A method for establishing a high-efficiency regeneration system of plectranthus barbatus somatic embryos

Abstract

The application discloses a method for establishing a high-efficiency regeneration system of Pseudolarix kaempferi somatic embryos, which comprises the following steps of seed disinfection and germination, induction of embryogenic callus, induction and proliferation of embryogenic callus sprouting, rooting culture and the like. The method can not only solve the problem of low proliferation efficiency in the current tissue culture technology, but also realize mass breeding of high-quality seedlings, and is more beneficial to laying a foundation for the researches on the preservation, propagation, genetic improvement and increase of the yield of secondary metabolites of the Pseudolarix kaempferi excellent germplasm resources.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture somatic cell regeneration technology, specifically relating to a method for establishing a highly efficient regeneration system for Pistacia chinensis somatic cell embryos. Background Technology

[0002] Picrorhiza scrophulariiflora is a commonly used herb in traditional Chinese medicine for clearing heat and deficiency. The 2020 edition of the Pharmacopoeia of the People's Republic of China specifies that its medicinal material comes from Picrorhiza scrophulariiflora, a plant of the Scrophulariaceae family. Picrorhiza scrophulariiflora The dried rhizome of Pennell. Coptis chinensis is bitter and cold in nature, entering the liver, stomach, and large intestine meridians. It has the effects of reducing deficiency heat, eliminating infantile malnutrition heat, and clearing damp heat. Modern pharmacological studies have shown that Coptis chinensis has hepatoprotective, anti-inflammatory, antioxidant stress-relieving, and immunomodulatory pharmacological effects, and is clinically used to treat liver diseases such as hepatitis, fatty liver, and cirrhosis. Currently, more than 90 components have been isolated and identified from Coptis chinensis, mainly iridoids, phenylethyl glycosides, cucurbitacins, phenolic glycosides, and aromatic acids.

[0003] Wild Picrorhiza scrophulariiflora resources in my country are on the verge of extinction and have been listed as a Class II protected rare plant. Currently, the biggest problem in the cultivation of Picrorhiza scrophulariiflora seedlings is the low seedling survival rate of tissue-cultured seedlings in the wild, difficulty in rooting, and slow rhizome growth. Coupled with the use of pesticides, pest and disease attacks, weather changes, and differences in planting sites, the yield of Picrorhiza scrophulariiflora is low, resulting in low economic benefits, low farmer enthusiasm for planting, and significant difficulties in cultivation demonstration and promotion. Currently, there is limited research on tissue culture technology for Picrorhiza scrophulariiflora. In 2007, Xue Runguang et al. studied the tissue culture and plant regeneration of Picrorhiza scrophulariiflora, using tissue materials with bud points to induce bud growth, but its proliferation efficiency was low. In 2017, Zhang Gaoxiang et al. conducted preliminary research on the induction and rooting of stem segment buds in Picrorhiza scrophulariiflora, but the number of adventitious buds induced was limited. Therefore, research on the somatic embryo induction and regeneration system of Picrorhiza scrophulariiflora is essential. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for establishing a highly efficient regeneration system for inducing somatic embryos of Picrorhiza scrophulariiflora. The highly efficient regeneration system constructed by the method of the present invention solves the problem of low proliferation efficiency of Picrorhiza scrophulariiflora in tissue culture technology, and realizes the large-scale propagation of high-quality Picrorhiza scrophulariiflora seedlings.

[0005] The objective of this invention is achieved through the following technical solution: A method for establishing a highly efficient regeneration system for induced somatic cell embryos of *Picrorhiza scrophulariiflora* includes the following steps: (1) Seed disinfection and germination: Take Coptis chinensis seeds, disinfect them with 3% sodium hypochlorite in a clean bench, then wash them with sterile water, then disinfect them with 75% alcohol, wash them with sterile water, repeat the cycle twice, finally dry the moisture on the surface of the seeds, sow them on MS medium with 30g / L sugar + 6g / L agar, pH 5.8, and culture them at 22℃, 16h light + 8h darkness, light intensity 2000-2500Lx until they germinate and grow into seedlings.

[0006] (2) Induction of embryogenic callus: Sterile seedlings of Coptis chinensis that have grown for 10 days were taken out in a clean bench. The hypocotyl and stem segments were cut to 0.5±0.1cm and inoculated into embryogenic callus medium of MS + 30g / L sugar + 6g / L agar + 0.5mg / L 6-BA + 1.0mg / L NAA, pH 5.8. The culture was carried out at 22℃ in the dark until embryogenic callus tissue was obtained.

[0007] (3) Shoot induction: Embryogenic callus was inoculated into MS medium containing 30 g / L sugar, 6 g / L agar, and 0.5 mg / L 6-BA at pH 5.8 and cultured at 22°C, under 16 h light, and with a light intensity of 2000-2500 Lx until adventitious shoots appeared. The adventitious shoots were then transferred to 1 / 2 MS medium containing 30 g / L sugar, 6 g / L agar, and 0.5 mg / L 6-BA at pH 5.8 and cultured at 22°C, under 16 h light, and with a light intensity of 2000-2500 Lx for 20-60 days to obtain Pistacia chinensis embryonic seedlings.

