KR101214785B1 - Method for regeneration of high-frequency direct shoot from Drymaria cordata leaves and the plants thereof - Google Patents

Abstract

The present invention relates to a method for direct high frequency regeneration of shoots from dry maria codata leaves and to plants according to the present invention, and more particularly, to dry maria codata cordata ) NAA; BAP or TDZ; And high-frequency regeneration of shoots in MS medium containing sucrose. Cordata ) Direct high frequency regeneration method of shoots, drymaria codata ( Refreshmaria) cordata ) Provided is a method for producing a dry Maria codata plant regenerated from shoots, a re-dried Dry Maria codata plant prepared according to the above method, and seeds thereof.

Description

Method for regeneration of high-frequency direct shoot from Drymaria cordata leaves and the plants etc.

The present invention relates to a method for direct high frequency regeneration of shoots from dried Maria codata leaves and to plants according to the present invention. The present invention relates to the development of a method for the regeneration of negative shoots with high frequency and high yield, and a method for the production of re-divided dried Maria codea plants.

Drymaria Cordata Wild cordata Willd, Caryophyllaceae is a perennial herbaceous plant, distributed in pan-tropical regions, particularly in Asia, Africa, Oceania, Central America, South America (Dequan L, Gilbert MG, 2001 Flora of China 6: 5-6) and the Indian Himalayas. Distributed in the following regions (Srivastava TN, Kaphi BK, 1992 J Econo Tax Bot 16: 1-17). Dry Maria Kodata has been used medicinally for numerous purposes in the Himalayas in India and Nepal. Extracts prepared from the leaves of this plant were affected by diarrhea, vomiting, urinary problems, and venomous bite (Neogi et al., 1989 Economic Bot 43: 471-479), and skin diseases (Rao RR, 1981 Economic Botany 35: 4-9). ), Sore throat and constipation (Focho et al., 2009 African J Pharm and Pharmacol 3: 001-013). In addition, the extract was very effective in coughing, sinusitis and acute colds (Mukherjee et al., 1997 J Ethnopharm 56: 77-80). The antibacterial (Mukherjee et al., 1996 Phyto Res II: 249-250) and antitussive efficacy of dry maria codata have been well established. Dry Maria codata leaf extract contains diarrhea, antipyretic, cooling (Sikdar M, Dutta U, 2008 Ethno Med 2: 39-45) and anti-inflammatory benefits (Singh et al., 2008 Inter J Integra Biol 3: 57-72) Has been reported. Because of this medicinal importance, many biologically active compounds have been isolated from the leaves of this plant, cyclopeptides (Ding et al., 2000 Planta Med 66: 386-388), flavonoid glucosides (Ding et al., 1999 Planta Med) 65: 578-579), norditerpenes and norditerpene glucosides (Vargas et al., 1988 Phytochem 27: 1532-1534), and chemotherapy for many types of diseases Has been used. In addition, several other flavonoids and glucosides of medical value have been isolated from the plant: drymariatin B, drymariatin C, drymariatin D, and diandraflavones. (diandraflavone) and drymarin (1) (Veitch NC, Grayer RJ, 2008 Nat Prod Rep 25: 555-611). Among these secondary metabolites, drymaritin (1) has been reported to have an anti-HIV effect in H9 lymphocytes (Hsieh et al., 2004 J Nat Prod 67: 1175-1177).

Dry maria codata grows naturally through seeds. However, proliferation has been limited by low germination rates and poor seed viability. Thus, alternative methods of propagation may be beneficial for mass growth, improvement, and preservation of this plant. Plant tissue culture may also be applied for ex situ conservation and rapid proliferation of rare and endemic medicinal plants (Purohit et al., 1994 Plant Cell Tiss Org Cult 39: 93-96). However, there have been no reports of in vitro propagation of dry maria coda. For this reason, the development of effective recycling systems for these species is required. Moreover, genetic engineering techniques requiring rapid and effective regeneration methods are useful for obtaining improved genotypes (Sanyal et al., 2005 Plant Sci 168: 1135-1146).

