical scheme requires the use of fresh plants of red sheath ginger, which is not convenient for long-distance transportation and is restricted by seasons, and its cultivation range is greatly limited. Summary of the invention
[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for in vitro culture and rapid propagation of wild resource Zingiber officinale. Zingiber officinale seeds are used as explants for culture and propagation, with a high germination rate, which can significantly improve the breeding effect of Zingiber officinale.
[0006] The present invention relates to an in vitro culture and rapid propagation method of Zingiber officinale, comprising the following steps:
[0007] S1. Disinfecting the seeds of Gingerbread;
[0008] S2, using MS+NAA0.2 mg / L as the basic culture medium and adding 2.0 mg / L-4.0 mg / L 6-BA to induce germination of Ginger seeds;
[0009] S3, using cytokinins to multiply adventitious buds of Ginger;
[0010] S4. Use growth hormone to stimulate the rooting and strong seedlings of Ginger;
[0011] In step S1, the seeds of the ginger are disinfected, including using the ginger seeds as explants, wrapping the seeds with gauze, rinsing with tap water, shaking with a detergent dilution for 1 minute, rinsing with tap water, draining the water, rinsing with sterile water for 3 times on a clean bench, treating with 75% alcohol for 15 seconds, and rinsing with sterile water once; soaking and shaking with 5.0% NaClO for 8-12 minutes, and rinsing with sterile water for 5 times; step S2, inoculating the disinfected seeds into MS+NAA 0.2 mg / L as the basic culture medium, adding different 6-BA concentration gradients, setting 2.0, 3.0 and 4.0 mg / L treatments, and inducing the germination of the ginger seeds. The cytokinin formula described in step S3 is to use MS+NAA 0.2 mg / L as the basic culture medium, add different cytokinins 6-BA2.0 mg / L, TDZ 2.0 mg / L and KT 2.0 mg / L, screen out the suitable cytokinin as 6-BA, and further explore the effects of different concentration ratios of cytokinin 6-BA on the proliferation of adventitious buds of Ginger, including 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L6-BA; the growth hormone described in step S4 uses MS+IBA0.5 mg / L as the basic culture medium and adds different NAA concentration gradients, setting 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L treatments.
[0012] Furthermore, in step S1, the seeds were immersed and shaken with 5.0% NaClO for 8 min, and the contamination rate, germination potential and germination rate were 11.11%, 34.96% and 90.24%, respectively.
[0013] Further, in step S2, 6-BA was added to MS+NAA0.2 mg / L as the basic culture medium, the content of added 6-BA was 2.0 mg / L, the proliferation coefficient was 4.97, and the fresh weight reached 2.27 g.
[0014] Furthermore, the cytokinin is 2.0mg / L-3.0mg / L6-BA, and the proliferation coefficient reaches 6.10-6.23.
[0015] Furthermore, the growth hormone includes 1.0 mg / L-5.0 mg / L of NAA. When 1.0 mg / L of NAA is added, the rooting rate, number of roots, longest root length and plant height reach maximum values, which are 99.26%, 5.53 roots, 7.51 cm and 7.53 cm, respectively.
[0016] The present invention is beneficial in that:
[0017] 1. The ginger seeds are used as explants. Since the embryo part of the seeds is wrapped by the seed coat, the damage to the embryo part can be avoided during the disinfection process with alcohol and NaClO. The germination rate of the seeds reaches more than 90%, which can overcome the defect that the cultivation of ginger can only be achieved through living plants in the prior art. The ginger seeds are easy to transport, convenient for planning the cultivation site, and expand the cultivation range.
[0018] 2. The cultivation process is based on MS medium, which is the most commonly used medium at present. It has a high concentration of inorganic salts, which can ensure the mineral nutrition required for tissue growth and accelerate the growth of callus tissue. There are mature products on the market, which do not need to be configured separately, are simple to operate and low in cost.
[0019] 3. The explant, disinfection method, cytokinin and growth hormone in the present invention are all first-of-its-kind based on the characteristics of Zingiber officinale seeds. Zingiber officinale seeds are easy to operate, not easily damaged, and the cultivation environment is easy to control.
[0020] In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are specifically cited below and explained in detail as follows. DETAILED DESCRIPTION
[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution. 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 work are within the scope of protection of the present invention.
