CN110396495B

CN110396495B - Himalayan mirabilis jalapa callus, proliferation method and suspension cell propagation method

Info

Publication number: CN110396495B
Application number: CN201910749434.4A
Authority: CN
Inventors: 陈玉珍; 卢存福
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2019-08-14
Filing date: 2019-08-14
Publication date: 2021-02-26
Anticipated expiration: 2039-08-14
Also published as: CN110396495A

Abstract

The invention relates to the technical field of plant tissue culture, and particularly discloses a Himalayan Mirabilis jalapa callus, a propagation method and a suspension cell propagation method. The Himalayan Mirabilis jalapa callus induction proliferation method comprises the steps of carrying out induction culture on leaves of Himalayan Mirabilis jalapa aseptic seedlings by using an induction culture medium to form callus, and further carrying out proliferation culture on the callus by using a proliferation culture medium to obtain subculture callus; wherein, the hormones in the induction and proliferation culture medium are TDZ and NAA. The method has high inductivity, less differentiation, short growth cycle, and soft and fragile callus, and is beneficial for subsequent utilization. The invention also provides a Himalayan Mirabilis jalapa suspension cell propagation method, which is characterized in that the Himalayan Mirabilis jalapa callus obtained by preparation is subjected to suspension culture by using a liquid culture medium containing TDZ, NAA and hydrolyzed casein. The method has the advantages of simple operation, high cell proliferation rate, short growth cycle and low cost, and can realize industrialized annual production.

Description

Himalayan mirabilis jalapa callus, proliferation method and suspension cell propagation method

Technical Field

The invention relates to the technical field of plant tissue culture. In particular to a callus, a propagation method and a suspension cell propagation method of Mirabilis himalaica.

Background

The Qinghai-Tibet Plateau (Qinghai-Tibet Plateau) is the highest Plateau in China and the highest altitude in the world, and is called the world ridge and the third pole of the earth. The solar energy greenhouse has the advantages of strong radiation, more sunshine, low air temperature, less accumulated temperature, reduction of air temperature along with the rise of altitude and latitude, and large day and night temperature difference; clear wet and dry, rainy night; the dry and cold are long and the wind is strong in winter; the typical plateau climate characteristics such as cool and rainy summer and more hailstones are existed. Under the environment condition, only plants with special ecological-biological adaptation characteristics can survive, settle and propagate in the course of struggle with severe climatic conditions such as strong radiation, ice and snow, severe cold, strong wind, meteoric stones, barren and the like.

Tibetan medicine is a medicine system with the pharmacological characteristics and national features of the Tibet plateau formed by combining the traditional medical experience, Indian medicine, traditional Chinese medicine and other traditional medicines of Tibetan people in the long-term struggle with diseases. Due to the unique natural ecological conditions of the Qinghai-Tibet plateau, the abundant medicinal components contained in the special plateau plants become the material basis of Tibetan medicine.

Mirabilis himalaica (Mirabilis himalaica) is a perennial herb of Mirabilis (Mirabilis) of Mirabilis family (Nyctaginaceae), and the wild resource is mainly concentrated in mountain slope, wasteland or brush forest zones with elevation of 3000-3400m, in northwest Sichuan and in India.

Himalayan Mirabilis jalapa has a long history of use in Tibetan medicines, is recorded in the Tibetan medicine famous works of 'four medical classics' and 'Jingzhu materia Medica', and is the most commonly used medicinal material in the Tibetan medicine book in China. The Tibetan medicine is prepared from roots, has the effects of warming kidney, tonifying kidney, promoting granulation, promoting urination, removing calculus and the like, is called five roots of a traditional Tibetan medicine together with rhizoma polygonati (Polygonatum verticillatum), Tribulus terrestris (Tribulius terrestris), Pimpinella javanica (Pleurospermum Hookeri) and Asparagus cochinchinensis (Asparagguscochinchinensis), mainly appears in a Tibetan medicine formula with a nourishing effect, and is one of main raw materials of dosage forms of Tibetan medicines, namely fifteen-flavor infantile tea pills, Bashen ghuangyou pills, twenty-five-flavor rhizoma podophylli pills, heat Barubu ointment and the like.

