A method for in vitro propagation using red ginseng leaves

Abstract

The present invention provides a method for in vitro propagation using red ginseng leaves, comprising: using 2-3 leaves at the top of the red ginseng as explants, and respectively adopting improved primary culture medium, secondary culture medium, and rooting culture medium for cultivation; after experimental verification, the culture medium with the best tissue culture effect of the red ginseng leaves is screened out, wherein the primary culture medium is MS+0.2mg / L KT+0.8mg / L 6-BA+0.5mg / LNAA, the secondary culture medium is MS+0.5mg / L 6-BA+0.5mg / L NAA, and the rooting culture medium is MS+NAA0.2mg / L+IBA0.5mg / L. The in vitro tissue culture method of the red ginseng leaves provided by the present invention has good induction effect, less callus tissue, high survival rate, developed root system, and the cultured red ginseng plants grow robustly and have dense leaves. It is an effective way to quickly and efficiently propagate red ginseng.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture and rapid propagation, and in particular to a method for in vitro propagation using red ginseng leaves. Background Art

[0002] Red ginseng, scientific name Cyclocodon lancifolius (Roxb.) Kurz, belongs to the Campanulaceae family (Campanulaceae) Cyclocodon genus (Cyclocodon), a perennial herb, growing in forests, bushes and grasslands below 1500m above sea level. Its fruit is in the shape of abacus beads, purple-red when ripe, and the calyx persists around the fruit, shaped like a "spider". It is also known as spider fruit, abacus fruit, meat abacus, mountain water chestnut, etc. The stems, leaves and roots can be used as medicine, with the effects of moistening the lungs and relieving cough, regulating qi and replenishing deficiency, removing blood stasis and relieving pain. It is used to treat injuries caused by falls, qi deficiency and fatigue, intestinal colic, tuberculosis cough, hernia, constipation and other diseases. Modern research shows that it mainly contains a variety of chemical components such as flavonoids, alkaloids, volatile oils, polysaccharides, etc.; the crude fiber content is high, and it can be stored for more than one month at room temperature. The ripe fruit is usually collected and eaten as fruit. Its flesh is purple, crisp, juicy, residue-free and delicious. It is a new resource for medicine and food that combines medicinal, edible and health functions, and has a very broad prospect for development and utilization.

[0003] Red ginseng, carrot-shaped root; usually the whole plant is glabrous, the stem is hollow, with many and long branches, flat or drooping; leaves are opposite, occasionally 3 in whorls, and have short petioles, leaf blades are ovate, ovate-lanceolate to lanceolate; flowers are usually solitary, terminal and axillary; pedicel or peduncle, with a pair of filiform bracts in the upper middle part of the pedicel or at the base of the indus; calyx is only attached to the lower part of the ovary, with 5 lobes usually far apart, filiform or linear, with branched and slender teeth on the edge; corolla is white or light red, tubular and bell-shaped; stamens are 5-6, filaments are as long as anthers, the base of the filaments is wide and lamellar, and the edges are hairy; style is with or without hair; berry is spherical with 5-6 chambers, purple-black when ripe; seeds are extremely numerous, polyhedral; flowering period is July-October every year; fruiting period is October to February of the following year every year.

[0004] Red ginseng is suitable for cultivation in warm and humid environments, and is widely distributed in Yunnan, Guizhou, Sichuan and other places in the southwestern mountainous areas of my country. However, it is currently difficult to cultivate, the volume is small, and the variety differences are large. Summary of the invention

[0005] The purpose of the present invention is to provide a method for in vitro propagation using red ginseng leaves.

[0006] A method for in vitro propagation using red ginseng leaves, comprising:

[0007] The leaves of Panax ginseng were used as explants and cultured using primary culture medium, secondary culture medium and rooting culture medium respectively.

[0008] Among them, the primary culture medium is MS+0.2mg / LKT+0.8mg / L 6-BA+0.5mg / LNAA.

[0009] Furthermore, in the above-mentioned method for in vitro propagation using red ginseng leaves, the subculture culture medium is: MS+0.5mg / L 6-BA+0.5mg / LNAA.

[0010] Furthermore, in the above-mentioned method for in vitro propagation using leaves of red ginseng, the rooting medium is: MS+0.2mg / LNAA+0.5mg / L IBA.

[0011] Furthermore, in the above-mentioned method of in vitro propagation using leaves of red ginseng, the top 2-3 true leaves of red ginseng are used as explants for tissue culture.

