Tissue culture method of haematoxylum

Abstract

This invention discloses a tissue culture method for *Gynostemma pentaphyllum*, comprising the following steps: S1: Pretreatment of *Gynostemma pentaphyllum* before the inflorescence emerges, obtaining sterilized inflorescences through aseptic operation; S2: Induction of callus tissue from the inflorescence stalk segment retaining the base of the flower bud, inoculated into a callus induction medium to obtain white callus tissue; S3: Greening of callus tissue, after removing the inflorescence stalk segment from the white callus tissue obtained in S2, transferred to a callus greening medium; S4: Induction and proliferation of shoot clusters, transferring the green callus tissue obtained in S3 to a shoot cluster induction and proliferation medium to cultivate *Gynostemma pentaphyllum* seedlings; S5: Rooting culture of robust seedlings to obtain rooted *Gynostemma pentaphyllum* plants. The advantages of this invention compared to existing technologies are: it provides a tissue culture method for inducing callus tissue and plant regeneration from the inflorescence stalk segment retaining the base of the flower bud of *Gynostemma pentaphyllum*, providing an effective tissue culture system for the commercial in vitro propagation of *Gynostemma pentaphyllum* seedlings.

Description

Technical Field

[0001] This invention relates to plant tissue culture methods, specifically a tissue culture method for red ginger flowers. Background Technology

[0002] *Hedychium neocarneum*, belonging to the genus *Hedychium* in the family Zingiberaceae, is native to Yunnan, my country. It grows to a height of 1–2 m and flowers from September to December. The flowers are white with bright red stamens and a strong fragrance (Hu Xiu et al., 2010; Wu Delin et al., 2016). Currently, *Hedychium neocarneum* is not widely cultivated, but it can be used for potted cultivation and ground planting in gardens, flower beds, and other flower borders for ornamental purposes. With its prominent, neat inflorescences and vibrant colors, *Hedychium neocarneum* is a valuable ornamental plant resource within the *Hedychium* genus.

[0003] In addition, the flowers of *Ginger sibirica* are highly fragrant and can be used to extract essential oils and fragrances, finding wide application in the daily chemical and cosmetic industries. *Ginger sibirica* has a low wild population, and its propagation methods primarily rely on rhizomes and seeds.

[0004] Because the number of tillers in red ginger lily is small, and its large rhizomes result in slow propagation, and because it takes two to three years for seeds to flower from sowing, both of these propagation methods are inefficient and cannot provide enough seedlings for the commercial production of red ginger lily.

[0005] Tissue culture techniques, through the induction of callus tissue, can provide an effective method for the large-scale propagation of seedlings. Tissue culture studies of *Hedychium* species have focused on *Hedychium praecox*, *Hedychium serratum*, *Hedychium rubrum*, *Hedychium laurentii*, *Hedychium laurentii*, *Hedychium dwarfum*, *H. muluense*, and *H. bousigonianum*.

[0006] Callus was induced using the shoot apical meristem of *H. muluense* as explants. The callus was pre-cultured in suspension culture, followed by somatic embryo induction. Salicylic acid and silver nitrate were then used to regulate the proliferation and somatic embryo induction process of *H. bousigonianum* callus, which had a certain effect on increasing the frequency of somatic embryogenesis.

[0007] Hypocotyls or adventitious buds of sterile seedlings germinated from mature seeds of *Hedychium coronarium* were used as explants and cultured on MS medium containing 1 mg / L 6-BA and 0.2 mg / L NAA, inducing clustered buds and producing complete plants. Immature filaments of *Hedychium coronarium* were used as explants and cultured on MS medium containing 4 mg / L 2,4-D, 4 mg / L NAA, and 1 mg / L 6-BA, inducing callus tissue after 180 days of culture. Pseudostem segments of *Hedychium truncatum* were used as explants and cultured on MS medium containing 3–5 mg / L 6-BA, 2 mg / LTDZ, and 0.01 mg / L NAA, inducing protocorms.

[0008] Using the inflorescence axis and bracts of *Gynostemma pentaphyllum* as explants, they were inoculated on MS medium containing 1 mg / L 6-BA and 2 mg / L NAA, and regenerated plants were obtained by inducing callus formation into adventitious shoots. Using the filaments and anthers of *Gynostemma pentaphyllum* as explants, they were inoculated on MS medium containing 4 mg / L 2,4-D, 4 mg / L NAA, 1 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, and callus was induced after 120 days of culture.

[0009] Adventitious shoots were induced by inoculating the 0.5–2.0 cm bud tips of the rhizomes of *Ginger lily* with MS medium supplemented with 5.0 mg / L 6-BA and 0.2 mg / L NAA. Seeds of *Ginger lily* were sown on callus induction medium (MS medium supplemented with 6.0–9.0 mg / L 6-BA, 6.0–8.0 mg / L TDZ, 0.03–0.05 mg / L NAA, 25.0–30.0% coconut milk, 35–40 g / L sucrose, and 13.0–13.5 g / L carrageenan powder) until white callus formation occurred on the hypocotyls of sterile seedlings.

[0010] During their tissue culture studies of *Gynostemma pentaphyllum*, the inventors discovered that when filaments and anthers of *Gynostemma pentaphyllum* were used as explants and cultured on the callus induction medium (MS + 4 mg / L 2,4-D + 4 mg / L NAA + 1 mg / L 6-BA) used in the literature, no callus germination occurred. This indicates that the culture medium formulation listed in the literature is unsuitable for callus induction culture of *Gynostemma pentaphyllum* filaments and anthers. Furthermore, when rhizome axillary buds of *Gynostemma pentaphyllum* were used as explants and cultured on the adventitious bud induction medium (MS + 5.0 mg / L 6-BA + 0.2 mg / L NAA) used in the literature, the rhizome axillary buds were relatively thick and contained endophytic bacteria, resulting in complete contamination of the explants.