Although the Qinghai-Tibet plateau breeds plant resources with rich varieties, the Qinghai-Tibet plateau faces a series of problems that the resource reserves of species are limited, the population updating speed is slow, the recovery is difficult after the resource collection, the resource exhaustion, the ecological environment damage and the like cannot be continuously utilized and the like are brought about successively by the industrial development. According to the field investigation in recent years, with the development of the Tibetan medicine industry, due to excessive collection of people, the wild resource of the most common medicinal material Himalayan mirabilis has been continuously reduced, the wild resource is gradually exhausted, a special discussion conference is called in the Tibetan science and technology hall at the end of 2005, and the medicinal material is classified as the first-class endangered Tibetan medicinal material.

Besides being not influenced by natural environment (such as climate, season, region, natural disasters and the like), the plant tissue and the cell culture have stable yield and quality of metabolites, can save cultivated land, and solve the problems of shortage and endangerment of plant resources. The method has the advantages of exploring a new synthetic route and obtaining a new product, particularly having irreplaceable advantages for the production of medicinal plants which are endangered to extinction, slow in growth, long in growth period, bad in growth environment and high in cultivation cost and plant resources with high economic value, and having very important application values from ecological benefits, economic benefits and social benefits. At present, over 60% of anticancer drugs and 75% of drugs for treating diseases are derived from natural products. Plant tissue culture has been widely applied in the fields of agriculture, forestry, medicine, chemical industry and the like, nearly 1000 plants producing secondary metabolites are produced, and more than 600 active ingredients are directly provided by plant cell culture.

Although great progress is made in the artificial planting of Himalayan Mirabilis jalapa in recent years, the medicinal part of Himalayan Mirabilis jalapa is a perennial root, the growth period is long, the influence of environmental conditions is easy to occur, and the great demand of Tibetan medicine industrialization on Himalayan Mirabilis jalapa is difficult to meet. Therefore, a more effective way of propagating Mirabilis himalaica is urgently needed to be found.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a Himalayan Mirabilis jalapa callus induced proliferation method which is high in induction rate, quick in growth, less in differentiation and stable in growth, and a light yellow, semitransparent, soft and fragile Himalayan Mirabilis jalapa callus prepared by the method.

The invention also aims to provide a Himalayan Mirabilis jalapa suspension cell propagation method with good dispersibility and high growth speed.

In order to realize the purpose of the invention, the technical scheme provided by the invention is as follows:

a Himalayan Mirabilis jalapa callus induction proliferation method comprises:

carrying out induction culture on the leaves of the Himalayan Mirabilis jalapa aseptic seedlings by using an induction culture medium to form callus, and further carrying out proliferation culture on the callus by using a proliferation culture medium to obtain subculture callus;

wherein the hormones in the induction medium and the proliferation medium are both TDZ and NAA.

According to the invention, a large number of researches show that the primary callus of the Himalayan Mirabilis jalapa can be successfully and efficiently induced by taking TDZ and NAA as hormones in an induction culture medium, and further, the TDZ and NAA are taken as hormones in a proliferation culture medium, so that the primary callus can quickly and stably grow, excessive differentiation is avoided, and the preparation of the high-efficiency and high-quality Himalayan Mirabilis jalapa callus (secondary callus) is realized.

Preferably, the induction medium is: MS +0.5-3.0mg/L TDZ +0.5-4.0mg/L NAA, more preferably: MS +1.5mg/L TDZ +2.0mg/L NAA.

The invention adopts MS as a basic culture medium, and the MS is matched with 0.5-3.0mg/L TDZ and 0.5-4.0mg/L NAA to induce the Himalayan mirabilis jalapa primary callus, so that the dedifferentiation effect is better, and when the preferable formula is MS +1.5mg/L TDZ +2.0mg/L NAA, higher induction rate (the cure rate is 96.67%) can be further obtained.

