A hormone-free method for the proliferation of Dendrobium protocorm stem cells

Abstract

This invention relates to a hormone-free proliferation method for Dendrobium protocorm stem cells. The method uses Dendrobium protocorm stem cells obtained through induction from Dendrobium seeds or stems as the culture medium. Aseptic plant tissue culture technology is employed, and the Dendrobium protocorm stem cells are inoculated into a hormone-free proliferation medium under sterile conditions. The cells are cultured for 30–45 days at a temperature of 25±2℃, a total light intensity of 2000–2500 Lx under white light plus monochromatic red light, and a photoperiod of 10–12 h / d. During the culture process, the differentiation rate of Dendrobium protocorm stem cells is less than 1%, and the proliferation rate is 7–9 times the inoculum size. This invention is applicable to the proliferation of Dendrobium huoshanense protocorm stem cells as well as Dendrobium officinale protocorm stem cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a hormone-free proliferation method of dendrobium protocorm stem cells. BACKGROUND

[0002] The dendrobium protocorm is a kind of embryonic tissue obtained by taking the seeds or stems of dendrobium as explants through plant tissue culture. The dendrobium protocorm cell has the ability of replication and proliferation and the ability of differentiation into a complete plant, has the characteristics of stem cells, and is a kind of plant stem cell.

[0003] On February 29, 2024, the National Health Commission issued a notice that the dendrobium protocorms derived from dendrobium huoshanense and dendrobium officinale were included in new food raw materials. The food safety index of the dendrobium protocorm requires that the content of plant growth regulators in the dry product is less than or equal to 0.05 mg / kg. However, in the existing tissue culture technology of the dendrobium protocorm, exogenous plant hormones, mainly auxins and cytokinins, need to be added to the culture medium to regulate the proliferation or differentiation of the dendrobium protocorm cells by adjusting the addition ratio of auxins and cytokinins. Exogenous plant hormones belong to plant growth regulators. Adding exogenous plant hormones to the culture medium will increase the content of plant growth regulators in the dendrobium protocorm, which cannot meet the limited requirements of the food safety index of the dendrobium protocorm on plant growth regulators.

[0004] Since the dendrobium protocorm cell is a kind of plant stem cell, if it is not regulated during the culture process, it will grow in both the proliferation and differentiation directions. Differentiation will reduce the proliferation speed of the dendrobium protocorm stem cells, reduce the yield and increase the cost.

[0005] In the technology of plant cell tissue culture, the composition of the culture medium and the light quality are two main factors affecting the growth, proliferation and differentiation of plant cells. The composition of the culture medium plays a key supporting role in the growth of tissue-cultured plant cells. The composition of the culture medium not only provides essential nutrients for the growth of plant cells, but also plays a regulating role in the proliferation or differentiation of plant cells. The light quality also plays an extremely important role in the proliferation and differentiation of tissue-cultured plant cells. Different light qualities have different effects on different plant cells. The possible reason is that the chromophore and apo-protein of the photosensitive pigment molecules in plant cells are diverse, which makes the photosensitive pigment exist in different active states in plant cells, resulting in different responses of different plant cells to light quality in inducing proliferation. The present application combines the factors of the composition of the culture medium and the light quality to provide a hormone-free rapid proliferation method of the dendrobium protocorm stem cells suitable for industrial production. SUMMARY

[0006] The present application aims to solve the following problems existing in the existing Dendrobium protocorm stem cell proliferation culture technology: if exogenous hormones are not used for regulation during the culture process, the Dendrobium protocorm stem cells will grow in both proliferation and differentiation directions, differentiation will reduce the proliferation speed of the Dendrobium protocorm stem cells, reduce the yield and increase the cost; if exogenous plant hormones are added in the culture medium, the content of the plant growth regulators in the Dendrobium protocorm will increase, which cannot meet the limited requirement of the plant growth regulators in the food safety index of the Dendrobium protocorm.

[0007] The present application increases the non-hormone components with the effect of promoting the division and proliferation of plant cells by optimizing the composition of the culture medium, and adjusts the light quality composition of the light source during the culture process, so as to promote the proliferation of the Dendrobium protocorm stem cells and inhibit the differentiation of the Dendrobium protocorm stem cells, effectively improve the proliferation speed of the Dendrobium protocorm stem cells; no exogenous plant hormones are used during the culture process, which will not cause the increase of the content of the plant growth regulators in the Dendrobium protocorm, and provides a new way for the large-scale rapid propagation of the Dendrobium protocorm stem cell new food raw material.