[0011] Furthermore, the literature only reports the process of regenerating plants from callus induction in white ginger flower, red ginger flower, dwarf ginger flower, H. muluense, and H. bousigonianum. Currently, there is still a lack of reports on tissue culture techniques for regenerating plants from callus induction in red ginger flower.

[0012] Therefore, it is necessary to invent a tissue culture method for red ginger flowers to solve the above problems. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to overcome the above-mentioned technical defects and provide a tissue culture method for inducing callus tissue and plant regeneration by retaining the inflorescence stalk segment at the base of the flower bud of *Gynostemma pentaphyllum*. This method can provide an effective tissue culture system for the commercial in vitro propagation of *Gynostemma pentaphyllum* seedlings.

[0014] To solve the above-mentioned technical problems, the present invention provides a method for tissue culture of red ginger flowers, comprising the following steps: S1: Pretreatment of unemerged inflorescences of red ginger flower: Select the swollen but unemerged inflorescences at the top of the leaf and stem of red ginger flower, remove the leaves, place them in a clean bench, and perform aseptic operation to obtain sterilized inflorescences. S2: Inducing callus by retaining the inflorescence stalk segment at the base of the pink ginger flower bud, removing the upper part of the flower bud on the sterilized inflorescence in S1, and retaining the flower bud base with a length of 0.2-0.5 cm; cutting the inflorescence stalk into segments with a length of 0.5-1.0 cm along the base of the flower bud, and inoculating them into the callus induction medium to obtain white callus; S3: Callus greening: After removing the inflorescence stalk segment from the white callus obtained in S2, the callus was transferred to callus greening medium. S4: Induction and proliferation of clustered shoots. The green callus tissue obtained in S3 was transferred to the induction and proliferation medium of clustered shoots to obtain clustered shoot clumps, which were then cultivated to obtain flesh-red ginger flower seedlings. S5: Seedling rooting culture. Select seedlings with a height of more than 5cm and 3-5 leaves from the red ginger flower seedlings obtained in S4. Cut off the buds on a clean bench and inoculate them into the seedling rooting culture medium to obtain rooted plants of red ginger flower.

[0015] Preferably, the aseptic operation in S1 includes first treating the surface of the inflorescence with an alcohol solution, then wiping the surface of the inflorescence with a sterile cotton, cutting off the outer bracts of the inflorescence with a blade, and then disinfecting with a mercuric chloride solution. Wash with sterile water 4-5 times, 1-2 minutes each time, then use sterile gauze to absorb the moisture on the surface of the inflorescence and air dry at room temperature for 5-10 minutes.

[0016] Preferably, the alcohol solution in S1 is a 70% volume concentration alcohol solution, which is sprayed from top to bottom onto the surface of the inflorescence until droplets are formed on the surface of the inflorescence. The mercuric chloride solution is a 0.1% mercuric chloride solution by mass; the disinfection time of the mercuric chloride solution is 16-18 minutes. The ambient temperature for air drying is 20–25°C.

[0017] Preferably, the callus induction medium in S2 is based on modified MS medium, with the addition of 3.5-4.5 mg / L of 6-benzylaminopurine, 3.5-4.5 mg / L of thidiazuron, 15.0-20.0% of coconut juice, 30-35 g / L of sucrose, 6.5-7.0 g / L of agar powder, and pH 5.8-6.0.

[0018] Preferably, the callus greening medium in S3 is based on modified MS medium, with the addition of 3.5-4.5 mg / L 6-benzylaminopurine, 0.35-0.45 mg / L thidiazuron, 0.035-0.045 mg / L naphthaleneacetic acid, 10.0-15.0% banana juice, 30-35 g / L sucrose, 6.5-7.0 g / L agar powder, and pH 5.8-6.0.

[0019] Preferably, the bud induction and proliferation medium in S4 is based on modified MS medium, with the addition of 3.0-3.5 mg / L 6-benzylaminopurine, 0.3-0.35 mg / L thidiazuron, 0.03-0.035 mg / L naphthaleneacetic acid, 10.0-15.0% banana juice, 30-35 g / L sucrose, 6.5-7.0 g / L agar powder, and pH 5.8-6.0.

[0020] Preferably, the seedling rooting culture medium in S5 is: modified MS medium + naphthaleneacetic acid 0.5-1.0 mg / L + 6-benzylaminopurine 0.2-0.5 mg / L + banana juice 10.0-15.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.0.

[0021] Preferably, the modified MS culture medium in S2 to S5 consists of ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, disodium EDTA 37.30 mg / L, ferrous sulfate heptahydrate 27.80 mg / L, nicotinic acid 0.50 mg / L, pyridoxine hydrochloride 0.50 mg / L, thiamine hydrochloride 0.10 mg / L, inositol 100 mg / L, glycine 2 mg / L, and sterile water as the solvent.

[0022] Preferably, in S2 to S5: The sterilization conditions for the culture medium are: sterilization at 125℃ for 25-30 min, and the pH of the culture medium is adjusted by using a 2 mol / L sodium hydroxide solution. The cultivation environment was as follows: cultivation temperature 25±1℃, light intensity 2000~2400lx, and light duration 14h / d.

