CN110100732A - A kind of black nightshade method for in-vitro rapid propagation - Google Patents

Abstract

A method for in vitro rapid propagation of nightshade nightshade, which belongs to the field of plant tissue culture. The invention uses nightshade nightshade seeds as test materials to establish an aseptic line, selects cotyledons and hypocotyls of aseptic seedlings as explants, and uses MS as the basic culture Add plant growth regulator 6-BA, NAA or IAA to induce callus, and then successfully obtain a large number of regenerated plants in a short period of time through differentiation culture, rooting culture, hardening and transplanting, and establish rapid propagation of Solanum nigrum tissue culture technology system to promote its large-scale development. The advantages of the present invention are: the present invention uses Solanum nigrum seeds as test materials, successfully obtains Solanum solanum in vitro regenerated plants through processes such as callus induction culture, differentiation culture, rooting culture, seedling hardening and transplanting, and establishes Solanum nigrum tissue culture The rapid propagation technology system provides sterile seedlings with stable genetic performance and high uniformity for the extraction of effective components of Solanum nigrum.

Description

A kind of Solanum solanum in vitro rapid propagation method

technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a rapid in vitro propagation method of nightshade nightshade.

Background technique

Solanum nigrum ( Solanum nigrum L.) belongs to the family Solanaceae, its berries and leaves are edible, and it is an annual herbaceous plant. According to the "Compendium of Materia Medica", the whole plant of Solanum nigrum is used as medicine, which can treat carbuncle, swelling, poison and bruises, and can also clear away heat and detoxify, diuretic and lower blood pressure. But the leaves contain a large amount of alkaloids, which can be detoxified only after being cooked, and are distributed almost all over the country. When the nightshade fruit is ripe, it is purple-black, sweet and slightly sour, rich in vitamins and amino acids, the content is several times that of tomato, and also contains a small amount of solanine, solanine and vitamin C, which can sterilize and reduce inflammation. Inflammation, prostatitis, acute nephritis, etc. have a certain effect. Solanum nigrum is more toxic when immature, and less toxic when mature. Heating can also decompose most of the toxic substances. Solanum nigrum seedlings can be eaten as wild vegetables, and nightshade fruit is a potential fruit resource because of its rich amino acids and vitamins. In addition, food pigments can also be extracted from Solanum nigrum, and its development and utilization have attracted more and more attention.

Solanum nigrum grows luxuriantly at 22-30°C, and it is suitable for flowering and has a high seed setting rate at 15-20°C. Solanum nigrum does not have very strict requirements on the soil, and the suitable soil pH value is 5.5-6.5. If the soil has strong fertility, water retention, and rich organic matter content, solanum nigrum will grow well in this environment. If the soil is poorly ventilated and lacks organic matter, the plant growth of Solanum nigrum is relatively weak in this environment, and its root system growth is not perfect, so it cannot be used as a processing material for commodities. And nightshade is difficult to grow in the summer and autumn of high temperature and high humidity, and the plant growth is slow in winter and spring, and its tender shoots are easy to fiber aging, and the commerciality is poor, which seriously hinders the needs of its large-scale production. At present, nightshade generally adopts the seeds of wild nightshade plants to sow, which is often limited by seasonal conditions and cannot be cultivated and produced on a large scale. Therefore, it has become a top priority to obtain sterile plants and establish a complete set of high-quality seedling breeding system through plant tissue culture technology.

Contents of the invention

The purpose of the present invention is to provide a method for rapid propagation of nightshade in vitro in order to solve the limitation of season conditions for sowing of existing nightshade plants.

In order to achieve the above object, the technical scheme that the present invention takes is as follows:

A method for in vitro rapid propagation of Solanum nigrum, said method may further comprise the steps:

Step 1: Establishment of the sterile line: select the full-bodied Solanum nigrum seeds as explants, first soak the Solanum nigrum seeds in sterile water for 24 hours, change the sterile water 3 times during this period, and then use volumetric Soak Solanum solanum seeds in 75% alcohol for 30 s, wash them with sterile water for 4 times, then soak Solanum nigrum seeds in 10% NaClO (sodium hypochlorite) solution for 10 min, and finally wash them with sterile water for 4 times. Sunflower seeds were inoculated on MS medium, placed under the light intensity of 1000 lx~2000 lx for 16 h, and cultured at a culture temperature of 20 °C and a relative air humidity of 70%~80%. Callus culture was carried out when the height was 5 cm;

Step 2: callus induction: the cotyledons and hypocotyls of the aseptic seedlings obtained in step 1 were used as explants for dedifferentiation culture, wherein the cotyledons were directly cut out, and the hypocotyls were cut out 1 cm. The lower and hypocotyl cuts were inoculated downwards into the induction medium for callus induction culture. After inoculation, they were placed under the light intensity of 1000 lx~2000 lx for 10 h. The culture temperature was 20 °C and the relative air humidity was 70%~80. 15 days under the condition of %;

