High-throughput propagation method of panacis quinquefolii seeds through direct organogenesis approach

Abstract

The application discloses a high-flux propagation method of Panacis ginseng C.A.Meyer seedlings mediated by a direct organogenesis pathway, and comprises the following steps: after the surface of a current-year tender stem section of a fine strain of Panacis ginseng C.A.Meyer is disinfected, the stem section is cut into sections, and the sections are inoculated into an axillary bud germination induction culture medium to be cultured; leaf blades obtained in the culture are used as explant materials, the explant materials are cut into sections, and the sections are inoculated into an adventitious bud induction culture medium to be cultured for induction of the adventitious buds; the sections are transferred into an adventitious bud proliferation and elongation culture medium to be cultured for proliferation and elongation of the adventitious buds; the obtained elongated buds are separated from the bud clusters, immersed into a solution added with K-IBA and IAA to be treated, and then transferred into an adventitious root induction culture medium to be cultured for induction of the adventitious roots; after domestication, washing and surface disinfection, the seedlings are transplanted to be cultured. The application provides a rapid, efficient and high-quality Panacis ginseng C.A.Meyer tissue culture propagation method which can keep the fine characteristics of the mother plant, and provides certain technical support for germplasm preservation, large-scale propagation and variety improvement of the fine strain of Panacis ginseng C.A.Meyer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, and in particular to a high-throughput propagation method of Campanumoea lancifolia seedlings mediated by a direct organogenesis pathway. BACKGROUND

[0002] Campanumoea lancifolia (Roxb.) Merr, also known as long-leafed Campanumoea, is a perennial erect or vine herbaceous plant of Campanulaceae family, which is an important medicinal and edible plant and also has strong ornamental value. As an important medicinal plant, Campanumoea lancifolia is used as a root medicine, with a sweet and slightly bitter taste, and a flat nature, which has the effects of tonifying deficiency, benefiting qi, relieving phlegm and relieving pain, and is mainly used for treating fatigue, qi deficiency, weakness, sprains and injuries, and intestinal colic. At the same time, the fruits of Campanumoea lancifolia contain sugars, alkaloids, flavonoids, phenols, anthraquinones, steroids, triterpenes, and organic acids, among which the polysaccharide content is as high as 45.80%, which can effectively scavenge hydroxyl radicals and superoxide anion radicals, and is of great benefit to patients with high cholesterol and diabetes. The above-mentioned multiple effects make Campanumoea lancifolia a new, natural, healthy, green and antioxidant high-grade food with a broad market prospect.

[0003] Seed seedlings are the source of Campanumoea lancifolia industry development, and the quality of the seedlings directly affects the yield and quality of Campanumoea lancifolia. At present, Campanumoea lancifolia is mainly propagated by sowing in production, but due to the slow growth of seed seedlings and large variation of offspring, and the method of seedling raising is limited by the germination season, it is difficult to carry out large-scale, standardized (genotype background consistent) planting and large-area promotion of high-quality varieties. As a modern biotechnology, plant tissue culture technology has the advantages of high propagation efficiency, short propagation cycle, and no limitation of season and time, which has important significance in economic crop resource germplasm preservation and industrialized breeding. At present, there have been reports on the research of Campanumoea lancifolia tissue culture and rapid propagation, which mainly focuses on the rapid propagation of Campanumoea lancifolia seed, seed seedling stem tip and wild stem section as explant materials. The research based on seed and seed seedling as materials for tissue culture and rapid propagation is difficult to maintain the excellent characteristics of the mother plant; the research based on wild stem section as material has the problems of low propagation coefficient and high cost; for example, the Chinese patent application document with publication number CN115943889A discloses a tissue culture and rapid propagation method for inducing multiple buds from Campanumoea lancifolia stem sections, which includes the steps of explant acquisition, cleaning, disinfection, multiple bud induction, proliferation culture and rooting culture. Although it has the advantages of short production cycle, low production cost and better seedling quality, and can effectively avoid the variation risk caused by the influence of external factors on callus, it still has the problem of low propagation coefficient; at present, there is no research on the use of Campanumoea lancifolia excellent strain leaves as explants for plant regeneration.