[0008] (4) Rooting culture: Transfer the Cordyceps militaris embryos to a rooting medium of 1 / 2 MS + 30 g / L sugar + 6 g / L agar + 0.2 mg / L 6-BA, pH 5.8, and culture at 22℃, 16 h light, and light intensity of 2000-2500 Lx. Rooting will occur in 6-10 days.

[0009] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to establish a mature and complete in vitro regeneration technology for Picrorhiza scrophulariiflora somatic embryos, a rare and endangered medicinal plant distributed in northwestern Yunnan Province. This technology can not only solve the problem of low proliferation efficiency in current tissue culture technology, but also enable the large-scale propagation of high-quality seedlings. It is also conducive to laying the foundation for research on the preservation, propagation, genetic improvement, and increase of secondary metabolite yield of Picrorhiza scrophulariiflora.

[0010] 2. Existing technologies for tissue culture of Coptis chinensis use the "bud-to-bud" method, which is relatively traditional. One bud can only generate one plant, resulting in low reproductive efficiency. This invention induces regeneration of somatic embryos, enabling one tissue to produce multiple buds, thereby generating multiple plants and greatly improving the efficiency of plant growth.

[0011] 3. The method described in this invention simplifies the plant regeneration process and saves a significant amount of time in preparing tissue culture materials. Furthermore, somatic cell clonal mutations have a low frequency, making somatic embryogenesis more suitable for market applications than organogenesis. In addition, mature somatic embryos can be stored in liquid nitrogen and, upon activation, can still grow into healthy plants, retaining embryogenetic characteristics at both the genetic and epigenetic levels, thus ensuring the sustainability of the material.

[0012] 4. The method described in this invention has a high differentiation efficiency, which allows the embryonic cells in the embryonic cell mass to develop fully, thereby improving the regeneration efficiency and budding efficiency of somatic embryos and providing a foundation for production applications. Attached Figure Description

[0013] Figure 1 The aseptic seedlings of Coptis chinensis grown for 10 days are from Example 1.

[0014] Figure 2 This refers to the callus tissue formed after 20 days of induction of the hypocotyl of Coptis chinensis in Example 1.

[0015] Figure 3 This refers to the embryogenic callus induced to form after 30 days in Example 1.

[0016] Figure 4 The tissue is the embryogenic callus-induced bud from Coptis chinensis in Example 1.

[0017] Figure 5 For example 1, from somatic cell tissue mass ( Figure 4 Adventitious buds obtained from part A of the middle section.

[0018] Figure 6 The embryonic seedlings cultured to 25 days in Example 1.

[0019] Figure 7 The embryonic seedlings cultured to 40 days in Example 1.

[0020] Figure 8 The embryonic seedlings cultured to 60 days in Example 1.

[0021] Figure 9 The phenotype is that of the rooted cultured for 10 days in Example 1.

[0022] Figure 10 This refers to the phenotype of the rooted cultured organism in Example 1 for 22 days, where part B is the phenotype of the organism. Figure 9 The same plant. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the embodiments. Example 1

[0024] like Figures 1-10 As shown, a method for establishing a highly efficient regeneration system for *Picrorhiza scrophulariiflora* cell embryos includes the following steps: (1) Seed disinfection and germination Seeds were extracted from mature Picrorhiza scrophulariiflora pods and screened using water selection. The screened seeds were then sterilized in a clean bench with sodium hypochlorite (3%) for 5 min, washed three times with sterile water, sterilized with 75% alcohol for 1 min, washed three times with sterile water, and the sodium hypochlorite + alcohol sterilization cycle was repeated twice. Finally, the seeds were washed five times with water. The surface moisture was blotted dry with sterile absorbent paper. The seeds were then sown on MS medium containing 30 g / L sugar and 6 g / L agar, pH 5.8 (sterilized at 121℃ for 25 min). Ten seeds were sown per bottle and cultured at 22℃, 16 h light + 8 h dark, light intensity 2000-2500 Lx. Germination was visible to the naked eye after 3-5 days. Cultivation continued until seedlings were formed, which were then used as material for subsequent stages.

[0025] (2) Induction of embryonic callus Remove sterile seedlings of Picrorhiza scrophulariiflora that have grown for 10 days from the clean bench (e.g., Figure 1 As shown in the image, hypocotyls and stem segments were cut into lengths of 0.5 ± 0.1 cm. Six to nine hypocotyls were inoculated into each bottle of MS medium containing 30 g / L sugar, 6 g / L agar, 0.5 mg / L 6-BA, 1.0 mg / L NAA, and pH 5.8. The culture was then incubated at 22°C in the dark for 30 days. Callus tissue obtained after 20 days of culture was shown in the image. Figure 2 As shown, the embryogenic callus tissue obtained after 30 days of culture is as follows: Figure 3 As shown.

