CN109792955B - Preparation method of rapidly-growing grosvenor momordica embryonic callus - Google Patents

Abstract

The invention discloses a preparation method of fast-growing Luo Han Guo embryogenic callus, comprising: preparation of stable callus: inducing seeds, leaves or stem segments of Luo Han Guo to obtain stable callus; new callus Preparation: culture the stable callus for 70-80 days, and inoculate the browned cell mass into the induction medium for subculture to obtain new callus; preparation of suspended embryogenic callus: selective growth The rapid, yellowish-white new callus was subcultured in a new medium to obtain suspendable new callus. The invention conducts induction and subculture on the basis of stable callus, shortens the growth delay period and suspension culture period of callus, and improves the growth of Luo Han Guo seedlings.

Description

A kind of preparation method of fast-growing Luo Han Guo embryogenic callus

technical field

The invention relates to the technical field of tissue culture, in particular to a preparation method of rapidly growing Luo Han Guo embryogenic callus.

Background technique

Luo Han Guo is a perennial vine of the Cucurbitaceae family. The main sweet component of Luo Han Guo fruit is Mogroside V, which is 300 times sweeter than sucrose, and its sweetness curve is similar to that of sucrose. It is widely loved because of its high sweetness, low calorie, and good taste. It is the most suitable sweetener and health care product for special groups such as obesity, hypertension and diabetes patients, and has a wide range of application values.

Mogroside V is mainly extracted from natural Luo Han Guo fruit. However, the inherent mogrosides in natural monk fruit are extremely low, the utilization rate of the prior art for the monk fruit and the extraction rate of the mogrosides are not high, resulting in a great waste of raw materials. At the same time, due to the influence of natural factors such as land resources and climate, the output of natural Luo Han Guo is limited and cannot meet the growing market demand. At present, most of the cultivation of Luo Han Guo adopts the method of tissue culture, and the stable callus is cultivated, but the stable callus material grows slowly, and the preparation of inoculated cells for suspension culture requires a large workload. Long and low efficiency, the output of Luo Han Guo still cannot meet the market demand.

Therefore, how to provide a kind of Momordica grosvenori embryogenic callus with rapid growth and short cultivation period is an urgent problem to be solved by those skilled in the art.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a preparation method of fast-growing Luo Han Guo embryogenic callus, which can induce and subculture on the basis of stable callus, shorten the growth delay period of callus, and shorten the period of time. The suspension culture cycle improves the growth of Luo Han Guo seedlings.

In order to achieve the above object, the present invention adopts the following technical solutions:

A preparation method of fast-growing Luo Han Guo embryogenic callus, comprising the following steps:

1) Preparation of stable callus: inducing seeds, leaves or stem segments of Luo Han Guo to obtain stable callus;

2) Preparation of new callus: the stable callus obtained in the step 1) is cultured for 70-80 days, and the browned cell mass is inoculated into an induction medium for subculture to obtain a new callus organize;

3) Preparation of suspendable embryogenic callus: select the fast-growing, yellowish-white new callus in step 2) to subculture into a new medium for subculture to obtain suspended new callus.

The technical effects achieved by the above technical solutions are as follows: the present invention discloses a method for obtaining the embryogenic callus of Luo Han Guo which can be suspended cultured and rapidly grown. Compared with the primary callus, the obtained neonatal callus grows faster, a large amount of suspension cell inoculation material is easy to prepare, the culture period is shortened from 21 days to 15 days, and the growth lag period is short, and the growth amount is also greatly increased. It also grows well with a small inoculum. When the same inoculum amount was 50g/L (fresh weight), after one cycle of culture, the cell dry weight of primary callus was 6.73g/L, and the dry weight of new callus could reach 15.77g/L. At the same time, the yield of mogroside V after the induction agent was comparable to that of primary callus.

Optionally, the specific process of induction in the step 1) includes: (1) preparation of primary callus: after sterilizing the Luo Han Guo seeds, leaves or stem segments, insert into a primary induction culture with a pH of 5-7 In the base, the temperature is 20-30 ℃ and no light conditions are induced and cultured for 15-21 days to obtain primary callus; (2) Preparation of stable callus: the primary callus obtained in the step (1) The callus was subcultured to obtain callus with stable characters.