Preferably, the multiplication medium is: MS +0.5-2.0mg/L TDZ +1.0-3.0mg/L NAA, more preferably: MS +1.0mg/L TDZ +2.0mg/L NAA;

the invention adopts MS as a basic culture medium, and the MS is matched with 0.5-2.0mg/L TDZ and 1.0-3.0mg/L NAA to carry out secondary multiplication on the Himalayan Mirabilis jalapa initial callus, so that the multiplication of the callus is fast, the cell differentiation is less, the cell growth is stable, and when the preferable formula is MS +1.0mg/L TDZ +2.0mg/L NAA, a better multiplication effect can be further obtained.

As a preferred embodiment of the present invention, the method of the present invention comprises:

(1) culturing sterile Mirabilis himalaica;

(2) inoculating the leaves of the aseptic seedlings into the induction culture medium, and performing induction culture for 30-40 days at 25 +/-2 ℃ in the dark to obtain the callus;

(3) inoculating the callus into the proliferation culture medium, and performing proliferation culture for 10-20 times at 25 + -2 deg.C in dark for 20-30 days each time to obtain subculture callus.

Preferably, the Mirabilis himalaica seeds are sterilized and then used for culturing aseptic seedlings. The sterilization method may be any known method, such as sterilization with a combination of 75% alcohol and 2% sodium hypochlorite, and the present invention is not particularly limited.

Preferably, the method for culturing the sterile seedlings comprises the following steps: culturing Himalayan Mirabilis jalapa seeds in MS culture medium at 25 + -2 deg.C under illumination intensity of 2500-.

More preferably, Mirabilis himalaica seeds are cultured for 25 days at 25 deg.C under illumination intensity of 3000lx and illumination time of 16 hr/day. The mode is more beneficial to the growth of the sterile seedlings of the himalayan Mirabilis jalapa.

Preferably, the seedling leaves of the sterile plantlets are inoculated.

Preferably, callus induction culture is performed in the dark at 25 ℃ for 35 days, which is more advantageous for callus generation from Himalayan Mirabilis jalapa explants.

Preferably, callus proliferation culture is performed 15 times in the dark at 25 ℃ for 25 days, which is more favorable for the formation of pale yellow, translucent, soft and friable callus.

Preferably, the induction medium and the proliferation medium further comprise 25-30g/L sucrose, 6.5-7.0g/L agar. The water in the induction medium and the multiplication medium is deionized water.

The invention uses sucrose to provide energy for callus and uses agar as a support.

Preferably, the pH of the induction medium and the multiplication medium is 5.8-6.0, more preferably 5.8.

The invention also provides a Himalayan Mirabilis jalapa callus prepared by the method.

The calli of the Himalayan Mirabilis jalapa is light yellow, semitransparent, soft and fragile, is beneficial to stable subculture, and is more beneficial to suspension cell culture and expanded production compared with the conventional calli obtained by only primary induction.

The invention also provides a Himalayan Mirabilis jalapa suspension cell propagation method, which comprises the following steps: and performing suspension culture on the callus of the Himalayan Mirabilis jalapa prepared in the above step by using a liquid culture medium, wherein the liquid culture medium comprises TDZ, NAA and hydrolyzed casein.

Preferably, the suspension culture is performed for 5-10 times, each time for 15-30 days, so as to obtain suspension cells with good growth, good dispersibility and high growth speed.

More preferably, the number of suspension cultures is 10, each for 20 days.

Preferably, the suspension cell propagation is carried out on a shaking table, and the rotation speed of the shaking table is 80-120 r/min; and/or, the suspension cell propagation is performed under dark conditions at 25 ± 2 ℃.

Preferably, the inoculation amount of the Himalayan mirabilis jalapa callus in the liquid culture medium is 60-80 g/L.