DETAILED DESCRIPTION OF THE INVENTION The present invention is derived from the above demands, and the present invention is a rare and endemic, endangered species of dry maria codata ( Drymaria cordata ) In addition to the development of an effective and high frequency negative shoot regeneration method by comparing the response of leaf explants to various concentrations of plant growth regulators (PGR) in vitro for the on-site preservation and rapid proliferation of plants, The present invention has been completed by developing an optimal medium and culture conditions for renal and rooting to develop a method for producing re-differentiated dry maria coda plants.

In order to solve the above problems, the present invention is NAA by cutting the leaves of seedlings seeded by germinating Drymaria cordata seeds; BAP or TDZ; And high-frequency regeneration of shoots in MS medium containing sucrose. cordata ) provides a direct method for high frequency reproduction of shoots.

In another aspect, the present invention provides a method for producing a dry maria coda plant re-divided from the dry maria cordata shoots reproduced with the high frequency.

The present invention also provides a re-differentiated dry maria cordata plant prepared according to the above method.

The present invention also provides a seed of the plant.

The present invention for the first time invented a protocol for the production of high frequency negative shoots directly from the leaf sections of dry maria codata. Rapid in vitro propagation of dry maria codata according to the method of the present invention can be used for the production of secondary metabolites, germplasm conservation and genetic transformation, as well as efficiency, time and cost of plant breeding. You can expect a dramatic effect.

1 shows in vitro proliferation of dry maria coda via direct shoot regeneration. (A) Blind eye initiation (arrow) from cut sections of leaf explants after 3 weeks in regeneration medium (Bar = 0.2 cm). (B) High frequency regeneration of a plurality of shoots from leaf explants after 5 weeks in regeneration medium (Bar = 1 cm). (C) Well developed and elongated shoots after 4 weeks in kidney medium (Bar = 1 cm). (D) Rooting of shoots regenerated (bar = 1 cm) in MS medium with 1 mg / l IBA added for 30 days. (E) Syringe with well-developed root system (bar = 1 cm). (F) Purified newsletter growing in the greenhouse (bar = 1 cm).
Figure 2 shows the results of observation of the developmental order of shoot eye from leaf explants of dry maria codata by histological and SEM micrographs. (A) SEM micrograph (bar = 20 μm) showing the initial state of ocular primitive cells (arrowheads) from meristmatic dome after 8 days of culture. (B) SEM micrograph (bar = 50 μm) showing shoot primitive cells from leaf explants after 15 days of culture. (C) Regenerated young plants (hair = 50 μm) with hairy leaves (arrowheads) on the leaf explant surface in regeneration medium after 23 days of culture. (D) Explant sections showing meristem zones (arrowheads) after 2 weeks of culture (Bar = 200 μm). (E) Longitudinal section of the eye connected to leaf explants showing leaf primitive cells and shoot apical meristem (bar = 200 μm).

In order to achieve the object of the present invention, the present invention

(1) Drymaria codata cordata ) sterilizing the seeds to germinate;

(2) cutting off the leaves of seedlings from the germinated seeds; And

(3) a section of the cut leaf is NAA (Naphthalene acetic acid); 6-benzylaminopurine (BAP) or Thidiazuron (TDZ); And regeneration of shoots at high-frequency in Murashige and Skoog (MS) medium containing sucrose ( Drymaria coda) cordata ) provides a direct method for high frequency reproduction of shoots.

In the method according to an embodiment of the present invention, the leaf of step (2) preferably comprises a petiole (petiole). The shoot induction rate was higher in the leaf-bearing leaves than in the leaf-free leaves.

In the method according to an embodiment of the present invention, the step (3) is 0.05 ~ 0.1mg / L NAA; 1-2 mg / l BAP or 1.5-2.5 mg / l TDZ; And shoots can be regenerated at high frequency in MS medium containing 2.5-3.5% sucrose. As plant growth regulators, it is preferable to use BAP in combination with NAA or TDZ in combination with NAA, and these concentrations are 1 to 2 mg / L BAP or 0.05 to 0.1 mg in combination with 0.05 to 0.1 mg / L NAA. Preferred is 1.5-2.5 mg / l TDZ in combination with / l NAA, 1.0 mg / l BAP in combination with 0.1 mg / l NAA or 2.0 mg / l TDZ in combination with 0.1 mg / l NAA More preferred.