[0022] A method for in vitro culture and rapid propagation of Zingiber officinale in a preferred embodiment of the present invention comprises the following steps:
[0023] S1. Disinfecting the seeds of Gingerbread;
[0024] S2, using MS+NAA0.2 mg / L as the basic culture medium and adding 2.0 mg / L-4.0 mg / L 6-BA to induce germination of Ginger seeds;
[0025] S3, using cytokinins to multiply adventitious buds of Ginger;
[0026] S4. Use growth hormone to stimulate the rooting and strong seedlings of Ginger;
[0027] In step S1, the seeds of the ginger are disinfected, including using the ginger seeds as explants, wrapping the seeds with gauze, rinsing with tap water, shaking with a detergent dilution for 1 minute, rinsing with tap water, draining the water, rinsing with sterile water for 3 times on a clean bench, treating with 75% alcohol for 15 seconds, and rinsing with sterile water once; soaking and shaking with 5.0% NaClO for 8 minutes, and rinsing with sterile water for 5 times; step S2, inoculating the disinfected seeds into MS+NAA 0.2 mg / L as the basic culture medium, adding different 6-BA concentration gradients, setting 2.0, 3.0 and 4.0 mg / L treatments, and inducing the germination of the ginger seeds. The cytokinin formula described in step S3 is to use MS+NAA 0.2 mg / L as the basic culture medium, add different cytokinins 6-BA 2.0 mg / L, TDZ 2.0 mg / L and KT 2.0 mg / L, screen out the suitable cytokinin as 6-BA, and further explore the effects of different concentration ratios of cytokinin 6-BA on the proliferation of adventitious buds of Ginger, including 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L6-BA; the growth hormone described in step S4 uses MS+IBA0.5 mg / L as the basic culture medium and adds different NAA concentration gradients, setting 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L treatments.
[0028] In the above embodiment, in step S2, 6-BA is added to MS+NAA0.2 mg / L as the basic culture medium, and the content of the added 6-BA is 2.0 mg / L.
[0029] In the above embodiment, the cytokinin is 2.0 mg / L 6-BA.
[0030] In the above embodiment, the growth hormone includes 2.0 mg / L-3.0 mg / L NAA.
[0031] In order to demonstrate the implementation effect of this application, the following experiments were conducted.
[0032] 1 Materials and methods
[0033] 1.1 Test materials
[0034] 1 Test materials
[0035] The mature and healthy seeds of Ginger were collected from Yuntou Village, Longhu Town, Ding'an County, Hainan Province, latitude: 19.555278, longitude: 110.408611.
[0036] 1.2 Test methods
[0037] 1.2.1 Sterilization method of ginger seeds
[0038] Use Ginger seeds as explants, wrap the seeds with gauze, rinse with tap water, shake with detergent dilution for 1min, and rinse with tap water. After draining the water, rinse with sterile water 3 times on the clean bench, treat with 75% alcohol for 15s, and rinse with sterile water once; soak and shake with 5.0% NaClO, set 3 time gradients of 8, 10, and 12min respectively, and rinse with sterile water 5 times. Set 3 seeds per bottle, 15 bottles per treatment, and repeat three times. Take the radicle length of 0.1cm as the germination mark, and record the number of germinated seeds and contaminated seeds every day. Count the seed germination potential on the 10th day, and the contamination rate and germination rate on the 30th day. Germination potential (%) = total number of seeds germinated within 10 days / total number of seeds × 100, (total number of seeds: total number of original seeds - number of contaminated seeds); contamination rate (%) = number of contaminated seeds / total number of original seeds × 100; germination rate (%) = number of germinated seeds / total number of seeds × 100, (total number of seeds: total number of original seeds - number of contaminated seeds).
[0039] 1.2.2 Effects of different 6-BA concentration ratios on seed bud induction of Ginger
[0040] MS+NAA0.2 mg / L was used as the basic culture medium, and different 6-BA concentration gradients were added to set up 2.0 mg / L, 3.0 mg / L and 4.0 mg / L treatments. Three seeds were inoculated in each bottle, 15 bottles were treated for three times. The bud induction of each sample was recorded at 30 days. The fresh weight was recorded with 3 induced buds as the unit.
[0041] 1.2.3 Effects of different cytokinin ratios on adventitious bud proliferation of Zingiber officinale
[0042] Using MS+NAA0.2 mg / L as the basic culture medium, different cytokinins 6-BA2.0 mg / L, TDZ2.0 mg / L and KT2.0 mg / L were added to carry out comparative experiments. Each bottle was inoculated with 3 single buds (buds induced by seeds), 15 bottles were inoculated in each treatment, and three replicates were performed. The proliferation and growth of each sample were recorded for 30 days.