Preferably, the liquid culture medium is MS +0.5-2.0mg/L TDZ +1.5-3.0mg/L NAA +100-300mg/L hydrolyzed casein, preferably: MS +1.0mg/L TDZ +2.0mg/L NAA +200mg/L hydrolyzed casein;

the invention adopts MS as basic culture solution, and the MS is matched with 0.5-2.0mg/L TDZ and 1.5-3.0mg/L NAA to propagate suspension cells, so that the suspension cells can grow stably, disperse well and proliferate quickly, and the preferable formula is MS +1.0mg/L TDZ +2.0mg/L NAA +200mg/L hydrolyzed casein, so that the propagation effect can be further improved. Wherein, a certain proportion of hydrolyzed casein has the promotion effect on cell suspension culture.

Preferably, the liquid medium further comprises 25-30g/L sucrose; and/or the pH value of the liquid medium is 5.8-6.0, preferably 5.8.

Preferably, the water in the liquid medium is deionized water.

The invention has the beneficial effects that:

the method can quickly obtain a large amount of callus with good growth and stable genetic characteristics of the Mirabilis himalaica, and has the advantages of high inductivity, less differentiation, short growth period and low cost.

The callus of Mirabilis himalaica obtained by the invention is in a light yellow, semitransparent, soft and fragile state, is beneficial to suspension cell screening and subculture, and is an ideal raw material for subsequent suspension cell propagation.

The suspension cell propagation method has the advantages of simple operation, high cell proliferation rate, short growth cycle and low cost, can provide pharmaceutical raw materials of Himalayan Mirabilis jalapa Mirabilis in Tibet without limit, realizes industrialized anniversary production of Himalayan Mirabilis jalapa Mirabilis, realizes development and utilization of resources, is beneficial to reducing dependence on wild Himalayan Mirabilis jalapa Mirabilis resources, can solve the problem of development of Tibetan medicine industry in China, and has important theoretical significance and wide market application prospect.

Drawings

FIG. 1 is a Himalayan Mirabilis jalapa plant of the present invention;

FIG. 2 is a partial callus obtained in step (2) of example 1 of the present invention;

FIG. 3 is a diagram showing a part of the secondary callus obtained in step (3) of example 1 of the present invention;

FIG. 4 shows suspension cells obtained in step (4) of example 1 of the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The materials used in the invention are: MS medium (Phytotechnology Laboratories, Inc., USA); 6-BA (6-benzyladenine), NAA (naphthylacetic acid), TDZ (thidiazuron), 2,4-D (2, 4-dichlorophenoxyacetic acid), hydrolyzed casein, and coconut milk (Beijing Byeldi Biotech Co., Ltd.).

Example 1

(1) Cultivation of aseptic seedlings

Taking mature seeds of Mirabilis himalaica (plant morphology is shown in figure 1), sterilizing according to a conventional sterilization method combining 75% alcohol and 2% sodium hypochlorite, inoculating on MS culture medium, and culturing aseptic seedlings. The culture conditions are as follows: the culture medium is illuminated under 3000lx light for 16h and dark for 8h every day, the set temperature is 25 ℃, and the culture time is 25 days.

(2) Induction of callus

Cutting the leaves (seedling leaves) of the aseptic seedlings cultured in the step (1) into 0.5cm multiplied by 0.5cm, inoculating the cut leaves into an induction culture medium for callus induction culture, and obtaining primary callus. The formula of the culture medium is as follows: MS +1.5mg/L TDZ +2.0mg/L NAA. Energy substance: 30g/L of sucrose; support material: 6.5g/L of agar; pH value: pH5.8; solvent: the water in the induction culture medium is deionized water; the culture conditions are as follows: dark, setting the temperature at 25 ℃; culturing time: for 35 days.

After the explant is inoculated for 10 days, the thickening phenomenon of the cut of the leaf explant can be seen, and the visible yellow-green compact granular callus can appear in 35 days, which is shown in figure 2.