[0008] The specific operation steps of the Dendrobium protocorm stem cell hormone-free proliferation method are as follows:

[0009] (1) the Dendrobium protocorm stem cells obtained by inducing the Dendrobium seeds or stems are used as the culture object, which are inoculated into the hormone-free proliferation culture medium under the sterile condition, and are cultured for 30-45 days under the conditions of temperature 25±2℃, total light intensity 2000-2500Lx, and light time 10-12h / d;

[0010] (2) the hormone-free proliferation culture medium formula is: MS culture medium+50-100g / L honey+60-90mg / L histidine+1-5mg / L vitamin B 12 +5-6g / L agar, pH 5.8-6.0;

[0011] (3) the light conditions during the culture process are as follows: the light uses white light+monochromatic red light, wherein the white light intensity is 1500-2000Lx, the monochromatic red light intensity with the wavelength of 630-730nm is 500-1000Lx, the total light intensity is 2000-2500Lx, the white light source is a white light LED lamp, and the red light source is a monochromatic red light LED lamp;

[0012] (4) the Dendrobium protocorm stem cells can be the Dendrobium huoshanense protocorm stem cells or the Dendrobium candidum protocorm stem cells.

[0013] The beneficial technical effects of the present application are embodied in the following aspects:

[0014] (1) the present application increases the honey, histidine and vitamin B 12The monosaccharide, disaccharide and other components in the honey can provide carbon source for the proliferation of the dendrobium protocorm stem cells, and the oligosaccharide, small molecule organic compound and other components in the honey can promote the proliferation of the dendrobium protocorm stem cells; the histidine can promote the synthesis of cell division factor of the plant cells, and can promote the division and proliferation of the dendrobium protocorm stem cells; the vitamin B 12 also has the effect of promoting the proliferation of the plant cells. The honey is a natural food, the histidine and the vitamin B 12 are nutrients, and the honey, the histidine and the vitamin B 12 are all non-plant hormone components, and cannot cause the increase of the content of the plant growth regulator in the dendrobium protocorm.

[0015] (2) The present application adds the honey, the histidine and the vitamin B 12 and other non-plant hormone components in the culture medium, and simultaneously enhances the light intensity of the red light in the culture process, effectively promotes the proliferation of the dendrobium protocorm stem cells, and after the culture for 30-45 days, the proliferation amount of the dendrobium protocorm stem cells can reach 7-9 times of the inoculation amount, effectively inhibits the differentiation of the dendrobium protocorm stem cells, and the differentiation rate of the dendrobium protocorm stem cells is less than 1% in the culture process. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The test result graph of the dendrobium protocorm stem cell proliferation. DETAILED DESCRIPTION

[0017] The present application will be further described below in combination with specific examples. The features and advantages of the present application will be more apparent with the description, but these exemplary examples are only used to illustrate the present application, and do not constitute any limitation on the scope of the present application.

[0018] Example 1

[0019] (1) The dendrobium protocorm stem cells obtained by inducing the seeds of Dendrobium huoshanense are taken as the culture object, and under the sterile condition, the same inoculation amount is respectively transferred into the same amount of the control group culture medium and the hormone-free proliferation culture group culture medium;

[0020] (2) The formula and culture condition of the control group culture medium are as follows: MS culture medium+30-50 g / L sucrose+5-6 g / L agar, the pH of the culture medium is 5.8-6.0; under the condition of temperature 25±2℃, total light intensity 2000-2500 Lx, light time 10-12 h / d, the light quality is white light, and the white light source is white light LED lamp;

[0021] (3) The formula and culture condition of the hormone-free proliferation culture group culture medium are as follows: MS culture medium+50-100 g / L honey+60-90 mg / L histidine+1-5 mg / L vitamin B 12+ 5-6 g / L agar, the pH of the culture medium is 5.8-6.0; under the conditions of temperature 25±2℃, total light intensity 2000-2500 Lx, light time 10-12 h / d, the culture is carried out for 45 d, wherein the light quality is white light + monochromatic red light, the white light intensity is 1500-2000 Lx, the monochromatic red light intensity with wavelength 630-730 nm is 500-1000 Lx, the white light source is white light LED lamp, and the red light source is monochromatic red LED lamp;

[0022] (4) The results are shown in Table 1 and the accompanying Figure 1 Figures, after 45 d of culture, the average proliferation amount of the protocorma of D. candidum in the control group is 3.69 times of the inoculation amount, and the average differentiation rate is 33.17%; the average proliferation amount of the protocorma of D. candidum in the hormone-free proliferation culture group is 7.92 times of the inoculation amount, and the average differentiation rate is 0.33%.