[0023] Preferably, the criteria for determining the cluster of buds in S4 are: the number of buds in a single bud cluster is ≥5, the bud height is 2-4cm, and the leaves are not yellowed.

[0024] The advantages of this invention compared to the prior art are: (1) High efficiency of sterility of unemerged inflorescence: This invention takes the swollen inflorescence at the top of the leaf stem of the red ginger flower and avoids the phenomenon that the inflorescence is easy to attract insects after it emerges and opens, which leads to incomplete disinfection and easy contamination during inoculation. This invention uses pruning shears to cut off the swollen inflorescence at the top of the leaf stem of the red ginger flower and peel off the leaves to expose the inflorescence. The inflorescence is placed in a clean bench, the surface of the inflorescence is sprayed with alcohol solution, the surface of the inflorescence is wiped with sterile cotton, the outer bracts on the inflorescence are cut off with a blade, then disinfected with mercuric chloride solution, and then placed in sterile water to wash 4 to 5 times, each time for 1 to 2 minutes. The inflorescence is taken out and placed in a sterile dish, the moisture on the surface of the inflorescence is absorbed with sterile gauze, and then air-dried at room temperature for 5 to 10 minutes to obtain the sterilized inflorescence of the red ginger flower. (2) Callus induction and plant regeneration of inflorescence peduncle segments with bud base retained: In this invention, inflorescence peduncle segments with bud base retained at 0.2-0.5 cm length are inoculated into callus induction medium (modified MS medium + 6-benzylaminopurine 3.5-4.5 mg / L + thidiazuron 3.5-4.5 mg / L + coconut juice 15.0-20.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.0), which can form firm, white callus at the bud base on the inflorescence peduncle segment. Then, it is gradually transferred to callus greening medium, shoot induction and proliferation medium to induce and proliferate shoots, and seedling rooting medium to obtain a large number of regenerated plants. This invention provides a method for inducing callus tissue and regenerating plants from the inflorescence stalk segment with the basal part of the flower bud in *Gynostemma pentaphyllum*, laying the foundation for rapid in vitro propagation and transgenic research of *Gynostemma pentaphyllum*; it is also beneficial for the large-scale promotion and application of *Gynostemma pentaphyllum* seedlings. Attached Figure Description

[0025] Figure 1 A photo of the inflorescence of the red ginger flower, after the outer bracts have been removed, being disinfected by soaking it in a mercuric chloride solution.

[0026] Figure 2 Photographs of callus induced to preserve the inflorescence stalk segment at the base of the pink ginger flower bud.

[0027] Figure 3 Photos showing the process of cutting off the inflorescence stalk of the red ginger flower, the greening of the remaining callus tissue, and the induction of a small number of clustered buds.

[0028] Figure 4 Photographs showing the continued induction and culture of clustered shoots of Ginger florets.

[0029] Figure 5 Photographs showing the continued propagation and cultivation of clustered buds of Ginger florets.

[0030] Figure 6 Photos of seedlings with 3-5 leaves and a height of over 5 cm obtained by continuing to cultivate the clustered shoots of Ginger florets.

[0031] Figure 7 Photographs showing the rooting and culturing of tissue culture seedlings of *Ginger lily*.

[0032] Figure 8 Photographs showing no germination of callus induced by the filaments of Ginger spp. (Comparative Example 1).

[0033] Figure 9 Photographs showing no germination of callus induced by the anthers of Ginger spp. (Comparative Example 2).

[0034] Figure 10 Photographs showing contamination of adventitious buds induced from the axillary buds of the rhizome of *Ginger florida* (Comparative Example 3). Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the accompanying drawings.