(3) Subculture of callus

And (3) carrying out subculture on the callus (primary callus) induced in the step (2) to obtain a secondary callus. The formula of the proliferation culture medium is as follows: MS +1.0mg/L TDZ +2.0mg/L NAA. Energy substance: 30g/L of sucrose; support material: 6.5g/L of agar; pH value: pH5.8; solvent: the water in the composition of the subculture medium is deionized water; the culture conditions are as follows: dark, 25 ℃ temperature, 25 days, cycle 15 times. The secondary callus was obtained, see FIG. 3.

(4) Suspension cell culture

And (4) inoculating 6g of the loose subculture callus proliferated in the step (3) into 100mL of liquid culture medium for multiple suspension subculture to obtain Himalayan Mirabilis jalapa suspension cells. The formula of the culture medium is as follows: MS +1.0mg/L TDZ +2.0mg/L NAA +200mg/L hydrolyzed casein. Energy substance: 30g/L of sucrose; pH value: pH5.8; solvent: the water in the suspension cell culture is deionized water; the culture conditions are as follows: in the dark, the rotation speed of the shaking table is 80r/min, the temperature is 25 ℃, the operation lasts 20 days, and the cycle is 10 times. The resulting suspension cells are shown in FIG. 4.

Example 2

Callus and suspension cell cultures were performed on Mirabilis himalaica in the same manner as in example 1, except that:

the culture condition of the aseptic seedlings is 2500lx of illumination, and the culture time is 30 days.

The callus induction culture medium comprises the following components: MS +3.0mg/L TDZ +4.0mg/L NAA, energy substance: 25g/L of sucrose; support material: 7.0g/L of agar; culturing time: for 30 days.

The subculture callus proliferation culture medium comprises the following components: MS +0.5mg/L TDZ +3.0mg/L NAA, energy substance: 25g/L of sucrose; support material: 7.0g/L of agar; culturing time: the circulation is carried out for 15 times in 20 days.

The suspension cell culture medium comprises the following components: MS +2.0mg/L TDZ +3.0mg/L NAA +300mg/L hydrolyzed casein, energy substance: 25g/L of sucrose; the culture conditions are as follows: the rotating speed of the shaking table is 120r/min, and the culture time is as follows: the circulation is 10 times for 15 days.

Example 3

the culture condition of the aseptic seedlings is 3500lx of illumination, and the culture time is 20 days.

The callus induction culture medium comprises the following components: MS +0.5mg/L TDZ +2.0mg/L NAA, culture time: for 40 days.

The subculture callus proliferation culture medium comprises the following components: MS +2.0mg/L TDZ +3.0mg/L NAA, culture time: the circulation is performed 20 times in 30 days.

The suspension cell culture medium comprises the following components: MS +0.5mg/L TDZ +1.5mg/L NAA +100mg/L hydrolyzed casein, culture time: cycle 5 times for 30 days.

Comparative example 1

Callus induction was performed on the sterile mirabilis jalapa himalayana seedling prepared in example 1 in the same manner as in example 1 to obtain primary callus, except that: the callus induction culture medium comprises the following components: MS +3.5mg/L TDZ +4.5mg/L NAA.

Comparative example 2

Callus induction was performed on the sterile mirabilis jalapa himalayana seedling prepared in example 1 in the same manner as in example 1 to obtain primary callus, except that: the callus induction culture medium comprises the following components: MS +1.5 mg/L6-BA +2.0mg/L NAA.

Comparative example 3

Callus induction was performed on the sterile mirabilis jalapa himalayana seedling prepared in example 1 in the same manner as in example 1 to obtain primary callus, except that: the callus induction culture medium comprises the following components: MS +1.5mg/L TDZ +2.0 mg/L2, 4-D.

Comparative example 4

The calli of Himalayan Mirabilis prepared in step (2) of example 1 (primary calli) were subcultured in the same manner as in example 1 except that: the formula of the proliferation culture medium is as follows: MS +2.5mg/L TDZ +3.5mg/L NAA.

Comparative example 5

The calli of Himalayan Mirabilis prepared in step (2) of example 1 (primary calli) were subcultured in the same manner as in example 1 except that: the formula of the proliferation culture medium is as follows: MS +1.0 mg/L6-BA +2.0mg/L NAA.

