Method for inducing vetiveria zizanioides callus and regenerating based on seeds

Abstract

The invention provides a method for inducing and regenerating vetiver grass calluses by taking seeds as explants. The method comprises the following steps: taking vetiver grass seeds as explants, and sequentially carrying out seed bran removal, disinfection, callus induction, subculture, differentiation culture and seedling transplanting to successfully establish an efficient vetiver grass regeneration system.

Description

Technical Field

[0001] The present application relates to the field of plant tissue culture, and in particular to a method for inducing and differentiating and regenerating vetiver callus. Background Art

[0002] Vetiveria zizanioides is a perennial grass plant native to tropical and subtropical regions. It is considered to be one of the herbs with the longest root system and is resistant to acid, alkali, waterlogging, barrenness, and heavy metals. It is not only a preferred material in the field of ecological restoration and soil and water conservation, but can also be used to extract vetiver oil or make paper. It is an important industrial raw material. However, current production has long relied on asexual reproduction, which has a low and unstable reproduction coefficient. Research on the regeneration system of vetiver began in the 1990s. Explants were mostly selected from stem segments, young leaves or stem nodes to induce callus tissue. The callus induction rate was low and easily contaminated. Therefore, it is of great significance to establish a set of efficient and stable vetiver regeneration and rapid propagation technologies. Summary of the invention

[0003] This application uses seeds as explants, optimizes seed pretreatment and callus induction processes, and then establishes an efficient regeneration system for vetiver, which can provide technical support for subsequent bioengineering such as gene editing and mutagenesis breeding.

[0004] Specifically, this application provides the following technical solutions.

[0005] In a first aspect, a method for inducing Vetiver callus is provided, comprising:

[0006] a) Providing vetiver seeds;

[0007] b) removing the bran from the vetiver seeds;

[0008] c) sterilizing the bran-removed vetiver seeds; and

[0009] d) subjecting the sterilized vetiver seeds to a callus induction treatment, which comprises culturing the sterilized vetiver seeds in a CI medium, wherein the CI medium comprises: 4.74 g / L MS powder, 30 g / L sucrose, 8 g / L agar and 0.1 mg / L 6-BA, with a pH value of 5.8-6.2, and about 0.5, about 2.0 or about 4.0 mg / L 2,4-D and about 0.5, about 1.0 or about 2.0 mg / L ABA are added to the CI medium.

[0010] In a preferred embodiment, about 4.0 mg / L 2,4-D and about 1.0 mg / L ABA are added to the CI medium.

[0011] In some embodiments of the first aspect, the bran removal treatment comprises shaking and soaking the vetiver seeds in a solvent of sulfuric acid and water in a volume ratio of 1:1 for about 5, about 10, about 20, about 30, or about 40 minutes.

[0012] In a preferred embodiment, the bran removal treatment comprises shaking and soaking the vetiver seeds in a solvent of sulfuric acid and water in a volume ratio of 1:1 for about 30 minutes.

[0013] In some embodiments of the first aspect, the disinfection treatment comprises soaking the vetiver seeds in alcohol for at least 30 seconds and then soaking them in NaClO or Chlorhexidine for about 5, about 10, about 20, or about 30 minutes.

[0014] In a preferred embodiment, the disinfection treatment comprises soaking the vetiver seeds in alcohol for at least 30 seconds and then soaking them in chlorhexidine for about 20 minutes.

[0015] In some embodiments of the first aspect, the callus induction treatment involves culturing the Vetiver seeds at 25° C. in the dark for at least 20 days.

[0016] In a preferred embodiment, the callus induction treatment involves culturing the Vetiver seeds at 25°C in the dark for about 30 days.