As a carbon source, sucrose is preferred, and the amount of sucrose may be 2.5 to 3.5% by weight, preferably 3% by weight sucrose. As the nutritional basal medium, MS, B5 or SH may be used, but is not limited thereto. Preferably, MS medium may be used.

The present invention also provides

(1) sterilizing and germinating the dry maria coda seeds;

(2) cutting off the leaves of seedlings from the germinated seeds;

(3) NAA section of the cut leaf; BAP or TDZ; And regenerating shoots at high frequency in MS medium containing sucrose;

(4) inducing rooting by incubating the regenerated shoots in MS medium to which IBA (Indole-3-butyric acid) is added; And

(5) Provides a method for producing a re-differentiated dry maria cordata plant comprising a purifying step of transplanting the rooted plant to the pot and cultivated.

In the method according to an embodiment of the present invention, the leaf of step (2) preferably comprises a petiole (petiole). The shoot induction rate was higher in the leaf-bearing leaves than in the leaf-free leaves.

In the method according to an embodiment of the present invention, the step (3) is 0.05 ~ 0.1mg / L NAA; 1-2 mg / l BAP or 1.5-2.5 mg / l TDZ; And shoots can be regenerated at high frequency in MS medium containing 2.5-3.5% sucrose. As plant growth regulators, it is preferable to use BAP in combination with NAA or TDZ in combination with NAA, and these concentrations are 1 to 2 mg / L BAP or 0.05 to 0.1 mg in combination with 0.05 to 0.1 mg / L NAA. Preferred is 1.5-2.5 mg / l TDZ in combination with / l NAA, 1.0 mg / l BAP in combination with 0.1 mg / l NAA or 2.0 mg / l TDZ in combination with 0.1 mg / l NAA More preferred.

As a carbon source, sucrose is preferred, and the amount of sucrose may be 2.5 to 3.5% by weight, preferably 3% by weight sucrose. As the nutritional basal medium, MS, B5 or SH may be used, but is not limited thereto. Preferably, MS medium may be used.

In the method according to one embodiment of the present invention, the plant growth regulator used in Drymaria cordata rooting induction medium, but not limited to IBA. Preferably it is IBA of 0.5-2.0 mg / L, More preferably, it is 1 mg / LIBA.

The present invention also provides a re-differentiated dry maria cordata plant and seeds thereof prepared according to the above method.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Way

1. Seed germination and Intercept ( explant ) Produce

Drymaria codata used in the experiment cordata seeds were obtained from Korea, Kangwon National University, and Bioherb Research Institute. The surface sterilization of the seed was immersed in 70% ethyl alcohol for 1 minute, then rinsed twice with sterile water, then dipped in a solution of 5% (v / v) sodium hypochlorite and gently stirred for 15 minutes. . It was washed six times in succession with sterile water. Surface sterile seeds were sown in MS (Murashige and Skoog) salts with 3% sucrose and 0.8% agar. Seeds were incubated at 25-27 ° C. with a 16 hour photoperiod at 36 μmol m ⁻² s ⁻¹ luminosity provided by cool white fluorescent tubes. After seed germination, young plants were grown in 1/2 MS medium. Leaves of seedlings grown in vitro (4-5 weeks after germination) were used as raw materials for the sections.

2. Culture conditions

To investigate the effect of culture medium on shoot regeneration, other basal mediums, such as MS (Murashige T, Skoog F, 1962 Plant Phyiol 15: 473-497), B5 (Gamborg et al., 1968 Exp Cell Res) 50: 151-158) and SH (Schenk RU, Hildebrandt AC, 1972 Can J Bot 50: 199-204). In addition, the effects of carbohydrate sources, namely sucrose, maltose and fructose (10, 20, 30 g / l), were investigated by adding to the medium for shoot induction from leaf sections, respectively. The pH of all media was adjusted to 5.8 before addition of 0.8% agar and autoclaved at 120 ° C. for 20 minutes.

3. Plant Regeneration

The leaves were cut to 4-6 week old seedlings by 0.5 cm, containing 30 g / l sucrose and 0.8% agar, alone or in combination, of different concentrations of auxin and cytokinin, for induction of negative shoots. Put into MS medium. All cultures were maintained at 25 ° C. with a 16 hour photoperiod with 36 μmol m ⁻² s ⁻¹ luminosity provided by cool white fluorescent lamps. Percentage of shoot initiation and average number of shoots per segment were recorded after 4 weeks.