[0026] (3) Bud induction The cultured callus tissue obtained from *Picrorhiza scrophulariiflora* (e.g.) Figure 3 (As shown) Inoculated into MS medium containing 30 g / L sugar, 6 g / L agar, and 0.5 mg / L 6-BA, pH 5.8, with 4-6 embryogenic callus tissues per medium, and cultured at 22℃, under 16-hour light conditions and a light intensity of 2000-2500 Lx, adventitious buds appeared after 20±5 days (e.g. Figure 4 (As shown). Adventitious buds (such as those separated from somatic cell masses) Figure 5(As shown) Transferred to 1 / 2 MS medium containing 30 g / L sugar, 6 g / L agar, and 0.5 mg / L 6-BA, pH 5.8, and cultured at 22℃ under 16-hour light intensity (2000-2500 Lx). Somatic embryos cultured for 25, 40, and 60 days are shown below. Figure 6 , Figure 7 , Figure 8 As shown, the *Hu Huang Conjoined Pisces* embryos were obtained.

[0027] (4) Rooting culture Transfer the 60-day-old somatic embryos to rooting medium containing 1 / 2 MS + 30 g / L sugar + 6 g / L agar + 0.2 mg / L 6-BA, pH 5.8. Place 4-6 embryos in each medium and culture at 22℃, 16h light, and a light intensity of 2000-2500 Lx. Rooting occurs in 6-10 days (e.g., ...). Figure 9 As shown), continue to grow until 22 days (as shown). Figure 10 As shown in the image. Example 2

[0028] This embodiment serves as a comparative example, differing from Example 1 in that: in step (2), during the induction of embryogenic callus, leaves from sterile seedlings of *Picrorhiza scrophulariiflora* grown for 10 days were selected for induction. The leaves were trimmed at the edges and cut into small cubes of 0.8cm × 0.8cm, and induction was performed under the same culture medium and conditions. The results showed that the growth rate induced by leaves was significantly lower than that induced by hypocotyls; therefore, hypocotyls were selected for induction in this invention.

Claims

1. A method for establishing a highly efficient regeneration system for *Picrorhiza scrophulariiflora* cell embryos, characterized in that, Includes the following steps: (1) Seed disinfection and germination: Take Coptis chinensis seeds, disinfect them, and sow them on MS medium with 30 g / L sugar and 6 g / L agar, pH 5.8, and culture under light until they germinate and grow into seedlings; (2) Induction of embryogenic callus: Seedlings were taken, the hypocotyls were cut into small pieces and inoculated into embryogenic callus medium of MS + 30 g / L sugar + 6 g / L agar + 0.5 mg / L 6-BA + 1.0 mg / L NAA, pH 5.8, and cultured in the dark until embryogenic callus tissue was obtained. (3) Shoot induction: Embryogenic callus was inoculated into MS medium containing 30 g / L sugar, 6 g / L agar, 0.5 mg / L 6-BA, and pH 5.

8. Adventitious shoots were induced by light. The adventitious shoots were then transferred to 1 / 2 MS medium containing 30 g / L sugar, 6 g / L agar, 0.5 mg / L 6-BA, and pH 5.8 and cultured for 20-60 days to obtain *Gnaphalium affine* embryonic seedlings. (4) Rooting culture: Transfer the Cordyceps militaris embryos to a rooting medium containing 1 / 2 MS + 30 g / L sugar + 6 g / L agar + 0.2 mg / L 6-BA, pH 5.8, and culture under light until roots are formed.

2. The method for establishing a highly efficient regeneration system for induced Pistacia chinensis cell embryos according to claim 1, characterized in that, The culture temperature in steps (1) to (4) is 22℃.

3. The method for establishing a highly efficient regeneration system for induced Pistacia chinensis cell embryos according to claim 1, characterized in that, The photoperiod for light cultivation in steps (1), (3), and (4) is 16 hours.

4. The method for establishing a highly efficient regeneration system for induced Pistacia chinensis cell embryos according to claim 1, characterized in that, The light intensity in steps (1), (3), and (4) is 2000-2500 Lx.

5. The method for establishing a highly efficient regeneration system for induced Pistacia chinensis cell embryos according to claim 1, characterized in that, The disinfection steps described in step (1) are as follows: first disinfect with 3% sodium hypochlorite for 5 minutes, rinse with sterile water, then disinfect with 75% alcohol for 1 minute, rinse with sterile water, repeat twice, and finally dry the surface of the seeds.

6. The method for establishing a highly efficient regeneration system for induced Pistacia chinensis cell embryos according to claim 1, characterized in that, In step (2), sterile seedlings of Coptis chinensis are taken and the hypocotyl is cut into 0.5±0.1cm pieces.

Images