[0017] In a second aspect, a method for regenerating vetiver seedlings is provided, comprising:

[0018] a) providing Vetiver callus induced by the method according to the first aspect;

[0019] b) subculturing the callus tissue of step a) in a subculture medium, wherein the subculture medium comprises: 4.74 g / L MS powder, 1.0 mg / L 2,4-D, 0.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar, pH 5.8, and the subculture medium is supplemented with about 0.5 g / L or about 1.0 g / L hydrolyzed casein; about 0.5 g / L or about 1.0 g / L tryptone; or about 5.0 g / L or about 10.0 g / L coconut juice; and

[0020] c) subjecting the callus tissue of step b) to differentiation culture in a differentiation medium to produce vetiver seedlings, wherein the differentiation medium comprises: 4.74 g / L MS powder, 30 g / L sucrose, 8 g / L agar and 0.1 mg / L NAA, with a pH of 5.8-6.2, and about 0.5, about 1.0 or about 2.0 mg / L 6-BA; or about 0.5, about 1.0 or about 2.0 mg / L ZT is added to the differentiation medium.

[0021] In some embodiments of the second aspect, about 10.0 g / L of coconut water is added to the subculture medium.

[0022] In some embodiments of the second aspect, about 1.0 mg / L ZT is added to the differentiation medium.

[0023] In some embodiments of the second aspect, the regimen further comprises:

[0024] d) transferring the vetiver grass seedlings produced in step c) into a cultivation medium for cultivation to produce vetiver grass seedlings, wherein the cultivation medium comprises peat soil, perlite and vermiculite in a weight ratio of 1:1:1.

[0025] In some embodiments of the second aspect, the subculture is performed at 25° C. in the dark for at least 28 days, and / or the differentiation culture is performed at a light intensity of about 600 μmol / m 2 / s, under the conditions of a photoperiod of 16.0 hours and an average temperature of 30°C / 25°C (day / night).

[0026] In some embodiments of the second aspect, the cultivation is carried out at a relative humidity of 65%, a PAR of 800 μmol / m 2 / s, a photoperiod of 16.0 hours, and an average temperature of 30°C / 25°C (day / night). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Normally germinated Vetiver seeds are shown.

[0028] Figure 2 Callus induced from Vetiver seeds is shown.

[0029] Figure 3 Shown are Vetiver seedlings after differentiation culture.

[0030] Figure 4 Vetiver seedlings are shown after regeneration. DETAILED DESCRIPTION

[0031] Example 1: Callus induction and plant regeneration of Vetiveria zizanioides

[0032] 1. Experimental Materials

[0033] Vetiver seeds were used as materials in this example, provided by Anhui Academy of Agricultural Sciences. All test agents, unless otherwise specified, were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0034] 2. Experimental methods

[0035] 2.1 Bran removal

[0036] Vetiver seeds were sieved (100 mesh) to remove impurities and then placed in a 50% sulfuric acid solution (1:1 volume ratio of sulfuric acid to water) and shaken for soaking (5, 10, 20, 30, 40 minutes). After soaking, they were washed three times with distilled water and rubbed to remove the bran. Each treatment contained 100 seeds. After the treatment, the seeds were transferred to moist sterile filter paper and cultured in a culture room under light (light intensity 800 μmol / m 2 / s, 30℃ / 25℃ (16.0h day / 8h night) for 10 days, and the germination rate was counted. As shown in Table 1, the germination rate of 50% sulfuric acid treatment for 30 minutes was the highest, significantly higher than that of other treatments.

[0037] Table 1 Effects of different bran removal treatments on germination rate of vetiver seeds

[0038]

[0039] 2.2 Vetiver Seed Disinfection

[0040] Rinse the vetiver seeds with bran removed in running water for 1 hour, transfer to a clean bench, soak in alcohol for 30 seconds, rinse with sterile water 1-2 times, and then soak in NaClO or Chlorhexidine for different lengths of time (5, 10, 20, 30 minutes). There were 8 treatments in total, 100 seeds in each treatment, repeated 3 times. Subsequently, rinse with sterile water 6-8 times. The sterilized seeds were inoculated into Murashige and Skoog (MS) medium, including: MS powder (Haibo Biotechnology, Qingdao, catalog number: HB8469) 4.74 g / L, sucrose 30 g / L, agar 8 g / L, pH 5.8-6.2. The seeds were transferred to the culture room for culture under light, with a light intensity of 800 μmol / m 2 / s, average temperature 30℃ / 25℃ (16.0h during the day / 8h at night). After 14 days of cultivation, the contamination rate (%) and germination rate (%) were calculated. As shown in Table 2, the seeds soaked in Sanisol for 20 minutes had the lowest contamination rate of 1.5%, and the germination rate was extremely high (Table 2).