4. Rooting ( rooting ) And plant purifying

Shoots regenerated from 2 to 2.5 cm in length with at least two expanded leaves were cut from the sections and placed in MS basal medium supplemented with different concentrations of Indole-3-butyric acid (IBA) and 30 g / l sucrose. 0.8% plant agar was added and transferred to firming rooting medium. The culture was carried out at 25 ° C. with a 16 hour photoperiod with a cool white fluorescent lamp (36 μmol m ⁻² s ⁻¹ ) luminous intensity. The experiment was repeated three times with 12 shoots per replicate. The number and length of root shoots were recorded 4 weeks after shoots were placed in root induction medium.

Regenerated plantlets with well-developed roots were gently washed with tap water to remove medium from their roots, and contained a mixture of sterile bed soil and perlite (3: 1). Transfer to a x 85 cm diameter plastic pot and soak in 1/2 MS salt. Each pot was covered with a polyethylene bag to maintain high humidity and incubated in a greenhouse at 25 ° C. with a 16 hour photoperiod with a reduced luminous intensity of 30 μmol m ⁻² s ⁻¹ . After two weeks, the bag of perforated ports was drilled to reduce humidity. The polyethylene bag was opened gradually and after 6 weeks the material was transferred to the shade (70%). It was then purified under greenhouse conditions and transferred to the field a month later. Data on plant survival rates were obtained after 6 weeks.

5. Scanning electron microscope observation

Samples were fixed with 3% glutaraldehyde and washed with 0.05 M potassium phosphate buffer (pH 7.0). Samples were dehydrated at incremental concentrations of ethanol at 10 minute intervals. The sample was dried to liquid critical point for 10 minutes with liquid carbon monoxide. After vacuum drying, the samples were fixed to aluminum stubs with double sided tape and coated with platinum (Pt) using an ion sputter apparatus (HITACHI E-1010). Samples were examined at an acceleration voltage of 15 kV using a HITACHI S-4300 Field Emission Scanning Electron Microscope (FESEM) at different resolutions and magnifications.

6. Histological investigation

During the incubation period, samples were taken periodically and fixed for 24 hours at room temperature with FAA solution (70% ethanol, 5% glacial acetic acid and 5% formaldehyde). After fixation, the sample was dehydrated with a gradual concentration of butanol solution and embedded in paraffin. For histological analysis, tissues were cut to 7 μm thickness using microtones (UCHIDA, JAPAN), stained for 3 minutes with hemooxylin solution, observed under an optical microscope (Olympus BX 50), Photographed with Olympus (Olympus, C4040 ZOOM).

7. Statistical Analysis

Each treatment consisted of 15-30 sections. All experiments were repeated at least three times. Data shown represent mean ± SD. The data was analyzed statistically using ANOVA, and the significant difference was determined by 0.05 probability.

Example  1. Negative shoots of (adventitious shoots)  Judo

To investigate the effect of plant growth regulators on the induction of negative shoots, dry Maria codata leaf fragments were divided into 2,4-D (Dicholorophenoxy acetic acid), NAA (Naphthalene acetic acid), BAP (6-benzylaminopurine) and TDZ. (Thidiazuron) was incubated in MS medium added alone or in combination at various concentrations. Leaf sections of the distal portions of the petiole were less regenerated than those close to the leaf disease. BAP concentrations lower than 1.0 mg / l reduced the number of shoots per culture (Table 1). Addition of TDZ to the medium had a significant effect on shoot induction. Of the various concentrations of TDZ tested, the 2.0 mg / l TDZ concentration added with 0.1 mg / l NAA was most effective in inducing multiple shoots (3.00 ± 0.63 per section). Direct shoot regeneration was significantly reduced when the TDZ concentration was decreased from 1.0 to 0.1 mg / l. Shoots were not induced when growth regulators were added to the culture medium alone. In the absence of BAP or TDZ, no regeneration of shoots was observed, which means that these compounds are very important for shoot regeneration in dry maria coda. The number of shoots increased significantly at 0.1 mg / l NAA in combination with 2.0 mg / l TDZ or 2.0 mg / l BAP, whereas increased levels of NAA induced complete inhibition and reduction of shoot formation. Induction of shoot buds also occurred when the axial surfaces of the leaf sections contacted the regeneration medium. Leaf explants, including the leaf disease, gave better results than no leaf disease (Table 2).