[0036] A tissue culture method for red ginger flowers includes the following steps: S1: Pretreatment of unemerged inflorescences of Ginger lily: Cut off the swollen and unemerged inflorescences at the top of the leaves and stems of Ginger lily with pruning shears, peel off the leaves to expose the inflorescences, place the inflorescences in a clean bench, spray the surface of the inflorescences with alcohol solution, wipe the surface of the inflorescences with sterile cotton, cut off the outer bracts on the inflorescences with a blade, disinfect with mercuric chloride solution, and then wash in sterile water 4-5 times, 1-2 minutes each time. Remove the inflorescences and place them in a sterile dish, absorb the moisture on the surface of the inflorescences with sterile gauze, and then air dry at room temperature for 5-10 minutes to obtain sterilized inflorescences of Ginger lily. S2: Callus induction from inflorescence stalk segments retaining the base of the flower buds of *Ginger indicum*: First, hold the sterilized inflorescence with tweezers, then cut off the upper part of the flower buds on the inflorescence with a blade, retaining a 0.2–0.5 cm long flower bud base; cut the inflorescence stalk into segments of 0.5–1.0 cm in length along the flower bud base, inoculate them on callus induction medium, and culture for 35–50 days until white callus forms at the flower bud base on the inflorescence stalk segments; the callus induction medium for the inflorescence stalk segments retaining the flower bud base is: modified MS medium + 6-benzylaminopurine 3.5–4.5 mg / L + thidiazuron 3.5–4.5 mg / L + coconut juice 15.0–20.0% + sucrose 30–35 g / L + agar powder 6.5–7.0 g / L, pH 5.8–6.0; S3: Callus Greening: On a clean bench, the white callus obtained in S2 was cut off with a blade, leaving the white callus intact. The cut was then transferred to a callus greening medium. After 40–55 days of culture, the callus clearly turned green. The callus greening medium consisted of: modified MS medium + 6-benzylaminopurine 3.5–4.5 mg / L + thidiazuron 0.35–0.45 mg / L + naphthaleneacetic acid 0.035–0.045 mg / L + banana juice 10.0–15.0% + sucrose 30–35 g / L + agar powder 6.5–7.0 g / L, pH 5.8–6.0. S4: Induction and proliferation of shoot clusters: On a clean bench, the green callus tissue from S3 was transferred to a shoot cluster induction and proliferation medium to induce and proliferate shoot clusters. After 50–60 days of culture, induced and proliferated shoot clusters were obtained. The shoot cluster induction and proliferation medium consisted of: modified MS medium + 6-benzylaminopurine 3.0–3.5 mg / L + thidiazuron 0.3–0.35 mg / L + naphthaleneacetic acid 0.03–0.035 mg / L + banana juice 10.0–15.0% + sucrose 30–35 g / L + agar powder 6.5–7.0 g / L, pH 5.8–6.0. S5: Seedling Rooting Culture: From the red ginger flower seedlings obtained in S4, select seedlings with a height of over 5 cm and 3-5 leaves. On a clean bench, cut buds and inoculate them into the seedling rooting culture medium. After 20-25 days of culture, rooted red ginger flower plants are obtained. The seedling rooting culture medium is: modified MS medium + naphthaleneacetic acid 0.5-1.0 mg / L + 6-benzylaminopurine 0.2-0.5 mg / L + banana juice 10.0-15.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.0. The specific steps for spraying the inflorescence surface with alcohol solution are as follows: first prepare a 70% alcohol solution, then put it into a spray bottle and spray the inflorescence surface from top to bottom until there are droplets on the inflorescence surface. The specific steps for disinfecting with mercuric chloride solution are as follows: soak the inflorescence in 0.1% mercuric chloride solution for 16 to 18 minutes. In S2, S3, S4, and S5, the modified MS medium composition is as follows: ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, disodium EDTA 37.30 mg / L, ferrous sulfate heptahydrate 27.80 mg / L, nicotinic acid 0.50 mg / L, pyridoxine hydrochloride 0.50 mg / L, thiamine hydrochloride 0.10 mg / L, inositol 100 mg / L, glycine 2... mg / L, solvent is sterile water, sterilization conditions of culture medium are 125℃ for 25-30 min, culture temperature is 25±1℃, light intensity is 2000-2400 lx, light time is 14 h / d, and pH of culture medium is adjusted with 2 mol / L sodium hydroxide solution. In S2, the white callus tissue formed at the base of the flower buds on the inflorescence stalk segment is a firm, white callus tissue; in S3, the callus tissue clearly turns green and becomes a firm, green tissue mass.

[0037] Example 1 In specific implementation, this invention includes the following steps: (1) Pretreatment of unemerged inflorescences of Ginger lily: Cut off the swollen and unemerged inflorescences at the top of the leaf stem of Ginger lily with pruning shears, peel off the leaves to expose the inflorescences, place the inflorescences in a clean bench, spray the surface of the inflorescences with alcohol solution, wipe the surface of the inflorescences with sterile cotton, remove the outer bracts on the inflorescences with a blade, and then disinfect the inflorescences by soaking them in 0.1% mercuric chloride solution for 16 min. Figure 1 Then, rinse the inflorescence in sterile water four times, one minute each time. Remove the inflorescence and place it in a sterile dish. Use sterile gauze to absorb the moisture on the surface of the inflorescence, and then air dry it at room temperature for 5 minutes to obtain the sterilized inflorescence of the red ginger flower.

[0038] (2) Callus induction from the peduncle segment retaining the base of the flower bud of *Ginger sibiricum*: First, hold the inflorescence with tweezers, then cut off the upper part of the flower bud on the inflorescence with a blade, leaving a 0.2 cm long bud base. Cut the peduncle along the base of the flower bud in segments, with each segment being 1.0 cm long. Inoculate the segments onto the callus induction medium and culture for 35 days until a firm, white callus forms at the base of the flower bud on the peduncle segment. Figure 2 The callus induction medium for the inflorescence peduncle segment with the bud base retained was: modified MS medium + 6-benzylaminopurine 4.5 mg / L + thidiazuron 4.5 mg / L + coconut juice 20.0% + sucrose 35 g / L + agar powder 7.0 g / L, pH 5.8.

[0039] (3) Callus turning green: On a clean bench, the white callus obtained in step (2) was cut off with a blade, leaving the white callus intact, and transferred to a callus turning green medium. After 40 days of culture, the callus turned green significantly. Figure 3 The callus greening culture medium described above is: modified MS medium + 6-benzylaminopurine 4.5 mg / L + thidiazuron 0.45 mg / L + naphthaleneacetic acid 0.045 mg / L + banana juice 15.0% + sucrose 35 g / L + agar powder 7.0 g / L, pH 6.0.

[0040] (4) Induction and proliferation of clustered shoots: On a clean bench, the callus tissue that had turned green in step (3) was transferred to a clustered shoot induction and proliferation medium to induce and proliferate clustered shoots. After 50 days of culture, induced clustered shoots were obtained. Figure 4 ) and increased clusters of shoots ( Figure 5 The bud induction and proliferation medium is: modified MS medium + 6-benzylaminopurine 3.5 mg / L + thidiazuron 0.35 mg / L + naphthaleneacetic acid 0.035 mg / L + banana juice 15.0% + sucrose 35 g / L + agar powder 6.5 g / L, pH 6.0.