Comparative example 6

The calli of Himalayan Mirabilis prepared in step (2) of example 1 (primary calli) were subcultured in the same manner as in example 1 except that: the formula of the proliferation culture medium is as follows: MS +1.0mg/L TDZ +2.0 mg/L2, 4-D.

Comparative example 7

Using Himalayan Mirabilis jalapa subculture callus prepared in step (3) of example 1 as a starting material, suspension cell culture was performed in the same manner as in example 1 except that: the suspension cell culture medium comprises the following components: MS +2.5mg/L TDZ +3.5mg/L NAA +350mg/L hydrolyzed casein.

Comparative example 8

Using Himalayan Mirabilis jalapa subculture callus prepared in step (3) of example 1 as a starting material, suspension cell culture was performed in the same manner as in example 1 except that: the suspension cell culture medium comprises the following components: MS +1.0mg/L TDZ +2.0mg/L NAA +350mg/L hydrolyzed casein.

Comparative example 9

Using Himalayan Mirabilis jalapa subculture callus prepared in step (3) of example 1 as a starting material, suspension cell culture was performed in the same manner as in example 1 except that: the suspension cell culture medium comprises the following components: MS +1.0mg/L TDZ +2.0mg/L NAA +50mg/L hydrolyzed casein.

Comparative example 10

Using Himalayan Mirabilis jalapa subculture callus prepared in step (3) of example 1 as a starting material, suspension cell culture was performed in the same manner as in example 1 except that: the suspension cell culture medium comprises the following components: MS +2.5mg/L TDZ +2.0mg/L NAA +200mg/L hydrolyzed casein.

Comparative example 11

Using Himalayan Mirabilis jalapa subculture callus prepared in step (3) of example 1 as a starting material, suspension cell culture was performed in the same manner as in example 1 except that: the suspension cell culture medium comprises the following components: MS +1.0mg/L TDZ +3.5mg/L NAA +200mg/L hydrolyzed casein.

Comparative example 12

Using Himalayan Mirabilis jalapa subculture callus prepared in step (3) of example 1 as a starting material, suspension cell culture was performed in the same manner as in example 1 except that: the suspension cell culture medium comprises the following components: the suspension cell culture medium comprises the following components: MS +1.0mg/L TDZ +2.0mg/L NAA +200mg/L coconut milk.

Experimental example 1

The primary callus obtained in examples 1 to 3 of the present invention and comparative examples 1 to 3 was observed and counted, and the results are shown in Table 1.

TABLE 1

	Number of inoculations	The cure rate%	Browning rate%
				Example 1	30	96.67	6.67
Example 2	30	93.30	6.67
				Example 3	30	83.30	6.67
Comparative example 1	30	56.67	16.67
				Comparative example 2	30	26.70	46.67
Comparative example 3	30	20.00	56.67

In table 1, the recovery rate (%) is the number of blocks induced to form compact callus/inoculation number × 100%; browning rate (%) — number of browned callus pieces/number of inoculated pieces × 100%.

From the data in table 1, it can be seen that: the healing rate of examples 1-3 is much higher than that of comparative examples 1-3; meanwhile, the browning rates of comparative examples 2 and 3 were much higher than those of examples 1 to 3.

Therefore, the induction effect of the combination of TDZ and NAA on the primary callus is better than that of the combination of 6-BA and NAA and the combination of TDZ and 2,4-D, and when the dosage of TDZ and NAA is too high, the healing rate is reduced, the browning rate is increased, and the ideal induction efficiency cannot be obtained.

After the himalayas Mirabilis leaf (explant) is inoculated for 10-20 days, the thickening phenomenon of the cut of the leaf explant can be seen, and after 30-40 days, visible yellow-green compact granular callus can be seen, and the healing rate is high and the browning rate is low.

Experimental example 2

The secondary callus obtained in examples 1 to 3 of the present invention and comparative examples 4 to 6 was observed and counted, and the results are shown in Table 2.