Callus began to form on the midveins at the wounded edges of the sections on day 6 after placing the sections in MS medium containing 2,4-D. After five weeks, the callus grew into a friable whitish or pale yellowish mass that completely covered the sections (FIG. 1A). 2,4-D concentrations below 1 mg / l produced small, slowly growing callus. Leaf sections cultured directly in MS medium with NAA combined with BAP or TDZ differentiated into multiple shoots directly from callus at the base of the leaf disease within 3-4 weeks after inoculation. Nerve shoot eyes initially appeared as small knob-like structures in the leaf base and later developed into new plants (FIG. 1B). Increased shoot number (10.67 ± 1.03 per section) was induced in MS medium with 0.1 mg / l NAA and 1.0 mg / l BAP added (FIG. 1C).

The present invention provides a method of high frequency in vitro negative shoot regeneration from leaf explants of dry maria coda. Low NAA concentrations are essential for direct shoot regeneration and multiple shoot formation. NAA concentrations of 0.1 mg / l or more in the regeneration medium resulted in decreased shoot regeneration. Direct shoot regeneration efficiency increased when BAP concentrations increased to 1.0 mg / l, while BAP concentrations below 1.0 mg / l decreased the number of sections forming shoots and shoots per section. In the absence of BAP or TDZ, a low shoot regeneration rate was observed, indicating that this plant growth regulator is important for shoot regeneration of dry maria coda.

In the present invention, BAP seems to be more effective than TDZ in induction of negative shoots. However, the combination of BAP and NAA is important in direct shoot regeneration.

(Data shows mean number of shoots per SD ± SD of three independent experiments. A-white, brittle; B-cream, brittle)

(Each number represents the mean ± (SD) of three independent experiments with 30 sections per experiment.)

Example  2. Organ differentiation organogenesis )in Carbon source  And impact of base badges

To investigate the effect of carbon sources on shoot regeneration from leaf sections, various concentrations of sucrose, fructose and maltose were added to the medium. The type and concentration of carbohydrates affected shoot growth (Table 3). Of the carbon sources tested, 3% sucrose was the most effective at the number of shoots per segment (11.33 ± 1.25). Reduction by 1% sucrose decreased shoot regeneration (2.00 ± 0.82). In comparison to sucrose, the inclusion of maltose resulted in fewer and shorter shoots with reduced leaf count per shoot. However, fructose inhibited the development and elongation of shoots. Shoot proliferation was not observed in all carbohydrate-deficient media.

(Each number represents the mean ± (SD) of three independent experiments with 30 sections per experiment.)

To investigate the effect of basal medium composition on shoot regeneration from leaf sections, MS, SH and B5 salts were added to the regeneration medium. As shown in Table 4, a significant difference in shoot regeneration was observed. Of the three basic salts, MS with 0.1 mg / l NAA, 1.0 mg / l BAP and 3% sucrose gave the highest shoot length average (4.63 ± 0.48 cm). In contrast, the medium containing B5 and SH had little induction of shoots and the mean length of shoots was 2.03 ± 0.31 and 2.03 ± 0.33 cm, respectively.

Plant tissues are known to contain and / or utilize other carbohydrates (Mello et al., 2001 Sci Agric 58: 481-485). According to the results of the present invention, the addition of 3% sucrose to the induction medium was more effective for shoot regeneration than the addition of fructose or maltose. However, less than 3% sucrose concentration showed reduced shoot regeneration. The addition of carbohydrates in the regeneration medium serves as an osmotic material for carbon sources, energy sources and organ differentiation (Verma DC, Dougall DK, 1977 Plant Physiol 59: 81-85).

(Each number represents the mean ± (SD) of three independent experiments with 30 sections per experiment.)