[0004] Therefore, on the basis of red fruit ginseng excellent strain breeding, in order to further expand the propagation method and path of red fruit ginseng, realize the factory production of red fruit ginseng excellent strain seedling, it is urgent to develop a kind of high-throughput propagation method of red fruit ginseng seedling through direct organogenesis pathway mediation, to meet the demand of rapid factory production and variety improvement of red fruit ginseng excellent seedling. SUMMARY

[0005] The technical problem to be solved by the present application is how to use plant tissue culture technology to high-throughput propagate red fruit ginseng seedlings.

[0006] The present application solves the above technical problems by the following technical means:

[0007] A high-throughput propagation method of red fruit ginseng seedlings through direct organogenesis pathway mediation, comprising the following steps:

[0008] S1, taking material of explant and inducing germination of axillary bud: taking the current year's young and tender stem segments with buds of red fruit ginseng excellent strain as the initial explant source, after surface disinfection, cutting into appropriate size segments, inoculating in axillary bud germination induction medium, and carrying out axillary bud germination induction culture;

[0009] S2, obtaining of aseptic leaf and inducing adventitious bud: taking the stretched leaf of S1 in the axillary bud germination induction culture as the explant material, cutting into appropriate size sections, inoculating in adventitious bud induction medium for adventitious bud induction culture;

[0010] S3, proliferation and elongation culture of adventitious bud: transferring the induced adventitious bud cluster in S2 to adventitious bud proliferation and elongation medium for proliferation and elongation culture of adventitious bud;

[0011] S4, induction culture of adventitious root: separating the elongated bud obtained from the bud cluster in S3, treating in a solution added with K-IBA and IAA, and then transferring to adventitious root induction medium for induction culture of adventitious root to obtain complete regenerated plants;

[0012] S5, acclimatization and transplanting of regenerated plants: after acclimatization, cleaning and surface disinfection of the complete regenerated plants obtained in S4, transplanting for seedling culture.

[0013] Preferably, in S1, the segments with axillary buds are cut into 0.5-1.0cm long; in S2, the sections are cut into 0.25-1.0cm 2 in size; in S4, the elongated bud is a red fruit ginseng elongated bud with a height of 2-3cm and accompanied by 2-3 mature leaves.

[0014] Preferably, in S1, the axillary bud germination induction medium is DKW medium added with 0.5-2.0 mg / L meta-topolin, 0.2-0.5 mg / L ZT, 30 g / L sucrose and 7 g / L agar.

[0015] Preferably, in S1, the germination induction culture time is 3 weeks; in S2, the adventitious bud induction culture time is 4 weeks; in S3, the adventitious bud proliferation and elongation culture time is 4 weeks; in S4, the adventitious root induction culture time is 2 weeks.

[0016] Preferably, in S2, the adventitious bud induction medium is DKW medium added with 0.5-2.0 mg / L TDZ, 1.0-3.0 mg / L meta-topolin, 30 g / L sucrose and 7 g / L agar.

[0017] Preferably, in S3, the adventitious bud proliferation and elongation medium is DKW medium added with 0.1-1.0 mg / L TDZ, 0.1-0.5 mg / L ZT, 30 g / L sucrose and 7 g / L agar.

[0018] Preferably, in S4, the treatment of immersing in the solution added with K-IBA and IAA refers to immersing the base of the elongated bud in the aqueous solution added with K-IBA at a final concentration of 500-1000 mg / L and IAA at a final concentration of 100-500 mg / L for 10-30 s.

[0019] Preferably, in S4, the adventitious root induction medium is 1 / 2 DKW medium added with 20 g / L sucrose and 7.0 g / L agar.

[0020] Preferably, in S5, the acclimatization is acclimatization under natural conditions for 2 weeks; the surface sterilization comprises immersing in an aqueous solution of potassium permanganate with a mass fraction of 0.1% to 0.3% for 5-10 min; the seedling culture is carried out using a mixed nutrient medium, and the mixed nutrient medium is a mixture of Pinus nutrient soil, vermiculite and garden soil at a volume ratio of 2-4:1-2:3-6.