Optionally, the induction medium in step 2) includes: choline 0.5-2.0g/L, vitamin C 0.5-3.0mg/L, sucrose 25-35g/L, agar 4-5g/L, muscle B5 solid medium with alcohol 80-120mg/L, 6-BA 0.5-2mg/L and NAA 0.5-1.5mg/L.

The technical effects achieved by the above technical solutions are: choline is a positively charged tetravalent base, which has beneficial effects on important physiological functions such as plant growth, yield, product quality, photosynthetic performance, membrane permeability, and plant resistance. Supplying choline to plants can improve their frost tolerance and lower the membrane phase transition temperature. At the same time, choline not only provides choline groups for membrane lipid components, but also has diverse biological effects on plants like phytohormones. It needs to be in a certain effective concentration range (0.5-2.0g/L) to exert its optimal regulatory effect. ;Vitamin C is an antioxidant that can help plants resist the stress caused by drought, ozone and ultraviolet rays. At the same time, as a relatively active substance, vitamin C can play a certain role in the regulation of plants. In the process of adversity and aging, vitamin C can help plants play a protective and delaying role.

Optionally, the conditions for the subculture in step 3) are: a temperature of 25-30° C., a light intensity of 4000 lux, and an illumination time of 12 h/d.

Optionally, the primary induction medium is composed of B5 basal saline solution medium, sucrose 25-35g/L, agar 4-5g/L, inositol 80-120mg/L, 6-BA 0.5-2mg/L and NAA. 0.5-1.5mg/L composition

Optionally, the B5 basal saline solution medium is composed of KNO ₃ 2500 mg/L, MgSO ₄ ·7H ₂ O 250 mg/L, CaCl ₂ ·2H ₂ O 150 mg/L, (NH ₄ ) ₂ SO ₄ 134 mg/L, NaH ₂ PO ₄ .H ₂ O 150 mg/L, KI 0.75 mg/L, H ₃ BO ₃ 3.0 mg/L, MnSO ₄ .4H ₂ O 10 mg/L, ZnSO ₄ .7H ₂ O 2.0 mg/L, Na ₂ MoO ₄ 2H ₂ O 0.25mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, Na ₂ -EDTA 37.3mg/L, FeSO ₄ 7H ₂ O 27.8mg/ L, niacin 1.0mg/L, pyridoxine hydrochloride 1.0mg/L and sulphur ammonium hydrochloride 10mg/L

As can be seen from the above-mentioned technical solutions, compared with the prior art, the present invention discloses and provides a preparation method for rapidly growing Luo Han Guo embryogenic callus, and the technical effect achieved is:

1) On the basis of primary callus, continue to induce it, the obtained suspendable embryogenic callus grows faster than primary callus, and a large number of suspended cell inoculation materials are easy to prepare, and the culture period is shortened from 21 days to 15 days, and the growth lag period is short, and the growth volume is also greatly increased.

2) It can also grow well with a small amount of inoculum. When the same inoculation amount was 50g/L (fresh weight), after one cycle of culture, the cell dry weight of the original callus was 6.73g/L, and the cell dry weight of the new callus could reach 15.77g/L.

3) Choline and vitamin C are added to the induction medium to improve the stress resistance of plants.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the provided drawings without creative work.

Fig. 1 accompanying drawing is a schematic diagram of the stable callus obtained by seed induction in Example 3 of the present invention;

Fig. 2 accompanying drawing is the schematic diagram of the browning cell mass of stable callus in Example 3 of the present invention;

Fig. 3 accompanying drawing is the schematic diagram of the new-born callus of embodiment 3 of the present invention;

Figure 4 is a schematic diagram of the suspended embryogenic callus in Example 3 of the present invention;

Figure 5 is a schematic diagram of the suspended embryogenic callus in Example 4 of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

The present embodiment is used for judging the optimal concentration ratio of choline and vitamin C, and specifically discloses a preparation method of fast-growing Luo Han Guo embryogenic callus, comprising the following steps:

1) Preparation of primary callus: after sterilizing the Luo Han Guo seeds, the seeds were immersed in a medium with a pH of 6, consisting of MS basal saline solution medium, 30 g/L of sucrose, 4.6 g/L of agar, 100 mg/L of inositol, 6- In an induction medium consisting of BA 1.0 mg/L and NAA 0.5 mg/L, the primary callus was obtained by inducing and culturing for 15 days under the condition of a temperature of 27 °C and no light;

2) Preparation of stable callus: subculture the primary callus obtained in the step 1) to obtain callus with stable characters;

3) Preparation of new-born callus: the stable callus obtained in the step 2) was cultured for 75 days, and the browned cell mass was inoculated to contain a. choline 0 g/L and vitamin C 0 mg/L respectively; b. Choline 0.5g/L, Vitamin C 0mg/L; c. Choline 1g/L, Vitamin C 0mg/L; d. Choline 2g/L, Vitamin C 0mg/L; e. Choline 0g/L, Vitamin C0 .5mg/L; f. choline 0.5g/L, vitamin C 0.5mg/L; g. choline 1g/L, vitamin C 0.5mg/L; h. choline 2g/L, vitamin C 0.5mg/L ;icholine 0g/L, vitamin C1mg/L; j.choline 0.5g/L, vitamin C1mg/L; k.choline 1g/L, vitamin C1mg/L; l.choline 2g/L, vitamin C1mg /L; m. choline 0g/L, vitamin C 2mg/L; n. choline 0.5g/L, vitamin C 2mg/L; o. choline 1g/L, vitamin C 2mg/L; p. choline 2g/L , Vitamin C. 2mg/L; q. Choline 0g/L, Vitamin C 3mg/L; r. Choline 0.5g/L, Vitamin C 3mg/L; s. Choline 1g/L, Vitamin C 3mg/L; t. Choline 2g/L, vitamin C. 3mg/L; other components are B5 solid with sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA 1.0mg/LNAA 0.5mg/L Subculture in medium to obtain new callus.

4) Preparation of suspendable embryogenic callus: select the fast-growing, yellowish-white new callus in step 3) to subculture into a new medium, at a temperature of 27° C., a light intensity of 4000 lux, and a light of 4000 lux. Subculture was carried out under the condition of 12h/d to obtain suspendable new callus.

The induction rate of the obtained neonatal callus and suspended embryogenic tissue is shown in Table 1:

Induction rate of new callus = number of new callus/browning callus × 100%

Induction rate of suspendable embryogenic callus = number of suspendable embryogenic callus clusters/number of new-born callus clusters × 100%

Table I

Example 2

This example is used to compare the technical effect difference between the suspension culture of the stable callus induced by seeds, leaves and stem segments and the suspension culture of the suspended embryogenic callus obtained by the technical scheme of the present invention. The specific steps are as follows. :

1) respectively sterilize Luo Han Guo seeds, leaves and stem sections, and dehull the seeds, and take seed embryos; insert seed embryos, sterilized leaves and sterilized stem sections into an induction medium with a pH value of 6, respectively, at a temperature of 6. Induction culture for 15 days at 20-30°C and no light conditions to obtain primary callus, wherein the induction medium consists of MS basal saline solution medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg /L, 6-BA 1.0mg/L, NAA 1mg/L composition.

2) Take the three kinds of primary callus obtained in step 1), and in a mixture containing sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA 1.0/L, NAA 0.5mg/L, pH is 6.0 B5 solid medium was subcultured, and three kinds of stable callus were selected which were subcultured for 4 consecutive times and had vigorous growth, loose texture and stable and uniform state.

3) Take the three kinds of stable callus obtained in step 2) and culture them for 75 days respectively and inoculate them to the cells containing 0.5 mg/L of vitamin C, 1 g/L of choline, 30/L of sucrose, 4.6 g/L of agar, and 100 mg of inositol. /L, 6-BA 1.0 mg/L, NAA 0.5 mg/L, and subculture on B5 solid medium with pH 6.

4) In the three media in step 3), the fast-growing, yellowish-white new callus on the 6th to 10th day was selected to be subcultured to a new medium (the same as the medium in step 1), and successively subcultured for 4 Second, the loose texture, vigorous and stable pale yellow suspended embryogenic callus were obtained.