[0043] 1.2.4 Effects of different 6-BA concentration ratios on adventitious bud proliferation of Zingiber officinale
[0044] MS+NAA0.5 mg / L was used as the basic culture medium, and different 6-BA concentration gradients were added to set up 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, 4.0 mg / L and 5.0 mg / L treatments. Three single buds were inoculated in each bottle, and 15 bottles were inoculated in each treatment, with three replicates. The proliferation and growth of each sample were recorded for 30 days.
[0045] 1.2.5 Effects of different NAA concentration ratios on rooting and seedling growth of Ginger
[0046] MS+IBA 0.5 mg / L was used as the basic culture medium, and different NAA concentration gradients were added to set up 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, 4.0 mg / L and 5.0 mg / L treatments. Two single buds were inoculated in each bottle, and 15 bottles were inoculated in each treatment, with three replicates. The rooting rate, number of roots, longest root length and plant height of each sample were recorded at 30 days.
[0047] 1.3 Data Analysis
[0048] Excel 2007 and SPSS21.0 software were used for data processing, analysis of variance and multiple comparisons (LSD, P<0.05).
[0049] 2 Results and analysis
[0050] 2.1 Evaluation of disinfection methods for Ginger seeds
[0051] Through analysis, it was found that the contamination rate and germination rate of the three time gradients of disinfection of Ginger seeds with 5.0% NaClO were statistically significant (P < 0.05), but there was no statistically significant difference in germination potential (P > 0.05) (Table 1). The contamination rate, germination potential and germination rate of the 8-min treatment were all the maximum, which were 11.11%, 34.96% and 90.24%, respectively. Pairwise comparisons using the LSD method showed that there were statistically significant differences in contamination rate between the 8-min treatment and the 10-min and 12-min treatments (P < 0.05); there was no statistically significant difference in germination potential among the three time gradient treatments (P > 0.05); as the disinfection time prolonged, the germination rate decreased, and there was no statistically significant difference in germination rate between the 8-min treatment and the 10-min treatment. In summary, the disinfection effect of Ginger seeds with 5.0% NaClO for 8 min was better. Table 1 Evaluation of disinfection methods for Ginger seeds
[0052]
[0053] Note: Different lowercase letters after the data in the same column indicate significant differences.
[0054] 2.2 Effects of different 6-BA concentration ratios on seed bud induction of Ginger
[0055] There were statistically significant differences in the proliferation coefficient and fresh weight of the seed bud induction of Ginger (P<0.05) under different 6-BA concentration ratios (Table 2). Pairwise comparisons using the LSD method revealed that there were statistically significant differences in the proliferation coefficients between the 6-BA2.0 mg / L+NAA0.2 mg / L treatment and the 6-BA3.0 mg / L+NAA0.2 mg / L treatment, between the 6-BA3.0 mg / L+NAA0.2 mg / L treatment and the 6-BA4.0 mg / L+NAA0.2 mg / L treatment, and between the 6-BA2.0 mg / L+NAA0.2 mg / L treatment and the 6-BA4.0 mg / L+NAA0.2 mg / L treatment (P < 0.05). Pairwise comparisons of the fresh weight of induced germination in Ginger seeds showed that there was a statistically significant difference in the fresh weight of 6-BA2.0 mg / L+NAA0.2 mg / L and 6-BA4.0 mg / L+NAA0.2 mg / L (P < 0.05). In general, as the concentration of 6-BA increased, the proliferation coefficient and fresh weight values decreased. Therefore, the suitable treatment for seed bud induction of Ginger was 6-BA2.0 mg / L+NAA0.2 mg / L.
[0056] Table 2 Effects of different 6-BA concentration ratios on germination induction in Zingiber officinale seeds
[0057]
[0058] Note: Different lowercase letters after the data in the same column indicate significant differences.
[0059] 2.3 Effects of different cytokinin ratios on adventitious bud proliferation of Zingiber officinale
[0060] The effects of different cytokinin ratios on adventitious bud proliferation showed statistically significant differences in proliferation coefficients (P < 0.05) (Table 3). The cytokinin ratio 6-BA2.0 mg / L+NAA0.2 mg / L treatment was superior to TDZ2.0 mg / L+NAA0.2 mg / L and KT2.0 mg / L+NAA0.2 mg / L treatments, with a proliferation coefficient of 5.77, and good growth, thick stems and green. KT2.0 mg / L+NAA0.2 mg / L treatment promoted callus growth, and good growth, green. Comprehensive comparison, 6-BA2.0 mg / L+NAA0.2 mg / L is suitable for the proliferation of adventitious buds of Ginger.
[0061] Table 3 Effects of different cytokinin ratios on the proliferation of adventitious buds of Ginger
[0062]
[0063]
[0064] Note: Different lowercase letters after the data in the same column indicate significant differences.
