CN104642106B - A kind of in vitro culture method of strange southern star - Google Patents

Abstract

The invention provides a method for in vitro cultivation of the strange southern star. The in vitro culture method of the strange southern star provided by the present invention comprises the following steps: cutting the petiole of the tissue-cultured aseptic seedling of the strange southern star to obtain the petiole segment; conducting callus induction culture on the petiole segment to obtain the callus ; Then the callus was cultured to obtain the regenerated plantlets of the strange southern star. Experiments have proved that the regenerated seedlings of A. chinensis can be successfully obtained by using the method for in vitro cultivation of A. schia.

Description

A kind of in vitro culture method of strange southern star

technical field

The invention relates to the field of biotechnology, in particular to an in vitro culture method of the strange southern star.

Background technique

Arisaemadecipiens, also known as iron lampstand and green foot lotus, is a plant of the genus Arisaema in the family Araceae. It is distributed in India, Guangxi, Yunnan and other places in mainland China, and grows at an altitude of 880 meters to 3400 meters. , mostly grows in hillside shrubs, and has not yet been artificially introduced and cultivated. The rhizome of this plant is used as medicine to treat unknown swelling, mastitis, carbuncle, venomous snake bites, bee scorpion stings, spider and mouse bites. In the settlements of the Dong ethnic group, the locals treat breast cancer by externally using the tubers of the strange southern star. At present, there are very few studies on the strange southern star. There is only one report on its chemical composition, and there is no report on its tissue culture.

Contents of the invention

One object of the present invention is to provide a method for in vitro culture of S. chinensis.

The in vitro culture method of the strange southern star provided by the present invention comprises the following steps: cutting the petiole of the tissue-cultured aseptic seedling of the strange southern star to obtain the petiole segment; conducting callus induction culture on the petiole segment to obtain the callus ; Then the callus was cultured to obtain the regenerated plantlets of the strange southern star.

In the above method, the medium used for the callus induction culture is prepared as follows: add 6-BA and 2,4-D to the MS medium, and make 6-BA in the callus induction medium The concentration in the medium is 1.0mg/L, and the concentration of 2,4-D in the callus induction medium is 1.0mg/L;

In the above method, the callus induction culture conditions are as follows: the temperature is 25±2°C, the light time is 12h·d ^-1 , and the light intensity is 30-40mmol·m ^-2 ·s ^-1 .

In the above method, the length of the petiole segment is 0.5 cm.

In the above method, the "cultivating the callus to obtain the regenerated plantlets of A. chinensis" includes the following steps: inducing and culturing the callus for adventitious buds to obtain seedlings with adventitious buds and adventitious roots.

In the above method, the medium for adventitious bud induction culture is prepared as follows: add IAA and 6-BA to MS medium, so that the concentration of IAA in the adventitious bud induction medium is 0.5-2.0mg/ L, specifically 0.5 mg/L or 2.0 mg/L, the concentration of 6-BA in the adventitious bud induction medium is 0.5-2.0 mg/L, specifically 0.5 mg/L or 2.0 mg/L;

In the above method, the conditions for the induction of adventitious buds are: the temperature is 25±2°C, the light time is 12h·d ^-1 , and the light intensity is 30-40mmol·m ^-2 ·s ^-1 .

In the above method, before the adventitious bud induction culture, the step of subculturing the callus is also included.

In the above method, the medium used for the subculture is prepared as follows: add 6-BA and 24-D to the MS medium, so that the concentration of 6-BA in the subculture medium is 0.5 ～4.0mg/L, specifically 0.5mg/L, 1.0mg/L or 4.0mg/L, the concentration of 2,4-D in the subculture medium is 0.5～4.0mg/L, specifically 0.5 mg/L, 1.0mg/L or 4.0mg/L;

In the above method, the subculture conditions are as follows: the temperature is 25±2°C, the light time is 12h·d ^-1 , and the light intensity is 30-40mmol·m ^-2 ·s ^-1 .

In the above method, the tissue-cultured sterile seedlings of the strange southern star are prepared according to the following method: the seeds of the strange southern star are cultivated in MS medium, illuminated for 12 hours a day, the culture temperature is 23-27°C, and the light intensity is 30-40mmol· m ^-2 ·s ^-1 , cultivated for 30 days to obtain the sterile vaccine.