5) The stable callus and suspendable embryogenic callus obtained in step 2) and step 4) were transferred to two groups containing sucrose 30g/L, inositol 100mg/L, 6 -BA 1.0/L, NAA 0.5mg/L, pH range of 5.5-6.0 in liquid medium, and in a rotating speed of 150r/min, culture temperature of 27 ℃, light intensity of 4000lux, light time of 12h/d Cultivated in a shaker to obtain 4 kinds of primary Luo Han Guo suspension cell systems.

6) Mature Monk fruit suspension cells were cultured with the four primary-generation Luo Han Guo suspension cell systems obtained in step 5), and 120 μmol/L of methyl jasmonate was added on the 6th day in the culture process.

7) On the 0-24th day of culture, extract the suspension cells of Luo Han Guo every other day, transfer the suspension cells of Luo Han Guo to a suction filter bottle containing an 8 μm filter membrane for suction filtration, rinse the cells three times with sterile water during the period, and suction filter to filter paper. until no more water drips. The weight at this time is the fresh weight (FCW) of the callus; the cells after suction filtration are dried in an oven at 60°C to a constant weight, and the weight at this time is the dry weight (DCW) of the callus, and the dry weight content is specific See Table 2:

Table II

It can be seen from Table 2 that when the stable callus induced by stem, leaf and seed is in suspension culture, the cell mass reaches the maximum on the 21st day; the cell dry weight of the suspended embryogenic callus reaches the maximum on the 15th day, The cultivation time is shortened. In addition, the growth index of suspended embryogenic callus was 0.8778 g/L.d, which was higher than that of stem segment-stable callus 0.1849 g/L.d, leaf-stable callus 0.2270 g/L.d, and seed-stable callus 0.2270 g/L.d. The 0.1914g/L·d was increased by 4.7, 3.8, and 4.6 times, respectively.

8) Weigh 1 g of dry weight cells of Luo Han Guo, add methanol at a ratio of 10:1, add methanol for ultrasonic extraction for 1 hour, and extract by shaking at 60°C for 2 hours. After repeated extraction for 3 times, the combined extract is concentrated under a nitrogen blower. to 1ml. The concentrated solution was filtered through a 0.22 μm filter membrane, and qualitative and quantitative analysis was carried out by high performance liquid chromatography.

Chromatographic conditions: the chromatographic column is a C18 reverse column, 4.6 mm×250 mm; the mobile phase is water-acetonitrile=78-22, the flow rate is 1.0 ml/min, and the detection wavelength is 203 nm.

The effects of different sources of Luohanguo callus on the synthesis of mogroside V in suspension culture are shown in Table 3.

table 3

In the process of suspension culture of callus cells from different sources, the content of mogroside V was extremely low when no inducer was added. After adding the inducer, the content of mogroside V was significantly increased. It can be seen from Table 3 that the mogroside V content of the new callus can be significantly increased by the inducer treatment. Combined with cell growth, the suspension culture period of callus induced by stem, leaves and seeds can be taken as 21 days, and the suspension culture period of new callus is 15 days, and the fermentation indices are 0.0864, 0.1146, 0.1342, and 0.3445 mg, respectively. /L·d. The fermentation index of the new callus was 4 times, 3 times and 2.5 times higher than that of the callus cells induced by stems, leaves and seeds, respectively.

Example 3

The present embodiment discloses a preparation method of rapidly growing Luo Han Guo embryogenic callus, comprising:

1) Preparation of primary callus: after sterilizing the Luo Han Guo seeds, the seeds were immersed in pH 5, B5 basal saline solution medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6- In the induction medium consisting of BA 1.0 mg/L and NAA 0.5 mg/L, the temperature is 20 ° C and no light conditions are induced and cultured for 15 days, that is, primary callus is obtained to obtain primary callus;

2) Preparation of stable callus: the primary callus obtained in the step 1) is subcultured to obtain callus with stable characters, and the specific results are shown in Figure 1;

3) Preparation of new-born callus: The stable callus obtained in the step 2) was cultured for 75 days, and the browned cell mass (see Figure 2) was inoculated into cells containing choline 0.5g/L, Subculture in B5 solid medium of vitamin C 1.0mg/L, sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA 1.0mg/L, NAA 0.5mg/L, and obtain new life callus; see Figure 3.