[0065] 2.4 Effects of different 6-BA concentration ratios on the proliferation of adventitious buds of Ginger
[0066] The effects of different 6-BA concentration ratios on the proliferation of adventitious buds of Ginger were statistically significant in the proliferation coefficient (P < 0.05) (Table 4). The LSD method was used to compare the two groups. It was found that there was no statistically significant difference between the treatment of 6-BA 2.0 mg / L + NAA 0.5 mg / L and 6-BA 3.0 mg / L + NAA 0.5 mg / L, and the treatment of 6-BA 4.0 mg / L + NAA 0.5 mg / L and 6-BA 5.0 mg / L + NAA 0.5 mg / L (P > 0.05). The differences between any two groups were statistically significant (P < 0.05). When the 6-BA concentration was in the range of 1.0 mg / L to 3.0 mg / L, the proliferation coefficient increased with the increase of concentration. Subsequently, with the increase of concentration, the growth of adventitious buds was inhibited and roots were grown, and the proliferation coefficient was as low as 3.37. Therefore, the suitable culture medium for the proliferation of adventitious buds of Ginger is 6-BA 2.0-3.0 mg / L + NAA 0.5 mg / L.
[0067] Table 4 Effects of different 6-BA concentration ratios on the proliferation of adventitious buds of Ginger
[0068]
[0069] Note: Different lowercase letters after the data in the same column indicate significant differences.
[0070] 2.5 Effects of different NAA concentration ratios on rooting and seedling growth of Ginger
[0071] The effects of different NAA concentration ratios on the rooting and strong seedlings of Ginger were statistically significant in terms of rooting rate, number of roots, longest root length, and plant height (P < 0.05) (Table 5). Overall, the rooting rate, number of roots, longest root length, and plant height of the NAA 1.0 mg / L + IBA 0.5 mg / L treatment reached the maximum values of 99.26%, 5.53 roots, 7.51 cm, and 7.53 cm, respectively. As the NAA concentration increased, the values of each indicator decreased, indicating that the increase in NAA concentration inhibited the rooting and strong seedlings of Ginger. In summary, the suitable culture medium for rooting and strong seedlings of Ginger is NAA 1.0 mg / L + IBA 0.5 mg / L.
[0072] Table 5 Effects of different NAA concentration ratios on rooting and seedling growth of Ginger
[0073]
[0074] Note: Different lowercase letters after the data in the same column indicate significant differences.
[0075] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of the present invention, there will be changes in the scope of the specific implementation methods. In summary, the content of this specification should not be understood as limiting the present invention.
Claims
1. A method for in vitro culture and rapid propagation of Zingiber officinale, characterized in that: The following steps are involved: S1. Disinfecting the seeds of Gingerbread; S2, using MS+NAA0.2 mg / L as the basic culture medium and adding 2.0 mg / L-4.0 mg / L 6-BA to induce germination of Ginger seeds; S3, using cytokinins to multiply adventitious buds of Ginger; S4. Use growth hormone to stimulate the rooting and strong seedlings of Ginger; In step S1, the seeds of the ginger are disinfected, including using the ginger seeds as explants, wrapping the seeds with gauze, rinsing with tap water, shaking with detergent dilution for 1 min, rinsing with tap water, draining the water, rinsing with sterile water 3 times on a clean bench, treating with 75% alcohol for 15 s, rinsing with sterile water once; soaking and shaking with 5.0% NaClO for 8-12 min, and rinsing with sterile water 5 times; In step S2, the sterilized seeds were inoculated into a medium with MS+NAA 0.2 mg / L as the basic medium, and different 6-BA concentration gradients were added, with 2.0, 3.0 and 4.0 mg / L treatments set to induce germination of the ginger seeds; In step S3, the cytokinin formula is to use MS+NAA 0.2 mg / L as the basic culture medium, add different cytokinins 6-BA 2.0 mg / L, TDZ 2.0 mg / L and KT 2.0 mg / L, screen out the suitable cytokinin as 6-BA, and further explore the effects of different concentration ratios of cytokinin 6-BA on the proliferation of adventitious buds of Ginger, including 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L 6-BA; In step S4, the growth hormone is treated with MS+IBA 0.5 mg / L as the basic culture medium and different NAA concentration gradients are added, with 1.0, 2.0, 3.0, 4.0 and 5.0 mg / L treatments being set.
2. The in vitro culture and rapid propagation method of Zingiber officinale according to claim 1, characterized in that: In step S1, the samples were immersed in 5.0% NaClO and shaken for 8 min.