[0041] Table 2 Effects of different disinfectants on callus induction rate in callus induction culture

[0042]

[0043] 2.3 Vetiver callus induction

[0044] After obtaining sterile seeds according to step 2.2, the seeds were inoculated into CI medium for callus induction ( Figure 2), CI medium is MS medium, including: MS powder (Haibo Biological, Qingdao, catalog number: HB8469) 4.74g / L, sucrose 30g / L, agar 8g / L, 6-BA 0.1mg / L, pH 5.8-6.2, and different concentrations of 2,4-D (0.5, 2.0 and 4.0mg / L) and ABA (0.5, 1.0 and 2.0mg / L) were added. There were 9 treatments in total, each containing 100 seeds, repeated 3 times. The callus induction environment was cultured in the dark at 25℃ for 20 days, and the callus tissue status was observed on the 20th day and the callus induction rate (%) was calculated. As shown in Table 3, 2,4-D was 4.0mg / L, ABA was

[0045] At 1.0 mg / L, the callus induction rate was the highest, at 73.4%, and it could inhibit seed germination and produce more transparent callus tissue.

[0046] Table 3 Effects of different 2,4-D and ABA concentrations on callus induction rate in callus induction culture

[0047]

[0048] 2.4 Subculture of Vetiver Callus

[0049] After about 30 days of induction, when the callus tissue grows to a diameter of 1.0-2.0 cm, the callus tissue is cut and transferred to a subculture medium containing different exogenous additives. Exogenous additives include acid hydrolyzed casein (0.5g / L, 1.0g / L), tryptone (0.5g / L, 1.0g / L) or fresh coconut juice (5.0g / L, 10.0g / L) (all three substances are purchased from Kingclone (Beijing) Biotechnology Co., Ltd.). The basic components of the subculture medium are: MS powder 4.74g / L, 2,4-D 1.0mg / L, NAA0.5mg / L, sucrose 30g / L, agar 8g / L, pH 5.8. After culturing for 28 days at 25℃ in the dark, the proportion of white solid granular embryonic callus tissue was counted. The embryonic callus ratio was calculated by the following formula: embryonic callus ratio = (number of callus blocks forming white solid embryonic callus / total number of callus blocks) × 100%. As shown in Table 4, the treatment of adding 10.0 g / L coconut juice significantly promoted the growth of callus and significantly increased the embryonic callus ratio.

[0050] Table 4 Effects of different exogenous additives on the proportion of embryonic callus

[0051]

[0052] 2.5 Vetiver callus differentiation

[0053] The white and firm embryonic callus was cultured on RM medium under light ( Figure 3 RM medium is MS medium, including: MS powder 4.74g / L, sucrose 30g / L, agar 8g / L, NAA 0.1mg / L, pH 5.8-6.2, and different concentrations of 6-BA (0.5, 1.0, 2.0mg / L) or ZT (0.5, 1.0, 2.0mg / L) (both substances were purchased from Jinclone (Beijing) Biotechnology Co., Ltd.). The callus tissue was transferred to culture under light conditions. The light intensity was about 600μmol / m 2 / s, photoperiod 16.0h, average temperature 25℃ / 20℃ (day / night), medium changed every 3 weeks. A total of 9 treatments, each treatment contained 100 callus tissues, repeated 3 times. The differentiation rate and reproduction coefficient of each callus tissue were calculated at 30 days. Callus differentiation rate = number of differentiable callus tissues / total number of callus tissues × 100%. As shown in Table 5, the callus differentiation rate of 1.0 mg / L ZT was the highest, at 98.3%.