[0041] (5) Seedling rooting culture: Among the red ginger flower seedlings obtained in step (4), select seedlings with a height of more than 5 cm and 5 leaves. Figure 6 On a clean bench, buds were cut and inoculated into a seedling rooting medium. After 20 days of culture, rooted plants of *Ginger lily* were obtained. Figure 7 The seedling rooting culture medium is: modified MS medium + naphthaleneacetic acid 1.0 mg / L + 6-benzylaminopurine 0.5 mg / L + banana juice 15.0% + sucrose 35 g / L + agar powder 6.5 g / L, pH 6.0.

[0042] In step (1) above, the specific steps for spraying the inflorescence surface with alcohol solution are as follows: first prepare a 70% alcohol solution, then put it into a spray bottle and spray it from top to bottom onto the inflorescence surface until there are droplets on the inflorescence surface.

[0043] In steps (2), (3), (4) and (5) above, the modified MS culture medium consists of ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, disodium EDTA 37.30 mg / L, ferrous sulfate heptahydrate 27.80 mg / L, nicotinic acid 0.50 mg / L, pyridoxine hydrochloride 0.50 mg / L, thiamine hydrochloride 0.10 mg / L, inositol 100 mg / L, glycine 2 mg / L, and sterile water as the solvent.

[0044] In steps (2), (3), (4) and (5) above, the sterilization conditions for the culture medium are 125℃ for 25 min.

[0045] In steps (2), (3), (4) and (5) above, the culture temperature is 26 ℃, the light intensity is 2400 lx, and the light exposure time is 14 h / d.

[0046] In steps (2), (3), (4) and (5) above, the pH of the culture medium is adjusted with 2 mol / L sodium hydroxide solution.

[0047] The rooting rate of the tissue culture seedlings after inoculation in Example 1 [Note: Rooting rate = (number of seedlings rooted 20 days after inoculation / total number of seedlings at the time of inoculation) × 100%) can reach over 95%; Example 1 can obtain a large number of rooted and strong tissue culture seedlings by pretreatment of the unemerged inflorescence, induction of callus tissue with the inflorescence stalk segment retaining the base of the flower bud, callus greening, induction and proliferation of clustered buds, and strong seedling rooting.

[0048] Example 2 A tissue culture method for ginger flowers with fleshy red color, comprising the following steps: (1) Pretreatment of unemerged inflorescences of Ginger florets: Cut off the swollen and unemerged inflorescences at the top of the leaves and stems of Ginger florets with pruning shears, peel off the leaves to expose the inflorescences, place the inflorescences in a clean bench, spray the surface of the inflorescences with alcohol solution, wipe the surface of the inflorescences with sterile cotton, remove the outer bracts on the inflorescences with a blade, then soak the inflorescences in 0.1% mercuric chloride solution for 18 min for disinfection, then rinse them in sterile water 5 times for 2 min each time, take out the inflorescences and place them in a sterile dish, dry the surface of the inflorescences with sterile gauze, and then air dry them at room temperature for 10 min to obtain sterilized inflorescences of Ginger florets.

[0049] (2) Callus induction of inflorescence stalk segments with the base of the flower buds of Ginger florets: First, hold the inflorescence with tweezers, then cut off the upper part of the flower buds on the inflorescence with a blade, leaving a 0.5 cm long flower bud base. Cut the inflorescence stalk segment along the base of the flower bud, with the inflorescence stalk segment being 0.5 cm long. Inoculate it on the callus induction medium and culture for 50 days until the base of the flower buds on the inflorescence stalk segment forms a firm, white callus. The callus induction medium with the base of the flower buds is: modified MS medium + 6-benzylaminopurine 3.5 mg / L + thidiazuron 3.5 mg / L + coconut juice 15.0% + sucrose 30 g / L + agar powder 6.5 g / L, pH 6.0.

[0050] (3) Callus turning green: On a clean bench, the white callus obtained in step (2) was cut off with a blade, leaving the white callus, and transferred to a callus turning green medium. After 55 days of culture, the callus turned green. The callus turning green medium was: modified MS medium + 6-benzylaminopurine 3.5 mg / L + thidiazuron 0.35 mg / L + naphthaleneacetic acid 0.035 mg / L + banana juice 10.0% + sucrose 30 g / L + agar powder 6.5 g / L, pH 5.8.

[0051] (4) Induction and proliferation of shoot clusters: On a clean bench, the callus tissue that turned green in step (3) was transferred to the shoot cluster induction and proliferation medium to induce and proliferate shoot clusters. After 60 days of culture, induced shoot clusters and proliferated shoot clusters were obtained. The shoot cluster induction and proliferation medium was: modified MS medium + 6-benzylaminopurine 3.0 mg / L + thidiazuron 0.3 mg / L + naphthaleneacetic acid 0.03 mg / L + banana juice 10.0% + sucrose 30 g / L + agar powder 7.0 g / L, pH 5.8.

[0052] (5) Seedling rooting culture: Among the seedlings of red ginger flower obtained in step (4), select seedlings with a height of more than 5 cm and 4 leaves. On a clean bench, cut off the buds and inoculate them into the seedling rooting culture medium. After 25 days of culture, rooted plants of red ginger flower are obtained. The seedling rooting culture medium is: modified MS medium + naphthaleneacetic acid 0.5 mg / L + 6-benzylaminopurine 0.2 mg / L + banana juice 10.0% + sucrose 30 g / L + agar powder 7.0 g / L, pH 5.8.