[0021] Preferably, in S1, S2, S3 and S4, the culture conditions are all as follows: the temperature is 25±2℃, the light intensity is 40-50 μmol m -2 s -1 , and the photoperiod is 14 / 10 h (light / dark); in S5, the seedling culture conditions are as follows: the temperature is 18-28℃, the humidity is 70%-85%, and the light intensity is 1800-4000 lx.

[0022] The present application has the following advantages:

[0023] The high-throughput propagation method of Panax ginseng C.A.Meyer seedlings mediated by a direct organogenesis pathway has the following outstanding advantages: first, the leaves are derived from axillary buds of current-year tender stem segments of P. ginseng excellent lines, and the direct induction of adventitious buds is adopted in the propagation process, which is rich in materials and the obtained regenerated plants can maintain the germplasm characteristics of the mother plants, so that rapid propagation of P. ginseng excellent single plants can be realized; second, the regeneration efficiency is high, the stem segment axillary bud germination rate is 100%, the leaf adventitious bud induction rate is as high as 98.4%, an average of 27.6 adventitious buds can be produced after each leaf cutting proliferation, and the rooting rate of the buds is as high as 95.7%, which provides important technical support for the germplasm preservation, large-scale propagation and later genetic improvement of P. ginseng excellent lines. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 P. ginseng stem segments cultured on the axillary bud germination induction medium for 1 week in Example 1 of the present application;

[0025] Figure 2 P. ginseng stem segments cultured on the axillary bud germination induction medium for 3 weeks in Example 1 of the present application (axillary buds elongated and accompanied by the formation of extended leaves);

[0026] Figure 3 P. ginseng leaves cultured on the adventitious bud induction medium for 2 weeks in Example 1 of the present application (adventitious bud points were induced at the cut);

[0027] Figure 4 P. ginseng leaves cultured on the adventitious bud induction medium for 4 weeks in Example 1 of the present application (a large number of adventitious buds were induced at the cut);

[0028] Figure 5 P. ginseng leaf adventitious bud clusters obtained after 3 weeks of culture on the proliferation and elongation medium in Example 1 of the present application;

[0029] Figure 6 P. ginseng regenerated plants obtained after 2 weeks of adventitious root induction culture in Example 1 of the present application;

[0030] Figure 7 P. ginseng regenerated plants obtained after 4 weeks of acclimatization and transplanting in Example 1 of the present application. DETAILED DESCRIPTION

[0031] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall into the scope of the present application.

[0032] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels unless otherwise specified.

[0033] The specific techniques or conditions not specified in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0034] Example 1

[0035] A high-throughput propagation method of Gynostemma pentaphyllum seedlings mediated by a direct organogenesis pathway, the specific operation being as follows:

[0036] (1) The current-year tender stem segments with buds of a Gynostemma pentaphyllum excellent strain were used as initial explant sources, washed with running water, cut into 3-4 cm long stem segments, wiped with 75% (v / v) alcohol, then sterilized with 75% (v / v) alcohol for 30 s, and then washed with sterile water for 4 times; then the stem segments were sterilized with 0.1% (w / v) mercury chloride solution for 2 min, and then washed with sterile water for 6 times. After the surface water of the stem segments was absorbed with filter paper, the stem segments with axillary buds were cut into 0.5-1.0 cm long segments, inoculated into DKW medium added with 1.0 mg / L meta-topolin, 0.3 mg / L ZT, 30 g / L sucrose and 7 g / L agar, and then subjected to rapid axillary bud germination induction culture in a greenhouse at a temperature of 25±2℃, a light intensity of 40-50 μmol m -2 s -1 , and a photoperiod of 14 / 10 h (light / dark). After illumination culture for 1 week, the axillary buds germinated Figure 1 ) and the germination rate reached 100%; after illumination culture for 3 weeks, the axillary buds germinated to about 2.3 cm long with 9.2 leaf blades Figure 2 ).