[0023] Table 1 Proliferation amount and differentiation rate of protocorma of D. candidum in different culture groups

[0024]

[0025] Note: * indicates significant difference from the control group, p<0.05.

[0026] Example 2

[0027] (1) The protocorma of D. candidum obtained by induction from the stems of D. candidum is taken as the culture object, and under sterile conditions, the same inoculation amount is respectively transferred into the same amount of culture medium in the control group and the hormone-free proliferation culture medium;

[0028] (2) The formula and culture conditions of the culture medium in the control group are as follows: MS culture medium + 30-50 g / L sucrose + 5-6 g / L agar, the pH of the culture medium is 5.8-6.0; under the conditions of temperature 25±2℃, total light intensity 2000-2500 Lx, light time 10-12 h / d, the culture is carried out for 45 d, wherein the light quality is white light, and the white light source is white light LED lamp;

[0029] (3) The formula and culture conditions of the culture medium in the hormone-free proliferation culture group are as follows: MS culture medium + 50-100 g / L honey + 60-90 mg / L histidine + 1-5 mg / L vitamin B 12 + 5-6 g / L agar, the pH of the culture medium is 5.8-6.0; under the conditions of temperature 25±2℃, total light intensity 2000-2500 Lx, light time 10-12 h / d, the culture is carried out for 45 d, wherein the light quality is white light + monochromatic red light, the white light intensity is 1500-2000 Lx, the monochromatic red light intensity with wavelength 630-730 nm is 500-1000 Lx, the white light source is white light LED lamp, and the red light source is monochromatic red LED lamp;

[0030] (4) Results are shown in Table 2 and Figure 2. Figure 1 As shown in Table 2 and Figure 2, the average proliferation amount of the protocorm stem cells of D. candidum in the control group was 4.19 times of the inoculation amount, and the average differentiation rate was 31.98% after 45 days of culture; the average proliferation amount of the protocorm stem cells of D. candidum in the hormone-free proliferation culture group was 8.26 times of the inoculation amount, and the average differentiation rate was 0.36%.

[0031] Table 2 Proliferation amount and differentiation rate of protocorm stem cells of D. candidum in different culture groups

[0032]

[0033] Note: * indicates significant difference from the control group, p < 0.05.

[0034] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A hormone-free proliferation method of Dendrobium protocorm stem cells, characterized by The method comprises the following steps: The Dendrobium candidum protocorm stem cells obtained from the Dendrobium candidum seeds or stems through induction are inoculated into hormone-free proliferation culture medium under sterile conditions; The hormone-free proliferation medium formula is: MS medium + 50-100 g / L honey + 60-90 mg / L histidine + 1-5 mg / L vitamin B 12 5-6 g / L agar, pH 5.8-6.0; under the conditions of temperature 25±2 ℃, total light intensity 2000-2500 Lx, light time 10-12 h / d, culture for 30-45 d.

2. The hormone-free proliferation method of Dendrobium protocorm stem cells according to claim 1, characterized in that The illumination condition is that white light and monochromatic red light are adopted, wherein the white light intensity is 1500-2000 Lx, the monochromatic red light intensity with a wavelength of 630-730 nm is 500-1000 Lx, the total light intensity is 2000-2500 Lx, the white light source is a white light LED lamp, and the red light source is a monochromatic red light LED lamp.

3. The hormone-free proliferation method of Dendrobium protocorm stem cells according to claim 1, characterized in that: The Dendrobium candidum protocorm stem cells are Dendrobium candidum protocorm stem cells or Dendrobium officinale protocorm stem cells, and the proliferation amount of the Dendrobium candidum protocorm stem cells is 7-9 times of the inoculation amount after 30-45 days of culture, and the differentiation rate of the Dendrobium candidum protocorm stem cells is less than 1% during the culture process.

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