Step 3: Differentiation culture: Take the callus obtained in step 2 and cut it into small pieces with a side length of 0.5 cm and inoculate it into the differentiation medium for differentiation culture. After inoculation, place it under the light intensity of 1000 lx~2000 lx for 16 h , cultured for 20 days at a culture temperature of 20°C and a relative air humidity of 70% to 80%, to obtain test-tube plantlets;

Step 4: Rooting culture: Inoculate the test-tube plantlets obtained from the three-differentiation culture from the base cutting step into the rooting medium to induce rooting. After inoculation, put them under the light intensity of 1000 lx~2000 lx for 10 h, and cultivate them at a temperature of 20 °C in air. Cultivate under the condition of 70%～80% relative humidity for 21 days to obtain bottle seedlings;

Step 5: Seedling hardening and transplanting: Harden the bottle seedlings obtained on the rooting medium with transplanting conditions. After the bottle seedlings are moved to room temperature for 4 to 5 days, open the sealing film for 2 to 3 days, and then wash them with clean water The culture medium remaining at the root is transplanted into a sterilized substrate containing fine sand. Before transplanting, the substrate is sprayed and disinfected with a potassium permanganate solution with a volume fraction of 5‰, and then rinsed with water for 3 to 5 times. When transplanting, the substrate needs to be compacted, and a single plant is transplanted, and sprayed in the morning and evening to ensure that the growth environment is humid.

Compared with the prior art, the beneficial effect of the present invention is: the present invention uses Solanum nigrum seeds as test materials, and successfully obtains Solanum nigrum in vitro regenerated plants through processes such as callus induction culture, differentiation culture, rooting culture, seedling hardening and transplanting. , to establish a rapid propagation technology system for Solanum nigrum tissue culture, to provide sterile seedlings with stable genetic performance and high uniformity for the extraction of active ingredients of Solanum nigrum.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the examples, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit scope of the technical solution of the present invention should be covered by the technical solution of the present invention. within the scope of protection.

The present invention uses Solanum nigrum seeds as test materials to establish an aseptic line, selects the cotyledon and hypocotyl of aseptic seedlings as explants, takes MS as the basic medium, and adds plant growth regulator 6-BA, NAA or IAA, first Induce callus, and then successfully obtain a large number of regenerated plants in a short period of time through differentiation culture, rooting culture, seedling hardening and transplanting, etc., establish a rapid propagation technology system for Solanum nigrum tissue culture, and promote its large-scale development.

Specific embodiment one: what this embodiment records is a kind of Solanum solanum in vitro rapid propagation method, and described method comprises the following steps:

Step 1: Establishment of the sterile line: select the full-bodied Solanum nigrum seeds as explants, first soak the Solanum nigrum seeds in sterile water for 24 hours, change the sterile water 3 times during this period, and then use volumetric The seeds were soaked in 75% alcohol for 30 s, washed with sterile water for 4 times, and then soaked with 10% NaClO solution for 10 min to disinfect the seeds, and finally washed with sterile water for 4 times. Solanum nigrum seeds were inoculated on MS medium, placed under the light intensity of 1000 lx~2000 lx for 16 h, and cultured at a culture temperature of 20 °C and a relative air humidity of 70%~80%. Callus culture was carried out when the plant height was 5 cm;

Step 2: callus induction: the cotyledons and hypocotyls of the sterile seedlings obtained in step 1 were used as explants for dedifferentiation culture, wherein the cotyledons were directly cut out, the hypocotyls were cut to a length of 1 cm, and the back of the cotyledon Inoculate the induction medium facing downward with the hypocotyl incision downward for callus induction culture. After inoculation, place it under the light intensity of 1000 lx~2000 lx for 10 h, at a culture temperature of 20°C and a relative air humidity of 70%~80 Under the condition of %, culture was carried out, and the induction situation was counted after 15 days;

Step 3: Differentiation culture: Take the callus obtained in step 2 and cut it into small pieces with a side length of 0.5 cm and inoculate it into the differentiation medium for differentiation culture. After inoculation, place it under the light intensity of 1000 lx~2000 lx for 16 h , the cultivation temperature is 20°C, and the relative air humidity is 70% to 80% for cultivation. After 20 days, test-tube plantlets are obtained, and the differentiation is counted;