[0037] (2) The leaf blades of the germinated Gynostemma pentaphyllum axillary buds in step (1) were cut into 0.25 cm 2 long sections, inoculated into DKW medium added with 1.0 mg / L TDZ, 2.0 mg / L meta-topolin, 30 g / L sucrose and 7 g / L agar, and then subjected to rapid axillary bud germination induction culture in a greenhouse at a temperature of 25±2℃, a light intensity of 40-50 μmol m -2 s -1Adventitious buds were induced and cultured in a greenhouse with a photoperiod of 14 / 10h (light / dark). After two weeks of light culture, adventitious buds formed at the leaf cut surfaces. Figure 3 After 4 weeks of light cultivation, numerous adventitious bud clusters were induced from the leaf cuts. Figure 4 The adventitious bud induction rate is as high as 98.4%, and an average of 8.7 adventitious buds can be produced per explant.

[0038] (3) The adventitious shoot clusters induced in step (2) were transferred to DKW medium supplemented with 0.5 mg / L TDZ, 0.2 mg / L ZT, 30 g / L sucrose and 7 g / L agar, and incubated at a temperature of 25 ± 2 °C and a light intensity of 40–50 μmol m. -2 s -1 Adventitious buds were propagated and elongated in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 3 weeks of light culture, an average of 27.6 adventitious buds were produced per explant after propagation and elongation culture. Figure 5 ).

[0039] (4) After culturing for another week, the elongated shoots of *Gynostemma pentaphyllum* with a height of 2-3 cm and 2-3 mature leaves were separated. The base of each shoot was immersed in a mixed aqueous solution containing 1000 mg / L K-IBA and 250 mg / L IAA for 20 seconds. Then, the shoots were directly inoculated into 1 / 2 DKW medium containing 20 g / L sucrose and 7.0 g / L agar at a temperature of 25 ± 2 °C and a light intensity of 40–50 μmol / m². -2 s -1 Adventitious root induction culture was conducted in a greenhouse with a photoperiod of 14 / 10h (light / dark). After one week of light culture, adventitious root primordia were induced at the base of the stem segments. After another week of culture, the adventitious root induction rate of the stem segments reached 95.7%, with an average of 5.6 adventitious roots produced per explant. Figure 6 ).

[0040] (5) Place the culture container containing the healthy, intact regenerated *Gynostemma pentaphyllum* plants obtained in step (4) under natural conditions for acclimatization for 2 weeks. During this period, slowly loosen the bottle opening every 3 days until the bottle opening is completely open. Then, remove the regenerated plants, wash away the culture medium from the plant roots with running water, and soak them in a 0.2% potassium permanganate solution for 7 minutes. Then, transplant them into seedling trays containing a mixed nutrient substrate and cultivate them in a greenhouse at a temperature of 18-28℃, a humidity of 70%-85%, and a light intensity of 1800-4000 lx. The mixed nutrient substrate is a mixture of Pinsbury potting soil, vermiculite, and garden soil in a volume ratio of 2:2:3. After 4 weeks of cultivation, the plants grow vigorously. Figure 7), the transplanting survival rate of the regenerated plants was as high as 97.7%.

[0041] Example 2

[0042] This example tests the effect of exogenous meta-topolin and ZT concentration on the induction of axillary bud germination of stem segments of excellent lines of Panax ginseng. The specific steps are as follows:

[0043] (1) The 0.5-1.0 cm stem segments with axillary buds cut in step (1) of Example 1 after surface sterilization are inoculated on DKW medium added with 0.5-2.0 mg / L meta-topolin and 0.2-0.5 mg / L ZT, 30 g / L sucrose and 7.0 g / L agar, and cultured for axillary bud germination induction at a temperature of 25±2℃, a light intensity of 40-50 μmol m -2 s -1 2, and a photoperiod of 14 / 10 h (light / dark) in a constant temperature culture room. The induction of axillary buds is observed in real time during the culture, and the induction rate, number of leaves and length of axillary buds are counted after 3 weeks of light culture. The results are shown in Table 1, which shows that the use of exogenous meta-topolin and ZT can promote the germination, elongation and increase in the number of leaves of axillary buds during the induction of axillary bud germination of Panax ginseng stem segments. Among them, the induction of axillary bud germination is best when the stem segments are inoculated in DKW medium added with 1.0 mg / L meta-topolin and 0.3 mg / L ZT, with an axillary bud germination induction efficiency of 100%, an average length of 2.3 cm per axillary bud and an average of 9.2 elongated leaves per explant.