4) Preparation of suspendable embryogenic callus: select the fast-growing, yellowish-white new callus in step 3) to subculture into a new medium, at a temperature of 25° C., a light intensity of 4000 lux, and a light of 4000 lux. Subculture was carried out under the condition of 12h/d to obtain suspended embryogenic callus, as shown in Figure 4.

Example 4

The present embodiment discloses a preparation method of rapidly growing Luo Han Guo embryogenic callus, comprising:

1) Preparation of primary callus: after sterilizing the Luo Han Guo seeds, the seeds were inserted into a medium with a pH of 5.5, a medium consisting of a B5 basal saline solution, 30 g/L of sucrose, 4.6 g/L of agar, 100 mg/L of inositol, 6- In the induction medium composed of BA 1.0 mg/L and NAA 0.5 mg/L, the temperature is 30 ° C and no light conditions are induced and cultured for 15 days, that is, primary callus is obtained to obtain primary callus;

2) Preparation of stable callus: subculture the primary callus obtained in the step 1) to obtain callus with stable characters;

3) Preparation of new callus: the stable callus obtained in the step 2) was cultured for 75 days, and the browned cell mass was inoculated into cells containing 0.5 g/L of choline and 1.0 mg/L of vitamin C, respectively. , sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA 1.0mg/LNAA, 0.5mg/L B5 solid medium for subculture to obtain new callus;

4) Preparation of suspendable embryogenic callus: select the fast-growing, yellowish-white new callus in step 3) to subculture into a new medium, at a temperature of 30° C., a light intensity of 4000 lux, and a light of 4000 lux. Subculture was carried out under the condition of 12 h/d to obtain suspended embryogenic callus, see Fig. 5 .

Example 5

This example is used to compare the induction effect of the stable callus induced by seeds, leaves and stem segments respectively, and the fresh callus without browning for 15-20 days and the browning callus which are cultured for 70-80 days . The technical effect of suspension culture of the suspended embryogenic callus obtained by the technical solution of the present invention is different, and the specific steps are as follows:

1) respectively sterilize Luo Han Guo seeds, leaves and stem sections, and dehull the seeds, and take seed embryos; insert seed embryos, sterilized leaves and sterilized stem sections into an induction medium with a pH value of 6, respectively, at a temperature of 6. Induction culture for 15 days at 20-30°C and no light conditions to obtain primary callus, wherein the induction medium consists of B5 basal saline solution medium, sucrose 30g/L, agar 4.6g/L, inositol 100mg /L, 6-BA 1.0mg/L, NAA 1mg/L composition.

2) Take the three kinds of primary callus obtained in step 1), and in a mixture containing sucrose 30g/L, agar 4.6g/L, inositol 100mg/L, 6-BA 1.0/L, NAA 0.5mg/L, pH is 6.0 B5 solid medium was subcultured, and three kinds of stable callus were selected which were subcultured for 4 consecutive times and had vigorous growth, loose texture and stable and uniform state.

3) Take the three kinds of stable callus obtained in step 2) and culture them for 75 days respectively and inoculate them to the cells containing 0.5 mg/L of vitamin C, 1 g/L of choline, 30/L of sucrose, 4.6 g/L of agar, and 100 mg of inositol. /L, 6-BA 1.0 mg/L, NAA 0.5 mg/L, and subculture on B5 solid medium with pH 6.

4) In the three media in step 3), the fast-growing, yellowish-white new callus on the 6th to 10th day was selected to be subcultured to a new medium (the same as the medium in step 1), and successively subcultured for 4 Second, the loose texture, vigorous and stable pale yellow suspended embryogenic callus were obtained.

5) Take the three kinds of stable callus obtained in step 2) and culture them for 15 days respectively, and inoculate them with vitamin C 0.5 mg/L, choline 1 g/L, sucrose 30/L, agar 4.6 g/L, inositol 100 mg /L, 6-BA 1.0 mg/L, NAA 0.5 mg/L, and subculture on B5 solid medium with pH 6.