TABLE 2

In table 2, differentiation rate (%) — number of differentiated blocks/number of inoculation × 100%; the browning rate (%) -% of the browned secondary callus pieces/number of inoculated pieces × 100%.

From the data in table 2, it can be seen that: the differentiation rate of the secondary callus of examples 1-3 was much lower than that of comparative examples 4-6; meanwhile, the browning rates of comparative examples 4 to 6 were much higher than those of examples 1 to 3.

Therefore, the combination of TDZ and NAA selected as hormones required by the callus proliferation of Mirabilis himalaica is superior to the combination of 6-BA and NAA and the combination of TDZ and 2,4-D, and when the dosage of TDZ and NAA is too high, the differentiation rate and the browning rate are increased, so that the ideal proliferation efficiency cannot be obtained.

After the Himalayan Mirabilis jalapa callus is subcultured for multiple times, the callus is continuously proliferated to finally form a pale yellow, semitransparent, soft and fragile callus (subcultured callus), and the differentiation rate and browning rate are low.

Experimental example 3

The results of observation and statistics of the suspension cells obtained in examples 1 to 3 of the present invention and comparative examples 7 to 12 are shown in Table 3:

TABLE 3

In table 3, the proliferation rate (%) (fresh weight after proliferation-fresh weight before proliferation)/fresh weight before proliferation × 100%, and the fresh weight was measured by removing the liquid medium from the cell suspension by vacuum filtration, washing with distilled water 3 times, and weighing the fresh mass after the removal of the water by suction filtration.

From the data in table 3, it can be seen that: the proliferation rate of suspension cells of examples 1-3 was much higher than that of comparative examples 7-12. When the amount of TDZ and/or NAA is too high (see comparative examples 7, 10 and 11), the suspension cells are slowly proliferated and are not suitable for the culture of Himalayan Mirabilis jalapa suspension cells. Too high or too low amounts of hydrolysed casein (see comparative examples 8-9) will slow the proliferation of suspension cells. The hydrolyzed casein had better growth and reproduction effects on Mirabilis himalaica suspension cells than coconut milk (see comparative example 12).

The Himalayan Mirabilis jalapa suspension cells obtained by the method have good dispersibility and high proliferation rate.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A Himalayan Mirabilis jalapa callus induction proliferation method is characterized by comprising the following steps:

wherein the hormones in the induction medium and the proliferation medium are both TDZ and NAA;

the induction culture medium is as follows: MS +0.5-3.0mg/L TDZ +0.5-4.0mg/L NAA;

the proliferation culture medium is as follows: MS +0.5-2.0mg/L TDZ +1.0-3.0mg/L NAA.

2. The method of claim 1, comprising:

(1) culturing sterile Mirabilis himalaica;

3. The method as claimed in claim 2, wherein the sterile plantlet is cultured by the following method: culturing Himalayan Mirabilis jalapa seeds in MS culture medium at 25 + -2 deg.C under illumination intensity of 2500-.

4. The method of any one of claims 1-3, wherein the induction medium and the multiplication medium further comprise 25-30g/L sucrose, 6.5-7.0g/L agar; and/or the pH value of the induction culture medium and the proliferation culture medium is 5.8-6.0.

5. A Himalayan Mirabilis jalapa suspension cell propagation method is characterized by comprising the following steps: suspension culture of Mirabilis himalaica callus obtained by the method of any one of claims 1-4 in a liquid medium comprising TDZ, NAA and hydrolyzed casein.

6. The method according to claim 5, wherein the number of suspension cultures is 5-10 times, each time for 15-30 days.

7. The method as claimed in claim 5 or 6, wherein the liquid medium is MS +0.5-2.0mg/L TDZ +1.5-3.0mg/L NAA +100-300mg/L hydrolyzed casein.

8. The method of claim 7, wherein the liquid medium further comprises 25-30g/L sucrose; and/or the pH value of the liquid culture medium is 5.8-6.0.