[0044] Table 1 Effect of exogenous meta-topolin and ZT concentration on the induction of axillary bud germination of Panax ginseng stem segments

[0045]

[0046] Note: The data are average values, each treatment contains 120 explants, and each treatment is repeated three times.

[0047] Example 3

[0048] This example tests the effect of exogenous TDZ and meta-topolin concentration on the induction of adventitious buds from leaves of excellent lines of Panax ginseng. The leaves obtained in step (1) of Example 1 are cut into 0.25 cm 2 2 sized pieces, inoculated in DKW medium added with different concentrations of TDZ (0.5-2.0 mg / L) and meta-topolin (1.0-3.0 mg / L) and 30 g / L sucrose and 7 g / L agar, and cultured for induction of adventitious buds at a temperature of 25±2℃, a light intensity of 40-50 μmol m -2s -1 The induction of adventitious buds was carried out in a greenhouse with a photoperiod of 14 / 10 h (light / dark). The induction of adventitious buds was observed in real time during the culture. After 4 weeks of light culture, the induction rate of adventitious buds and the number of adventitious buds produced per explant were counted. The results are shown in Table 2.

[0049] Table 2 Effect of exogenous TDZ and meta-topolin concentration on the induction of adventitious buds of G. rubrum leaves

[0050]

[0051]

[0052] Note: The data are the average values. Each treatment contained 120 explants, and each treatment was repeated three times.

[0053] The results show that exogenous TDZ and meta-topolin have a significant promoting effect on the induction of adventitious buds of G. rubrum leaves. When the leaves are inoculated in DKW medium with a final concentration of 1.0 mg / L TDZ and 2.0 mg / L meta-topolin, the induction effect of adventitious roots is the best, with an induction efficiency of 98.4% and an average of 8.7 adventitious buds per explant.

[0054] Example 4

[0055] In this example, the effect of exogenous K-IBA and IAA concentration on the induction of adventitious roots of G. rubrum elongated buds was tested. The G. rubrum elongated buds with a height of 2-3 cm and 2-3 mature leaves obtained in step (4) of Example 1 were separated, and the base was immersed in a mixed aqueous solution with different concentrations of K-IBA (500, 750 and 1000 mg / L) and IAA (100, 250 and 500 mg / L) for 20 s, and then inoculated in 1 / 2DKW medium with 20 g / L sucrose and 7.0 g / L agar at a temperature of 25±2°C, a light intensity of 40-50 μmol m -2 s -1 The induction of adventitious roots was carried out in a greenhouse with a photoperiod of 14 / 10 h (light / dark). The results after two weeks of culture are shown in Table 3. The results show that the exogenous K-IBA and IAA concentration has an important effect on the induction of adventitious roots of G. rubrum. Among the concentrations tested, the treatment with 1000 mg / L K-IBA and 250 mg / L IAA for 20 s is the most conducive to the induction of adventitious roots, with an induction rate of 95.7% and an average of 5.6 adventitious roots per explant

[0056] Table 3 Effect of exogenous K-IBA and IAA concentration on induction of adventitious roots of P. rubiginosa

[0057]

[0058] Note: Data are means, each treatment contained 120 explants, each treatment was replicated three times.