6) respectively select the fresh callus of the 6th-10th day in the three culture media in step 5) to be subcultured into a new culture medium (same as the culture medium in step 1), and successively pass 4 times to obtain loose texture, Vigorous and stable pale yellow suspended embryogenic callus.

7) The stable callus and suspendable embryogenic callus obtained in step 2), step 4) and step 6) were respectively transferred to two plants containing sucrose 30g/L and inositol 100mg at an inoculum amount of 50g/L. /L, 6-BA1.0/L, NAA 0.5mg/L, pH range of 5.5-6.0 B5 liquid medium, and in the speed of 150r/min, culture temperature of 27 ℃, light intensity of 4000lux, light Four kinds of first-generation Luo Han Guo suspension cell systems were obtained by culturing in a shaker with a time of 12 h/d. The dry weight and the content of glycoside V were measured in Table 4 after culturing for 24 days.

Table 4

It can be seen that the new induction medium only has a significant induction effect on callus cultured to browning, but has no obvious effect on the growth of fresh callus and the synthesis of glycoside V.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the various embodiments can be referred to each other. As for the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant part can be referred to the description of the method.

The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (5)

1. a preparation method of fast-growing Luo Han Guo embryogenic callus, is characterized in that, comprises the following steps: 1) preparation of stable callus: the seed, leaf or stem section of Luo Han Guo are induced, obtain stable callus. injured tissue; 2) Preparation of new callus: culture the stable callus obtained in the step 1) for 70-80 days, and inoculate the browned cell mass into the induction medium for subculture to obtain new callus Tissue; induction medium: containing choline 0.5-2.0g/L, vitamin C 0.5-2.0mg/L, sucrose 25-35g/L, agar 4-5g/L, inositol 80-120mg/L, 6 -B5 solid medium with BA0.5-2mg/L and NAA 0.5-1.5mg/L; 3) Preparation of suspendable embryogenic callus: select the fast-growing, yellowish-white new callus in the step 2) to subculture into a new medium for subculture to obtain a suspended new callus. 2. The preparation method of a fast-growing Luo Han Guo embryogenic callus according to claim 1, wherein the specific process induced in the step 1) comprises: (1) Preparation of primary callus: After sterilizing the seeds, leaves or stem segments of Luo Han Guo, they are placed in a primary induction medium with a pH of 5-7, and the temperature is 20-30° C. and no light conditions. Induction culture for 15-21 days to obtain primary callus; (2) Preparation of stable callus: subculture the primary callus obtained in the step (1) to obtain callus with stable characters. 3. The preparation method of a fast-growing Luo Han Guo embryogenic callus according to claim 1, characterized in that, the conditions of said step 3) subculture are as follows: the temperature is 25-30° C., the light intensity is It is 4000lux and the illumination time is 12 h/d. 4. the preparation method of a kind of fast-growing Luo Han Guo embryogenic callus according to claim 2, is characterized in that, described primary induction medium consists of B5 basal saline solution medium, sucrose 25-35g/L, agar 4-5g/L, inositol 80-120mg/L, 6-BA 0.5-2mg/L and NAA 0.5-1.5mg/L. 5. the preparation method of a kind of fast-growing Luo Han Guo embryogenic callus according to claim 4, is characterized in that, described B5 basal saline solution medium consists of KNO ₃ 2500mg/L, MgSO ₄ 7H ₂ O 250mg /L, CaCl ₂ ·2H ₂ O 150mg/L, (NH ₄ ) ₂ SO ₄ 134mg/L, NaH ₂ PO ₄ .H ₂ O 150mg/L, KI 0.75mg/L, H ₃ BO ₃ 3.0mg/L, MnSO ₄ ·4H ₂ O 10 mg/L, ZnSO ₄ ·7H ₂ O 2.0 mg/L, Na ₂ MoO ₄ ·2H ₂ O 0.25 mg/L, CoCl ₂ ·6H ₂ O 0.025 mg/L, CuSO ₄ ·5H ₂ O 0.025mg/L, Na ₂ -EDTA 37.3mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, niacin 1.0mg/L, pyridoxine hydrochloride 1.0mg/L and thiammonium hydrochloride 10mg/L .

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