[0053] In step (1) above, the specific steps for spraying the inflorescence surface with alcohol solution are as follows: first prepare a 70% alcohol solution, then put it into a spray bottle and spray it from top to bottom onto the inflorescence surface until there are droplets on the inflorescence surface.

[0054] In steps (2), (3), (4) and (5) above, the modified MS culture medium consists of ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, disodium EDTA 37.30 mg / L, ferrous sulfate heptahydrate 27.80 mg / L, nicotinic acid 0.50 mg / L, pyridoxine hydrochloride 0.50 mg / L, thiamine hydrochloride 0.10 mg / L, inositol 100 mg / L, glycine 2 mg / L, and sterile water as the solvent.

[0055] In steps (2), (3), (4) and (5) above, the sterilization conditions for the culture medium are 125℃ for 30 min.

[0056] In steps (2), (3), (4) and (5) above, the culture temperature is 24 ℃, the light intensity is 2000 lx, and the light exposure time is 14 h / d.

[0057] In steps (2), (3), (4) and (5) above, the pH of the culture medium is adjusted with 2 mol / L sodium hydroxide solution.

[0058] The rooting rate of the tissue culture seedlings after inoculation in Example 2 [Note: Rooting rate = (number of seedlings rooted 25 days after inoculation / total number of seedlings at the time of inoculation) × 100%) can reach over 97%; Example 2 can obtain a large number of rooted and strong tissue culture seedlings by pretreatment of the inflorescence without the flower buds emerging, induction of callus tissue with the flower bud base retained, callus greening, induction and proliferation of clustered buds, and strong seedling rooting.

[0059] Example 3 A tissue culture method for ginger flowers with fleshy red color, comprising the following steps: (1) Pretreatment of unemerged inflorescences of Ginger florets: Cut off the swollen and unemerged inflorescences at the top of the leaves and stems of Ginger florets with pruning shears, peel off the leaves to expose the inflorescences, place the inflorescences in a clean bench, spray the surface of the inflorescences with alcohol solution, wipe the surface of the inflorescences with sterile cotton, remove the outer bracts on the inflorescences with a blade, then soak the inflorescences in 0.1% mercuric chloride solution for 16 min for disinfection, then rinse them in sterile water 5 times, 1 min each time, take out the inflorescences and place them in a sterile dish, dry the surface of the inflorescences with sterile gauze, and then air dry at room temperature for 5 min to obtain sterilized inflorescences of Ginger florets.

[0060] (2) Callus induction of inflorescence stalk segments with the base of the flower buds of Ginger florets: First, hold the inflorescence with tweezers, then cut off the upper part of the flower buds on the inflorescence with a blade, leaving a 0.3 cm long flower bud base. Cut the inflorescence stalk segment along the base of the flower bud, with the inflorescence stalk segment being 0.8 cm long. Inoculate it on the callus induction medium and culture for 40 days until the base of the flower buds on the inflorescence stalk segment forms a firm, white callus. The callus induction medium with the base of the flower buds is: modified MS medium + 6-benzylaminopurine 4.0 mg / L + thidiazuron 4.0 mg / L + coconut juice 20.0% + sucrose 35 g / L + agar powder 7.0 g / L, pH 5.8.

[0061] (3) Callus turning green: On a clean bench, the white callus obtained in step (2) was cut off with a blade, leaving the white callus, and transferred to a callus turning green medium. After 45 days of culture, the callus turned green. The callus turning green medium was: modified MS medium + 6-benzylaminopurine 4.0 mg / L + thidiazuron 0.40 mg / L + naphthaleneacetic acid 0.04 mg / L + banana juice 15.0% + sucrose 35 g / L + agar powder 7.0 g / L, pH 6.0.

[0062] (4) Induction and proliferation of shoot clusters: On a clean bench, the callus tissue that turned green in step (3) was transferred to the shoot cluster induction and proliferation medium to induce and proliferate shoot clusters. After 55 days of culture, induced shoot clusters and proliferated shoot clusters were obtained. The shoot cluster induction and proliferation medium was: modified MS medium + 6-benzylaminopurine 3.5 mg / L + thidiazuron 0.3 mg / L + naphthaleneacetic acid 0.03 mg / L + banana juice 10.0% + sucrose 30 g / L + agar powder 6.5 g / L, pH 6.0.

[0063] (5) Seedling rooting culture: Among the seedlings of red ginger flower obtained in step (4), select seedlings with a height of more than 5 cm and 3 leaves. On a clean bench, cut off the buds and inoculate them into the seedling rooting culture medium. After 23 days of culture, rooted plants of red ginger flower are obtained. The seedling rooting culture medium is: modified MS medium + naphthaleneacetic acid 0.8 mg / L + 6-benzylaminopurine 0.3 mg / L + banana juice 15.0% + sucrose 35 g / L + agar powder 7.0 g / L, pH 6.0.

[0064] In step (1) above, the specific steps for spraying the inflorescence surface with alcohol solution are as follows: first prepare a 70% alcohol solution, then put it into a spray bottle and spray it from top to bottom onto the inflorescence surface until there are droplets on the inflorescence surface.

[0065] In steps (2), (3), (4), and (5) above, the modified MS culture medium consists of ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, disodium EDTA 37.30 mg / L, ferrous sulfate heptahydrate 27.80 mg / L, nicotinic acid 0.50 mg / L, pyridoxine hydrochloride 0.50 mg / L, thiamine hydrochloride 0.10 mg / L, inositol 100 mg / L, glycine 2 mg / L, and sterile water as the solvent.