Example  3. Shooting Kidney and Root Formation

Shoot regeneration in regenerated shoots was successful in 0.5 × MS medium without Plant Growth Regulator (PGR) (FIG. 1D). Regenerated shoots 2 to 2.5 cm in length were cut with at least two expanded leaves and transferred to rooting medium consisting of MS basal medium supplemented with varying concentrations of IBA (Table 5). Root initiation occurred within 7 days. An effective root system appeared after 4 weeks of culture (FIGS. 1E and 1F). The addition of 1.0 mg / l IBA to the medium was most effective in inducing root formation (21.33 ± 2.49 mm) without basic callusing.

Healthy roots were removed from the medium and transferred to pots containing vermiculite and pearlite (1: 1). The pot was covered with a polyethylene sheet the first week. The rooted plant was transferred to the pot of the growth chamber and gradually purified in the greenhouse. The purified news material was eventually planted in natural soil, and over 90% of the plants survived. There was no phenotypic difference between the regenerated plants. The shape of the newsletter was comparable to that of the seed-derived plant (FIG. 1G).

MS basal medium has been found to be the most preferred for organ differentiation from leaf explants of dry maria coda. Optimal rooting was observed at 1.0 mg / l IBA. 100% of regenerated shoots developed roots with an average number of roots per shoot of 25.67 ± 3.68 and an average root length of 21.33 ± 2.49 cm. In the present invention, the root induction rate and the average root length in the absence of plant growth regulators were significantly higher.

(Each number represents the mean ± (SD) of three independent experiments with 30 sections per experiment.)

Example  4. Shinshu  Scanning electron microscopy and histological analysis of regeneration

Analysis by scanning electron microscopy (SEM) showed the regeneration of several spherical structures on the explant surface after 8-10 days of culture (FIG. 2A). The structure, composed of small cells surrounded by extracellular matrix, developed several times after 14 days of explant culture and developed into shoot primordia (FIG. 2B). Usually, two leaf primitive cells were observed per regenerated bud, and fully developed shoots were closely associated with the explants (FIG. 2C). The presence of multiple negative shoots in such a proximity indicates the presence of competent cells in close proximity to each other.

Histological analysis provided morphological details to help explain the process of organ differentiation from dry maria coda leaf explants. During the first four days, no histological changes on the surface were observed. Examination of the sections at different stages of regeneration revealed that epithelial cells were the source of organ differentiation. The initial visible change was the dedifferentiation of epithelial cells along the section of the explant. After 8 days of culture in the regeneration medium, these cells formed clusters of meristematic structures that developed negative shoots by periclinal divisions. Meristem cells consisted of cells closely aligned with the dense cytoplasm, small vacuoles and distinct nuclei (FIG. 2D). The origin of shoot eye is direct, which is indicated by the presence of continuous vascular strands from the explant to the shoot eye (FIG. 1E).

In conclusion, the present invention for the first time invented a protocol for the production of negative shoots directly from the leaf explants of dry maria coda. In this way, rapid in vitro propagation of dry maria coda can be used for the production of secondary metabolites, germplasm conservation and genetic transformation.

Claims (8)

(1) sterilizing and germinating Drymaria cordata seeds;
(2) cutting off the leaves of seedlings from the germinated seeds; And
(3) a section of the cut leaf, 0.05-0.1 mg / l NAphthalene acetic acid (NAA); 1-2 mg / l of BAP (6-benzylaminopurine) or 1.5-2.5 mg / l of TDZ (Thidiazuron); And regenerating shoots at high-frequency in Murashige and Skoog (MS) medium containing 2.5-3.5% sucrose. The method of direct high frequency regeneration of Drymaria cordata shoots. The method of claim 1, wherein the leaf of the step (2) comprises a petiole (petiole), the method of direct high frequency regeneration of dry maria kodata shoots. delete (1) sterilizing and germinating the dry maria coda seeds;
(2) cutting off the leaves of seedlings from the germinated seeds;
(3) NAA at 0.05-0.1 mg / l of the cut leaf; 1-2 mg / l BAP or 1.5-2.5 mg / l TDZ; And regenerating shoots at high frequency in MS medium containing 2.5-3.5% sucrose;
(4) incubating the regenerated shoots in MS medium to which 0.5 ~ 2.0 mg / l of IBA (Indole-3-butyric acid) was added to induce rooting; And
(5) A method for producing a re-dried dry maria codata plant comprising a purifying step of transplanting and cultivating the rooted news product in a pot. delete delete delete delete

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