[0012] Furthermore, according to the method for in vitro propagation using red ginseng leaves as described above, before primary culture, the explants are first disinfected, and the disinfection measures are: the picked explants are soaked in a container with laundry detergent for 5 minutes, and rinsed with pure water 2-3 times. Before the explants are taken to the clean bench, the clean bench is irradiated with ultraviolet light for 30 minutes, the leaves are soaked in 75% alcohol for 20 seconds on the clean bench, rinsed with sterile water 3 times, soaked in 2% sodium hypochlorite for 30 seconds, and then rinsed with sterile water 3 times.

[0013] The in vitro tissue culture method of red ginseng leaves provided by the invention has good induction effect, less callus tissue, high survival rate, luxuriant root system, and the cultured red ginseng plants grow robustly and have dense leaves. The invention is an effective way to rapidly and efficiently propagate red ginseng. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] The invention selects high-quality varieties and adopts an in vitro tissue culture method to realize a high-efficiency tissue culture and rapid propagation system of red ginseng leaves, which not only retains the excellent traits of the varieties but also can provide a large number of high-quality seedlings on a large scale.

[0016] Experimental example:

[0017] Material

[0018] The seeds were collected in Maguan County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province in 2023, and sown in the greenhouse of Yunnan Agricultural University. When the plants grew to four to five true leaves, the top 2-3 true leaves were taken as explants for tissue culture experiments.

[0019] Explant preparation and sterilization

[0020] Soak the top leaves of the picked red ginseng in a container with a detergent for 5 minutes, rinse with pure water 2-3 times, and irradiate the top leaves of the red ginseng with ultraviolet light for 30 minutes before taking them to the clean bench. Soak the leaves in 75% alcohol for 20 seconds on the clean bench, rinse with sterile water 3 times, soak with 2% sodium hypochlorite for 30 seconds, and then rinse with sterile water 3 times. When the water on the leaves is dry, place them flat in the primary culture medium. All operations are carried out under the clean bench, and all the equipment used is sterilized by high-pressure sterilization.

[0021] Experimental equipment

[0022] Clean bench, tweezers, scalpel, 75% alcohol, 2% sodium hypochlorite, red ginseng leaves, culture medium, sterile water, display rack, glass bottle.

[0023] Medium preparation

[0024] This embodiment uses a standard MS culture medium, and different hormones are added to the primary culture, subculture and rooting culture medium to screen the culture medium formula with the best effect at each stage. All culture media are sterilized by high-pressure steam after preparation.

[0025] 1. Primary Culture

[0026] The concentration of KT used in the primary culture was 0.1 mg / L, 0.2 mg / L, and 0.5 mg / L, the concentration of 6-BA was 1.0 mg / L, 0.8 mg / L, and 0.2 mg / L, and the concentration of NAA was kept constant at 0.5 mg / L. A total of 6 groups of culture media were formed, with 5 replicates in each group:

[0027] CD1: MS+KT0.1mg / L+6-BA1.0mg / L+NAA0.5mg / L

[0028] CD2: MS+KT0.2mg / L+6-BA1.0mg / L+NAA0.5mg / L

[0029] CD3: MS+KT0.5mg / L+6-BA1.0mg / L+NAA0.5mg / L

[0030] CD4: MS+KT0.5mg / L+6-BA0.8mg / L+NAA0.5mg / L

[0031] CD5: MS+KT0.5mg / L+6-BA0.2mg / L+NAA0.5mg / L

[0032] CD6: MS+KT0.2mg / L+6-BA0.8mg / L+NAA0.5mg / L

[0033] 2. Subculture

[0034] Subculture mainly cultivates strong seedlings, and at the same time, the callus formed in subculture is reduced by adjusting the medium formula. This experiment used 2.0mg / L, 1.5mg / L, 1.0mg / L, 0.5mg / L of 6-BA and 0.5mg / L of NAA to form a subculture medium for induction, forming the following 4 groups of culture media, each with 5 replicates:

[0035] JD1: MS+6-BA2.0mg / L+NAA0.5mg / L

[0036] JD2: MS+6-BA1.5mg / L+NAA0.5mg / L

[0037] JD3: MS+6-BA1.0mg / L+NAA0.5mg / L

[0038] JD4: MS+6-BA0.5mg / L+NAA0.5mg / L

[0039] 3. Rooting culture

[0040] Rooting culture is suitable for forming less callus tissue and more roots, developing root system and strong plants. In this experiment, at room temperature of 25℃, two concentrations of NAA 0.5mg / L and 0.2mg / L were combined with IBA 0.5mg / L, 0.2mg / L, IAA 0.5mg / L and IAA 0.2mg / L to form rooting medium for induction, forming the following 4 groups of culture media, each with 5 replicates:

[0041] SG1: MS+NAA0.5mg / L+IAA0.2mg / L

[0042] SG2: MS+NAA0.2mg / L+IAA0.5mg / L

[0043] SG3: MS+NAA0.5mg / L+IBA0.2mg / L

[0044] SG4: MS+NAA0.2mg / L+IBA0.5mg / L

[0045] 4. Transplantation and domestication of test tube seedlings

[0046] Rooting culture is carried out at room temperature of 25°C, and the test tube seedlings are acclimated after about 30 days of cultivation. During the acclimation, the seedlings are placed in a greenhouse for 2-3 days. During this period, small holes are opened in the breathable membrane for ventilation to adapt to field cultivation in advance.

[0047] Mix peat and perlite in a ratio of 3:1 for the substrate, transplant the seedlings into a 72-hole plug tray and culture for about 20 days. Roots begin to grow in the tray, and then apply a certain concentration of 20:20:20 water-soluble fertilizer. When the roots wrap the substrate tightly, transplant them into the facility soil, and follow up on the seedling stage and subsequent maintenance.

[0048] Selection of the Primary Culture Medium for Callus Induction from Red Ginseng Leaves

[0049] The seeds obtained from Maguan County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province were sown directly in the greenhouse of Yunnan Agricultural University. After 2 months, when they grew to 4-5 true leaves, the top 2-3 true leaves were taken for tissue culture. They were cultured in standard MS medium and inoculated at room temperature at 25°C for 14 days under normal light. The induction results after 30 days are shown in Table 2.

[0050] Table 1 Callus induction status of different hormone ratios in primary culture of red ginseng

[0051]

[0052] According to Table 1 above, under the same culture conditions, the induction rate of CD6 culture medium in the primary culture can reach more than 70%, which can reach more than 60 seedlings, and the induction effect is significantly better than the other 5 groups of treatments. Therefore, the induction effect of red ginseng leaves is best in the medium of MS+0.2mg / LKT+0.8mg / L6-BA+0.5mg / LNAA, which is an ideal culture medium for inducing red ginseng leaves.

[0053] In summary, this experiment took the top 2-3 true leaves of the same variety of red ginseng as experimental materials to study the growth of red ginseng induced by different hormone ratios of red ginseng leaves on the basis of standard MS medium. After experiments and data screening, the best induction medium is: MS+0.2mg / LKT+0.8mg / L6-BA+0.5mg / LNAA.

[0054] The study found that soaking in 75% alcohol for 30 seconds and 2% sodium hypochlorite for 30 seconds has the best disinfection effect, which can eliminate bacteria to the greatest extent without damaging the explant itself, and the survival rate is over 80%.

[0055] Hormone ratio for subculture of red ginseng leaves

[0056] Based on the hormone ratio of the culture medium screened out from the primary culture of red ginseng, the hormone ratio was further adjusted to make the seedlings grow stronger and have less callus tissue. The MS standard culture medium was used, and the hormone types were changed to 6-BA and NAA to conduct the red ginseng subculture experiment. The specific growth conditions are shown in Table 2.

[0057] The specific parameters are shown in Table 2:

[0058] Table 2 Experiment on subculture induction of callus tissue of red ginseng leaves

[0059]

[0060] As can be seen from Table 2 above, the seedlings of red ginseng subcultured under the treatment of JD4 medium in subculture grew vigorously and had less callus tissue. The callus tissue amount in the hormone ratio of JD1 medium was too large and the growth state of the seedlings was weaker than that of the other treatments. Therefore, the hormone ratio of JD4 medium was the best ratio for subculture of red ginseng leaves. In subculture, high concentrations of 6-BA inhibited the growth of plants, and the morphology of callus was roughly the same, but the callus amount was optimal with low concentrations of 6-BA, that is, JD4 medium.

[0061] Based on the growth conditions of primary culture and subculture, different hormones have different effects on the growth of red ginseng tissue culture seedlings. Finally, the standard MS+0.2mg / LKT+0.8mg / L 6-BA+0.5mg / LNAA was selected as the best culture medium. Under the conditions of this culture medium, red ginseng plants can grow strong, have dense leaves and less callus tissue.