[0066] In steps (2), (3), (4) and (5) above, the sterilization conditions for the culture medium are 125℃ for 25 min.

[0067] In steps (2), (3), (4) and (5) above, the culture temperature is 25 ℃, the light intensity is 2300 lx, and the light exposure time is 14 h / d.

[0068] In steps (2), (3), (4) and (5) above, the pH of the culture medium is adjusted with 2 mol / L sodium hydroxide solution.

[0069] The rooting rate of the tissue culture seedlings after inoculation in Example 3 [Note: Rooting rate = (number of seedlings rooted 23 days after inoculation / total number of seedlings at the time of inoculation) × 100%) can reach over 92%; Example 3 can obtain a large number of rooted and strong tissue culture seedlings by pretreatment of the inflorescence without the flower buds emerging, induction of callus tissue with the flower bud base retained, callus greening, induction and proliferation of clustered buds, and strong seedling rooting.

[0070] Comparative Example 1 (1) Selection and disinfection of explants: Select flower buds on immature inflorescences of ginger lily, wipe the surface of the flower buds with a sterile cotton ball dipped in 75% alcohol solution, disinfect with 0.1% mercuric chloride solution for 8 min; then wash in sterile water 6 times, 4 min each time, peel off the outer bracts of the flower buds, separate the filaments in the flower buds, and cut them into small sections of 0.5-0.8 cm.

[0071] (2) Callus induction: On a clean bench, the small segments of the filaments of the red ginger flower obtained in step (1) were inoculated onto callus induction medium for culture: MS + 4 mg / L 2,4-D + 4 mg / L NAA + 1 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar. The sterilization temperature and time, culture temperature, light intensity and time of the medium in this comparative example were the same as those in Example 1. After 180 days of culture, there was no contamination, but the small segments of filaments did not sprout, and no callus appeared. Figure 8 ).

[0072] Comparative Example 2 (1) Selection and disinfection of explants: Select flower buds on immature inflorescences of ginger lily, wipe the surface of the flower buds with a sterile cotton ball dipped in 75% alcohol solution, disinfect with 0.1% mercuric chloride solution for 8 min; then wash in sterile water 6 times, 3 min each time, peel off the outer bracts of the flower buds, separate the filaments and anthers in the flower buds, and obtain individual anthers.

[0073] (2) Callus induction: On a clean bench, individual anthers of the red ginger flower obtained in step (1) were inoculated onto callus induction medium: MS + 4 mg / L 2,4-D + 4 mg / L NAA + 1 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar. The sterilization temperature and time, culture temperature, light intensity and time of the medium in this comparative example were the same as those in Example 1. After 120 days of culture, there was no contamination, but the anthers did not sprout and no callus appeared. Figure 9 ).

[0074] Comparative Example 3 (1) Selection and disinfection of explants: Take the axillary buds of the rhizomes of ginger flowers, place them in a clean bench, wipe the surface of the axillary buds of the rhizomes with a sterile cotton ball dipped in 75% alcohol solution, and then disinfect them with 0.1% mercuric chloride solution for 22 min; then wash them in sterile water 5 times, 4 min each time, take them out and put them in a sterile dish to dry the surface moisture, and obtain sterilized rhizomes of ginger flowers.

[0075] (2) Induction of adventitious buds: The axillary buds of the sterilized rhizomes of *Gingeria fraseri* obtained in step (1) were peeled off, and cut into small segments of 0.5–1.0 cm. These segments were then inoculated onto an adventitious bud induction medium: MS + 5.0 mg / L 6-BA + 0.2 mg / L NAA. The sterilization temperature and time of the medium in this comparative example, as well as the culture temperature, light intensity, and time, were the same as those in Example 1. After 15 days of culture, due to the thicker axillary buds containing endophytic bacteria, the explants were completely contaminated. Figure 10 As shown.

[0076] Figure 1 The image shows a photograph of the inflorescence of *Ginger florets* (with the outer bracts removed) disinfected by soaking in a mercuric chloride solution. Figure 2 Photographs of callus induced from the peduncle segment retaining the base of the pink ginger flower bud are provided. White callus tissue can be seen forming at the base of the flower bud on the peduncle segment. Figure 3 The image shows the cut-off segment of the red ginger flower inflorescence stalk, the remaining callus tissue turning green, and the induced small clusters of shoots. The greened, compact tissue clumps are visible. Figure 4 Photographs of the continued induction culture of the clustered shoots of the red ginger flower are provided. Figure 5 Photographs of the continued propagation and cultivation of clustered buds of the red ginger flower are provided. Figure 6 Photographs are provided showing seedlings with a height of over 5 cm and 3-5 leaves obtained by further cultivation of the clustered shoots of Ginger lily. Figure 7 Photographs of rooting and culturing tissue culture seedlings of *Ginger lily* are provided. Figure 8 Photographs showing no germination of callus induced by the filaments of Ginger florets are provided (Comparative Example 1). Figure 9 Photographs showing no germination of callus induced by anthers of Ginger spp. (Comparative Example 2) are provided. Figure 10 Photographs showing the contamination of adventitious buds induced by axillary buds of the rhizome of *Ginger florida* are provided (Comparative Example 3).