Step 4: Rooting culture: Inoculate the robust test-tube plantlets obtained from the base cutting step three-differentiation culture into the rooting medium to induce rooting. After inoculation, put them under the light intensity of 1000 lx~2000 lx for 10 h, and cultivate them at a temperature of 20 ° C. Cultivate under the condition of 70%～80% relative air humidity, after 21 days, obtain the bottle seedling, count the rooting situation;

Step 5: Seedling hardening and transplanting: Harden the bottle seedlings obtained on the rooting medium with transplanting conditions. After the bottle seedlings are moved to room temperature for 4 to 5 days, open the sealing film for 2 to 3 days, and then wash them with clean water The culture medium remaining at the roots should be transplanted into a sterilized substrate containing fine sand. Before transplanting, the substrate should be sprayed and disinfected with a potassium permanganate solution with a volume fraction of 5‰, and then rinsed with clean water for 3 to 5 times. , When transplanting, the substrate needs to be compacted, and a single plant is transplanted, sprayed in the morning and evening to ensure a humid growth environment, and the survival rate is counted 7 days after transplanting.

Specific embodiment two: a kind of Solanum nigrum in vitro rapid propagation method described in specific embodiment one, in step two, described induction medium is: MS+0.5 mg/L6-BA+2.0 mg/L IAA+30 g/L sucrose+7 g/L agar powder or MS+0.5 mg/L6-BA+1.0 mg/L IAA+30 g/L sucrose+7 g/L agar powder, the pH value is 5.8.

Specific embodiment three: a kind of solanum solanum in vitro rapid propagation method described in specific embodiment one, in step three, described differentiation medium is: MS+2.0 mg/L6-BA+0.1 mg/LNAA+30 g /L sucrose+7 g/L agar powder, the pH value is 5.8.

Specific embodiment four: a kind of Solanum nigrum in vitro rapid propagation method described in specific embodiment one, in step 4, described rooting medium is: 1/2MS+0.05mg/LNAA+15 g/L sucrose+7 g/L agar powder or 1/2MS+0.05mg/LIAA+15 g/L sucrose+7 g/L agar powder, the pH value is 5.8.

Example 1

(1) Establishment of the sterile line: select the seeds with plump fruits as explants, soak them in sterile water for 24 hours, change the sterile water three times during the period, and then soak them in alcohol with a volume fraction of 75% under sterile conditions For 30 s, rinse 4 times with sterile water, then sterilize the seeds with 10% NaClO solution for 10 min, and finally rinse the seeds 4 times with sterile water, inoculate the sterilized seeds on MS medium, place in The seeds were cultivated under the conditions of 1000 lx~2000 lx light intensity for 16 h at a cultivation temperature of 20 °C and a relative air humidity of 70%~80%, and the germination rate was counted.

(2) Callus induction: The cotyledons and hypocotyls of the sterile seedlings obtained in step (1) were selected as explants for dedifferentiation culture, wherein the cotyledons were cut directly, and the hypocotyls were cut to a length of 1 cm. Inoculate the callus induction medium with the back side down and the hypocotyl incision down. After inoculation, put it under the light intensity of 1000 lx~2000 lx for 10 h. The culture temperature is 20 ℃ and the relative air humidity is 70 %~80% of the culture, the induction rate was 85% after 15 days. The induction medium is: MS+0.5 mg/L6-BA+2.0 mg/L IAA+30 g/L sucrose+7 g/L agar powder (suitable for cotyledons) or MS+0.5 mg/L6-BA+1.0 mg/L IAA+30 g/L sucrose+7 g/L agar powder (suitable for hypocotyls), pH 5.8.

(3) Differentiation culture: Take out the callus obtained in step (2) and cut it into small pieces with a side length of about 0.5 cm and inoculate it into the differentiation medium for differentiation culture. After inoculation, place it under the light intensity of 1000 lx~2000 lx 16 h, cultured at a culture temperature of 20°C and a relative air humidity of 70%-80%, after 20 days, the differentiation rate of adventitious buds of cotyledons reached 89.2%, and the differentiation rate of adventitious buds of hypocotyls reached 67.3%; The number is 8, and the average number of hypocotyl adventitious buds is 3. The differentiation medium is: MS+2.0 mg/L6-BA+0.1 mg/LNAA+30 g/L sucrose+7 g/L agar powder, with a pH value of 5.8.

(4) Rooting culture: Cut the robust test-tube plantlets obtained from the differentiation culture in step (3) from the base and inoculate them into the rooting medium to induce rooting. ℃, and the relative humidity of the air is 70%~80%, and the rooting rate is over 90% after 20 days. The rooting medium is: 1/2MS+0.05 mg/LNAA+15 g/L sucrose+7 g/L agar powder or 1/2MS+0.05 mg/LIAA+15 g/L sucrose+7 g/L agar Powder, pH 5.8.