Another object of the present invention is to provide a callus induction medium and/or an adventitious bud induction medium used in the in vitro culture of Qiqinanxing.

The aforementioned Qiqinanxing callus induction medium was prepared as follows: 6-BA and 2,4-D were added to the MS medium so that the concentration of 6-BA in the callus induction medium was 1.0 mg/L, the concentration of 2,4-D in the callus induction medium is 1.0 mg/L.

The above-mentioned Adventitious Bud Induction Medium is prepared according to the following method: add IAA and 6-BA to the MS medium, so that the concentration of IAA in the adventitious bud induction medium is 0.5-2.0 mg/L, specifically: 0.5 mg/L or 2.0 mg/L, the concentration of 6-BA in the adventitious bud induction medium is 0.5-2.0 mg/L, specifically 0.5 mg/L or 2.0 mg/L.

Experiments have proved that the regenerated seedlings of A. chinensis can be successfully obtained by using the method for in vitro cultivation of A. schia.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, strange southern star in vitro propagation method

1. Plant material source and disinfection treatment

Qi Nanxing seeds were collected in Longbang Town, Jingxi County, Guangxi Province. Rinse the collected seeds with tap water for 1 hour, soak them in distilled water for 24 hours, and then sterilize the surface with 75% ethanol for 30 seconds in the ultra-clean workbench, then sterilize with 0.1% mercury liter for 7 minutes, and finally use aseptic Rinse with water 5 times.

2. Culture medium and culture conditions

Using MS medium as the basic medium, the medium was autoclaved at 121°C for 20 minutes. The sterilized seeds were used as explants. After inoculation, they were exposed to light for 12 hours a day and placed in a culture room with a temperature of 25±2°C and a light intensity of 30-40 mmol·m ^-2 ·s ^-1 for cultivation.

3. Obtaining sterile vaccines

About one week or so, the sterilized seeds inoculated on the MS medium began to germinate, and leaves grew after 15 days, and the height of the seedlings was 3-5 cm after 30 days, and sterile seedlings were obtained.

4. Callus induction and proliferation

In the ultra-clean workbench, cut the leaves of the aseptic seedlings of Qiqi Nanxing into pieces about 0.5×0.5cm in size, cut the petioles into 0.5cm long pieces, and inoculate them with different concentrations of 2,4-D and 6-BA medium for callus induction. Medium for callus induction: MS+6-BA1.0mg/L+2,4-D1.0mg/L. The callus was induced under the culture conditions of temperature 25±2°C, light time 12h·d ^-1 and light intensity 30-40mmol·m ^-2 ·s ^-1 . Callus can be induced after 2 weeks on the petiole, but the callus cannot be induced on the leaves, which are browned and dead.

The induced callus was inoculated in MS+6-BA (0.5-4.0) mg/L+2,4-D (0.5-4.0) mg/L medium for subculture. The culture conditions for subculture: the temperature is 25±2°C, the light time is 12h·d ^-1 , and the light intensity is 30-40mmol·m ^-2 ·s ^-1 .

The callus proliferated rapidly, and the multiplication ratio was 8.09-11.72 times. When the concentrations of 6-BA and 2,4-D are both 0.5mg/L, the callus multiplication factor is 9.34; when the concentrations of 6-BA and 2,4-D are both 4.0mg/L, the callus The tissue proliferation factor was 8.09. Among them, MS+6-BA1.0mg/L+2,4-D1.0mg/L medium had the best proliferation effect, and the callus multiplied by 11.72 times.

5. Induction of Adventitious Buds and Plant Regeneration

Adventitious buds are induced in a medium supplemented with IAA and 6-BA, and the medium for adventitious bud induction culture: MS+IAA (0.5-2.0) mg/L+6-BA (0.5-2.0) mg/L. The subcultured calli were placed under the culture conditions of temperature 25±2°C, light time 12h·d ^-1 , and light intensity 30-40 mmol·m ^-2 ·s ^-1 to induce adventitious buds.