[0059] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A high-throughput propagation method for *Gynostemma pentaphyllum* seedlings mediated by direct organogenesis, characterized in that: Includes the following steps: S1. Explant collection and axillary bud germination induction: Young, bud-bearing stem segments from superior *Gynostemma pentaphyllum* strains were used as the initial explant source. After surface disinfection, the segments were cut into appropriately sized pieces and inoculated into an axillary bud germination induction medium for axillary bud germination induction culture. The axillary bud germination induction medium was a DKW medium containing 0.5-2.0 mg / L metatopolin, 0.2-0.5 mg / L ZT, 30 g / L sucrose, and 7 g / L agar. S2. Obtaining sterile leaves and inducing adventitious buds: Leaves from the axillary bud germination induction culture in S1 were used as explant materials, cut into appropriately sized slices, and inoculated into adventitious bud induction medium for adventitious bud induction culture; the adventitious bud induction medium was DKW medium containing 0.5-2.0 mg / L TDZ, 1.0-3.0 mg / L meta-topolin, 30 g / L sucrose and 7 g / L agar; S3. Adventitious bud proliferation and elongation culture: The adventitious bud clusters induced in S2 were transferred to an adventitious bud proliferation and elongation culture medium for adventitious bud proliferation and elongation culture; the adventitious bud proliferation and elongation culture medium was DKW medium containing 0.1-1.0 mg / L TDZ, 0.1-0.5 mg / L ZT, 30 g / L sucrose and 7 g / L agar; S4. Induction and culture of adventitious roots: The elongated shoots obtained from S3 culture were separated from the shoot clusters, treated with a solution of 500-1000 mg / L K-IBA and 100-500 mg / L IAA, and then transferred to an adventitious root induction medium for induction and culture of adventitious roots to obtain complete regenerated plants; the adventitious root induction medium was 1 / 2 DKW medium of 20 g / L sucrose and 7.0 g / L agar; S5. Acclimatization and transplanting of regenerated plants: After acclimatizing, cleaning, and surface disinfecting the complete regenerated plants obtained in S4, transplant them for seedling cultivation.

2. The method for high-throughput propagation of *Gynostemma pentaphyllum* seedlings mediated by direct organogenesis according to claim 1, characterized in that: In S1, cut into 0.5-1.0 cm long segments with axillary buds; in S2, cut into 0.25-1.0 cm long segments. 2 Slices of varying sizes; in S4, the elongated bud is a red ginseng elongated bud that is 2-3 cm high and has 2-3 mature leaves.

3. The high-throughput propagation method for *Gynostemma pentaphyllum* seedlings mediated by direct organogenesis as described in claim 1, characterized in that: In S1, the germination induction culture time is 3 weeks; in S2, the adventitious bud induction culture time is 4 weeks; in S3, the adventitious bud proliferation and elongation culture time is 4 weeks; and in S4, the adventitious root induction culture time is 2 weeks.

4. The method for high-throughput propagation of *Gynostemma pentaphyllum* seedlings mediated by direct organogenesis according to claim 1, characterized in that: In S4, the immersion time in solutions with a concentration of 500-1000 mg / L K-IBA and a concentration of 100-500 mg / L IAA is 10-30 seconds.

5. The method for high-throughput propagation of *Gynostemma pentaphyllum* seedlings mediated by direct organogenesis according to claim 1, characterized in that: In S5, the acclimatization is carried out under natural conditions for 2 weeks; the surface disinfection includes soaking in a potassium permanganate aqueous solution with a mass fraction of 0.1% to 0.3% for 5-10 minutes; and seedling cultivation is carried out using a mixed nutrient substrate, which is a mixture of Pinsbury potting soil, vermiculite, and garden soil in a volume ratio of 2-4:1-2:3-6.

6. The method for high-throughput propagation of *Gynostemma pentaphyllum* seedlings mediated by direct organogenesis according to any one of claims 1-5, characterized in that: In S1, S2, S3, and S4, the cultivation conditions were all: temperature 25 ± 2 ℃, light intensity 40–50 μmol m² / min. -2 s -1 The photoperiod is 14 hours of light / 10 hours of darkness; in S5, the conditions for seedling cultivation are: temperature 18-28℃, humidity 70%-85%, and light intensity 1800-4000 lx.

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