[0062] Hormone ratio for rooting culture of red ginseng leaves

[0063] Rooting mainly depends on the callus tissue. The more roots, the better, and the thicker and more luxuriant they are, the best. Rooting culture is carried out after subculture of red fruit ginseng. The top 2-3 true leaves of subculture are taken for tissue culture. This application is based on MS standard culture medium, and 0.2mg / L, 0.5mg / L IAA and 0.2mg / L, 0.5mg / L IBA are added respectively. At the same time, 0.5mg / L NAA is added to each group of culture medium for rooting experiments. The growth status is observed after 15d and 30d respectively. The specific growth status and number of roots are shown in Table 3 below.

[0064] Table 3 Rooting culture induction experiment of red ginseng leaf callus

[0065]

[0066] This experiment used tissue culture seedlings in subculture, and added different concentrations of IAA and IBA to the standard MS medium. As can be seen from Table 3, at 15 days of rooting culture, the SG4 medium with an IAA concentration of 0.5 mg / L had the largest number of roots, reaching 25 roots, and the root length was 0.8 cm, and the rooting effect was significantly better than other treatments. Continue to culture until the 30th day, the SG4 medium still had the largest number of roots, but the SG1 medium with an IBA concentration of 0.5 mg / L had the longest root length, reaching 1.1 cm.

[0067] From the main purpose of the rooting medium, a comprehensive comparison of four rooting medium with different hormone levels showed that under the same environmental conditions, standard MS medium + 0.5 mg / L NAA + 0.5 mg / L IAA had significant advantages in root quantity and root length, and was the best rooting medium screened for this experiment.

[0068] The method for in vitro propagation using red ginseng leaves provided by the present invention uses the top 2-3 leaves of the red ginseng as explants and respectively adopts a primary culture medium, a secondary culture medium and a rooting culture medium for cultivation, thereby effectively retaining the excellent traits of the variety and simultaneously increasing the yield of high-quality red ginseng seedlings.

[0069] After verification, there is no case of propagating red ginseng by tissue culture. The present invention has completed the in vitro tissue culture of red ginseng leaves from excellent varieties under a series of experimental verifications, and optimized and screened the most suitable culture medium in primary culture, subculture and rooting culture, wherein the primary culture medium is MS+0.2mg / L KT+0.8mg / L 6-BA+0.5mg / L NAA, the subculture culture medium is: MS+0.2mg / LKT+0.8mg / L 6-BA+0.5mg / L NAA, and the rooting culture medium is: MS+0.5mg / L NAA+0.5mg / LIAA. The tissue culture seedlings complete outdoor seedling hardening and field cultivation and management, and the resulting plants grow strong, have a well-developed root system, and have thick leaves. In summary, the method provided by the present invention is an efficient and rapid in vitro tissue culture method for red ginseng.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for in vitro propagation using red ginseng leaves, characterized in that: include: The leaves of Panax ginseng were used as explants and cultured using primary culture medium, secondary culture medium and rooting culture medium respectively. Among them, the primary culture medium is: MS+0.2mg / L KT+0.8mg / L 6-BA+0.5mg / L NAA.

2. The method for in vitro propagation using red ginseng leaves according to claim 1, characterized in that: The subculture medium is: MS+0.5 mg / L 6-BA+0.5 mg / L NAA.

3. The method for in vitro propagation of red ginseng leaves according to claim 1, characterized in that: The rooting medium is: MS+NAA0.2mg / L+IBA0.5mg / L.

4. The method for in vitro propagation using red ginseng leaves according to claim 1, characterized in that: The top 2-3 true leaves of red ginseng were used as explants for tissue culture.

5. The method for in vitro propagation using red ginseng leaves according to claim 1, characterized in that: Before primary culture, the explants are first disinfected, and the disinfection measures are as follows: the picked explants are soaked in a beaker with laundry detergent for 5 minutes, and rinsed with pure water 2-3 times. Before the explants are taken to the clean bench, the clean bench is irradiated with ultraviolet light for 30 minutes, and the leaves are soaked in 75% alcohol for 20 seconds on the clean bench, rinsed with sterile water 3 times, soaked in 2% sodium hypochlorite for 30 seconds, and then rinsed with sterile water 3 times. When the water on the leaves is dried, they are placed flat in the primary culture medium.

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