The induction rate of adventitious buds is closely related to the concentrations of the two growth regulators. When the concentrations of IAA and 6-BA are both 0.5 mg/L, the induction rate of adventitious buds is the highest, up to 82%, and the growth status of adventitious buds Good, the petiole is thicker, the leaves are larger, and the growth is vigorous. When the concentrations of IAA and 6-BA were both 2.0mg/L, the induction rate of adventitious buds was 42.67%.

While inducing adventitious buds, the adventitious buds began to appear white adventitious roots, and then the root tips elongated, and the adventitious roots were distributed radially. Most of the roots were thicker and more root hairs. The rooting rate reached 100%, and the average rooting number was 10.35. The average root length is 7.43cm.

6. Hardening and transplanting

Open the mouth of the triangular flask to harden the seedlings. After 1 week, the regenerated seedlings with good growth and well-developed root system were taken out, the medium of the root was washed, and transplanted into a mixed substrate composed of peat soil, perlite and sand (4:3:2, v/v/v) , placed in an environment with a relative humidity of 80% to 90% and a temperature of about 20°C, watered regularly, and the survival rate reached 100% after 30 days.

Claims (8)

1. an in-vitro culture method for unusual Rhizoma Arisaematis, comprises the steps: that the petiole by the group training aseptic seedling of unusual Rhizoma Arisaematis carries out cutting, obtains petiole section；Petiole section is carried out induction of callus, obtains callus；Callus is carried out cultivation again and obtains unusual Rhizoma Arisaematis regrowth；Described " callus carries out cultivation again and obtains unusual Rhizoma Arisaematis regrowth " comprises the steps: described callus is carried out adventitious bud induction culture, obtains the seedling with adventitious bud and adventitious root；

Culture medium used by described induction of callus is prepared as follows and obtains: add 6-BA and 2 in MS culture medium, 4-D, to make 6-BA concentration in described callus inducing medium be 1.0mg/L, 2, the 4-D concentration in described callus inducing medium is 1.0mg/L；

The culture medium of described adventitious bud induction culture is prepared as follows and obtains: add IAA and 6-BA in MS culture medium, to make IAA concentration in described adventitious bud induction culture base be 0.5～2.0mg/L, the 6-BA concentration in described adventitious bud induction culture base is 0.5～2.0mg/L；

The condition of described adventitious bud induction culture is: temperature is 25 ± 2 DEG C, light application time is 12h d^-1, intensity of illumination be 30～40mmol m^-2·s^-1。

2. method according to claim 1, it is characterised in that:

The condition of described induction of callus is: temperature is 25 ± 2 DEG C, light application time is 12h d^-1, intensity of illumination be 30～40mmol m^-2·s^-1。

3. method according to claim 1 and 2, it is characterised in that: the length of described petiole section is 0.5cm.

4. method according to claim 1 and 2, it is characterised in that: IAA concentration in described adventitious bud induction culture base is 0.5mg/L or 2.0mg/L, the 6-BA concentration in described adventitious bud induction culture base is 0.5mg/L or 2.0mg/L.

5. method according to claim 1 and 2, it is characterised in that: described carry out adventitious bud induction culture before, also include the step that described callus is carried out successive transfer culture；Culture medium used by described successive transfer culture is prepared as follows and obtains: add 6-BA and 2 in MS culture medium, 4-D, to make 6-BA concentration in described successive transfer culture culture medium be 0.5～4.0mg/L, 2, the 4-D concentration in described successive transfer culture culture medium is 0.5～4.0mg/L.

6. method according to claim 5, it is characterised in that:

The condition of described successive transfer culture is: temperature is 25 ± 2 DEG C, light application time is 12h d^-1, intensity of illumination be 30～40mmol m^-2·s^-1。

7. method according to claim 5, it is characterised in that: 6-BA concentration in described successive transfer culture culture medium is 0.5mg/L, 1.0mg/L or 4.0mg/L；2,4-D concentration in described successive transfer culture culture medium is 0.5mg/L, 1.0mg/L or 4.0mg/L.

8. method according to claim 1 and 2, it is characterized in that: the group training aseptic seedling of described unusual Rhizoma Arisaematis is prepared as follows and obtains: by the seed MS culture medium culturing of unusual Rhizoma Arisaematis, illumination every day 12h, cultivation temperature is 23～27 DEG C, and intensity of illumination is 30～40mmol m^-2·s^-1, cultivate 30 days, obtain described aseptic seedling.