[0054] Table 5 Effects of different cytokinins on callus differentiation

[0055]

[0056] 2.6 Transplantation of Vetiver Grass Regeneration Seedlings

[0057] The 3-5 cm vetiver seedlings obtained in step 2.5 ( Figure 4 ) were transferred to 128-hole trays in the greenhouse. The greenhouse environment was 65% relative humidity, PAR800μmol / m2 / s, 16.0h photoperiod, and 30℃ / 25℃ average temperature (day / night). The seedlings were transferred to the cultivation medium. The cultivation medium formula was: peat soil, perlite: vermiculite (weight ratio 1:1:1). After 14 days of closed culture with plastic wrap, they can be transplanted to the field.

Claims

1. A method for inducing vetiver callus, comprising: a) Providing vetiver seeds; b) removing the bran from the vetiver seeds; c) sterilizing the bran-removed vetiver seeds; as well as d) subjecting the sterilized vetiver seeds to callus induction treatment, comprising culturing the sterilized vetiver seeds in a CI medium, wherein the CI medium comprises: 4.74 g / L MS powder, 30 g / L sucrose, 8 g / L agar and 0.1 mg / L 6-BA, with a pH of 5.8-6.2, and about 0.5, about 2.0 or about 4.0 mg / L 2,4-D and about 0.5, about 1.0 or about 2.0 mg / L ABA are added to the CI medium, preferably, about 4.0 mg / L 2,4-D and about 1.0 mg / L ABA are added to the CI medium.

2. The method according to claim 1, wherein the bran removal treatment comprises shaking and soaking the vetiver seeds in a solvent of sulfuric acid and water in a volume ratio of 1:1 for about 5, about 10, about 20, about 30, about 40 minutes, preferably about 30 minutes.

3. The method according to claim 1 or 2, wherein the disinfection treatment comprises soaking the vetiver seeds in alcohol for at least 30 seconds, and then soaking them in NaClO or Chlorhexidine for about 5, about 10, about 20, or about 30 minutes, preferably soaking them in Chlorhexidine for about 20 minutes.

4. The method according to claim 1 or 2, wherein the callus induction treatment comprises culturing the Vetiver seeds at 25°C in the dark for at least 20 days, preferably about 30 days.

5. A method for regenerating vetiver seedlings, comprising: a) providing Vetiver callus induced by the method according to any one of claims 1 to 4; b) subculturing the callus tissue of step a) in a subculture medium, wherein the subculture medium comprises: 4.74 g / L MS powder, 1.0 mg / L 2,4-D, 0.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar, pH 5.8, and the subculture medium is supplemented with about 0.5 g / L or about 1.0 g / L hydrolyzed casein; about 0.5 g / L or about 1.0 g / L tryptone; or about 5.0 g / L or about 10.0 g / L coconut juice; and c) subjecting the callus tissue of step b) to differentiation culture in a differentiation medium to produce vetiver seedlings, wherein the differentiation medium comprises: 4.74 g / L MS powder, 30 g / L sucrose, 8 g / L agar and 0.1 mg / L NAA, with a pH of 5.8-6.2, and about 0.5, about 1.0 or about 2.0 mg / L 6-BA; or about 0.5, about 1.0 or about 2.0 mg / L ZT is added to the differentiation medium.

6. The method according to claim 5, wherein about 10.0 g / L of coconut water is added to the subculture medium.

7. The method according to claim 5 or 6, wherein about 1.0 mg / L of ZT is added to the differentiation medium.

8. The method according to claim 5 or 6, further comprising: d) transferring the vetiver grass seedlings produced in step c) into a cultivation medium for cultivation to produce vetiver grass seedlings, wherein the cultivation medium comprises peat soil, perlite and vermiculite in a weight ratio of 1:1:

1.

9. The method according to claim 5 or 6, wherein the subculture is carried out at 25°C in the dark for at least 28 days, and / or the differentiation culture is carried out at a light intensity of about 600 μmol / m 2 / s, under the conditions of a photoperiod of 16.0 hours and an average temperature of 30°C / 25°C (day / night).

10. The method according to claim 8, wherein the cultivation is carried out at a relative humidity of 65%, a PAR of 800 μmol / m 2 / s, a photoperiod of 16.0 hours, and an average temperature of 30°C / 25°C (day / night).

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