[0077] As can be seen from the above comparative examples 1, 2 and 3, when the filaments and anthers of *Gynostemma pentaphyllum* were inoculated on the culture medium listed in the literature, no callus germination occurred; the axillary buds of the rhizome of *Gynostemma pentaphyllum* were relatively thick and contained endophytic bacteria, and the explants were completely contaminated, so it was not suitable as an explant for adventitious bud induction. In this embodiment, the inflorescence stalks, retaining the base of the flower buds of *Gynostemma pentaphyllum*, were segmented and inoculated onto callus induction medium (modified MS medium + 3.5–4.5 mg / L 6-benzylaminopurine + 3.5–4.5 mg / L thidiazuron + 15.0–20.0% coconut milk + 30–35 g / L sucrose + 6.5–7.0 g / L agar powder, pH 5.8–6.0). After culture, white callus tissue formed at the base of the flower buds on the inflorescence stalk segments. The inflorescence stalk segments were then cut off with a blade, retaining the white callus tissue, and transferred to callus greening medium, shoot induction and proliferation medium to induce and proliferate shoots, and seedling rooting medium to obtain regenerated *Gynostemma pentaphyllum* plants. This successfully established a rapid tissue culture propagation system for *Gynostemma pentaphyllum*. This invention lays an important foundation for the rapid tissue culture propagation and transgenic research of *Gynostemma pentaphyllum*.

[0078] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0079] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0080] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method of tissue culture of Hedychium coccineum, characterized by: Includes the following steps: S1: Pretreatment of unemerged inflorescences of *Gynostemma pentaphyllum*: Select swollen but unemerged inflorescences at the top of the leaf and stem of *Gynostemma pentaphyllum*, remove the leaves, place them in a clean bench, and perform aseptic procedures to obtain sterilized inflorescences; The aseptic procedures in S1 include first spraying the surface of the inflorescence with an alcohol solution from top to bottom until droplets form on the surface of the inflorescence, then wiping the surface of the inflorescence with sterile cotton, cutting off the outer bracts of the inflorescence with a blade, and then disinfecting with mercuric chloride solution; washing with sterile water 4-5 times, 1-2 minutes each time, absorbing the moisture on the surface of the inflorescence with sterile gauze, and air-drying at room temperature for 5-10 minutes; S2: Callus induction by retaining the inflorescence stalk segment at the base of the pink ginger flower bud. The upper part of the flower bud on the sterilized inflorescence in S1 is removed, retaining the flower bud base with a length of 0.2-0.5 cm. The inflorescence stalk is cut into segments with a length of 0.5-1.0 cm along the base of the flower bud and inoculated into the callus induction medium to obtain white callus. The callus induction medium in S2 consists of modified MS medium + 6-benzylaminopurine 3.5-4.5 mg / L + thidiazuron 3.5-4.5 mg / L + coconut juice 15.0-20.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.

0. S3: Callus greening: The white callus obtained in S2 was transferred to a callus greening medium after removing the inflorescence stalk segment; the callus greening medium in S3 consisted of modified MS medium + 6-benzylaminopurine 3.5-4.5 mg / L + thidiazuron 0.35-0.45 mg / L + naphthaleneacetic acid 0.035-0.045 mg / L + banana juice 10.0-15.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.0; S4: Shoot induction and proliferation. The green callus tissue obtained in S3 was transferred to the shoot induction and proliferation medium to obtain shoot clusters, which were then cultivated into red ginger flower seedlings. The shoot induction and proliferation medium in S4 consisted of modified MS medium + 6-benzylaminopurine 3.0-3.5 mg / L + thidiazuron 0.3-0.35 mg / L + naphthaleneacetic acid 0.03-0.035 mg / L + banana juice 10.0-15.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.

0. S5: Seedling rooting culture. Seedlings with a height exceeding 5cm and bearing 3-5 leaves obtained in S4 were selected. On a clean bench, buds were cut and inoculated into the seedling rooting culture medium to obtain rooted plants of *Gynostemma pentaphyllum*. The seedling rooting culture medium in S5 consisted of modified MS medium + naphthaleneacetic acid 0.5-1.0 mg / L + 6-benzylaminopurine 0.2-0.5 mg / L + banana juice 10.0-15.0% + sucrose 30-35 g / L + agar powder 6.5-7.0 g / L, pH 5.8-6.

0. The modified MS medium in S2-S5 consists of ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride dihydrate 220 mg / L, magnesium sulfate heptahydrate 185 mg / L, potassium dihydrogen phosphate 85 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, disodium EDTA 37.30 mg / L, ferrous sulfate heptahydrate 27.80 mg / L, nicotinic acid 0.50 mg / L, pyridoxine hydrochloride 0.50 mg / L, thiamine hydrochloride 0.10 mg / L, inositol 100 mg / L, glycine 2 mg / L, and sterile water as the solvent.

2. The method for tissue culture of ginger flower according to claim 1, characterized in that: The alcohol solution in S1 is a 70% alcohol solution by volume.

3. The method for tissue culture of ginger flower according to claim 1, characterized in that: The mercuric chloride solution is a 0.1% mercuric chloride solution by mass; the disinfection time of the mercuric chloride solution is 16-18 minutes; the ambient temperature for air drying is 20-25°C.

4. The method for tissue culture of ginger flower according to claim 1, characterized in that: In S2 to S5: The sterilization conditions for the culture medium are: sterilization at 125℃ for 25-30 min, and the pH of the culture medium is adjusted by using a 2 mol / L sodium hydroxide solution. The cultivation environment was as follows: cultivation temperature 25±1℃, light intensity 2000~2400lx, and light duration 14h / d.

5. The method for tissue culture of red ginger flower according to claim 1, characterized in that: The criteria for determining the clustered buds in S4 are: the number of clustered buds in a single bud cluster is ≥5, the bud height is 2-4cm, and the leaves are not yellowed.

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