(5) Seedling hardening and transplanting: Harden the seedlings obtained on the rooting medium and meet the transplanting conditions. After the bottle seedlings are moved to room temperature for 4 to 5 days, open the sealing film for 2 to 3 days, and then wash them with clean water. The culture medium remaining at the roots should be transplanted into a sterilized substrate containing fine sand. Before transplanting, the substrate should be sprayed and disinfected with a potassium permanganate solution with a volume fraction of 5‰, and then rinsed with water for 3 to 5 times. , When transplanting, the substrate needs to be compacted, and a single plant should be transplanted, sprayed in the morning and evening to ensure a humid growth environment, and the survival rate can reach 95% after 10 days of transplanting.

Claims (4)

1. a kind of black nightshade method for in-vitro rapid propagation, it is characterised in that: the described method comprises the following steps:

Step 1: the foundation of aseptic strain: selecting the black nightshade seed of completely filled fruit as explant, impregnates dragon using sterile water first During which 24 h of certain herbaceous plants with big flowers seed replaces 3 sterile waters, then aseptically first impregnate 30 s with the alcohol that volume fraction is 75%, With sterile water wash 4 times, then the NaClO solution for being 10% with volume fraction impregnates 10 min of black nightshade seed, finally uses sterile water again Cleaning 4 times, black nightshade seed is inoculated on MS culture medium, 16 h of illumination under the lx intensity of illumination of 1000 lx ~ 2000 is placed in, is training 20 DEG C of temperature is supported, relative air humidity cultivates black nightshade seed under conditions of being 70% ~ 80%, until plant height carries out when being 5 cm Callus tissue culture；

Step 2: callus induction: the cotyledon for the aseptic seedling that step 1 is obtained and hypocotyl carry out de- point as explant Change culture, wherein cotyledon is directly cut, and hypocotyl cuts 1 cm length, by the back side of cotyledon downward, hypocotyl notch connects downwards Kind carries out induction of callus to induced medium, and inoculation is placed on illumination 10 under the lx intensity of illumination of 1000 lx ~ 2000 H, at 20 DEG C of cultivation temperature, relative air humidity is cultivated 15 days under conditions of being 70% ~ 80%；

Step 3: differentiation culture: the fritter that side length size is 0.5 cm is cut into the callus taking-up that step 2 obtains and is inoculated with Differentiation culture is carried out to differential medium, inoculation is placed on 16 h of illumination under the lx intensity of illumination of 1000 lx ~ 2000, in culture temperature 20 DEG C of degree, relative air humidity are cultivated 20 days under conditions of being 70% ~ 80%, obtain test tube seedling；

Step 4: culture of rootage: from base portion cut the test tube seedling that obtains of step 3 differentiation culture be seeded in root media into Row root induction, inoculation is placed on 10 h of illumination under the lx intensity of illumination of 1000 lx ~ 2000, at 20 DEG C of cultivation temperature, air phase It is cultivated 21 days under conditions of being 70% ~ 80% to humidity, obtains bottle seedling；

Step 5: acclimatization and transplants: the bottle seedling for having transplanting condition obtained on root media is subjected to hardening, bottle seedling moves to often It after lower 4 ~ 5 days of temperature, opens sealed membrane 2 ~ 3 days, then cleans the remaining culture medium in root with clear water, be transplanted to containing of having sterilized In the matrix of fine sand, matrix is sprayed with the liquor potassic permanganate that volume fraction is 5 ‰ before transplanting and is sterilized, is then rushed again with clear water Matrix need to be compacted by leaching 3 ~ 5 times, when transplanting, and carry out single plant transplanting, sooner or later spraying, guarantee that growing environment is moist.

2. a kind of black nightshade method for in-vitro rapid propagation according to claim 1, it is characterised in that: described in step 2 Induced medium are as follows:+7 g/L agar powder of MS+0.5 mg/L6-BA+2.0 mg/L IAA+30 g/L sucrose or MS+0.5 mg/ + 7 g/L agar powder of L6-BA+1.0 mg/L IAA+30 g/L sucrose, pH value is 5.8.

3. a kind of black nightshade method for in-vitro rapid propagation according to claim 1, it is characterised in that: described in step 3 Differential medium are as follows:+7 g/L agar powder of MS+2.0 mg/L6-BA+0.1 mg/LNAA+30 g/L sucrose, pH value 5.8.

4. a kind of black nightshade method for in-vitro rapid propagation according to claim 1, it is characterised in that: described in step 4 Root media are as follows:+7 g/L agar powder of 1/2MS+0.05mg/LNAA+15 g/L sucrose or 1/2MS+0.05mg/L IAA+15 + 7 g/L agar powder of g/L sucrose, pH value 5.8.