WO2016138688A1

WO2016138688A1 - Rhynchostylis lateral bud inducing medium and culture method therefor

Info

Publication number: WO2016138688A1
Application number: PCT/CN2015/076219
Authority: WO
Inventors: 陈永得
Original assignee: 佛山市顺德区今日景艺生物科技有限公司
Priority date: 2015-03-03
Filing date: 2015-04-10
Publication date: 2016-09-09
Also published as: CN104620992B; CN104620992A

Abstract

The present invention provides a rhynchostylis lateral bud inducing medium and a culture method therefor. An improved MS medium is used as a basic medium in the medium, and isopentennyladenine, indolebutyric acid, polyvinylpyrrolidone, agar powder and white granulated sugar are added. The inducing medium can promote growth and development of a rhynchostylis lateral bud, and reduce browning.

Description

Fox bud lateral bud induction medium and culture method thereof

Technical field

The invention relates to the technical field of orchid tissue culture, in particular to a bud-inducing medium and a culture method of foxtail blue.

Background technique

Rhynchostylis is a perennial herb of the genus Orchidaceae. It is distributed in tropical Asia and is a typical tropical epiphytic orchid. It is distributed in China and is one of the fine germplasm resources of wild orchids. The leaves of the foxtail are thick and green, the flower poses are charming or clear, the flowers are dense and dense, and the temperature is about 20-28 °C during the growing season. The flowering period is before and after the traditional Chinese New Year, which is an excellent New Year's greeting flower. The masses are favored.

Since the foxtail orchid only blooms once a year, and the seeds are extremely difficult to germinate under natural conditions, at present, the culture of the foxtail orchid is mainly based on cutting propagation or tissue culture sowing. Cutting the ramets to breed, as long as the stem segments with aerial roots are cut together with the top buds, they can be planted separately, and the buds of the original plants are cut off, and then the lateral buds are differentiated at the leaf stalks, but The plant breeding method also has defects such as long breeding cycle, low reproductive rate, and slow quantitative production process. Tissue culture sowing and propagation method is a common method in the orchid planting industry. However, after the artificial pollination of foxtail orchid, due to the small amount of seeds, there are often empty fruit pods without seeds, the germination rate is low, and the buds of the original bulbs are developed. The size of the body, the quality of the seedlings, and the quality of the seedlings are uneven, far from meeting the market demand.

Summary of the invention

Based on this, an object of the present invention is to provide a bud-inducing medium for foxtail blue, which can promote the growth and development of side buds of foxtail blue, and has the advantages of short induction time, low browning rate, and high induction survival rate.

Another object of the present invention is to provide a method for inducing lateral bud induction of foxtail blue, which uses the bud of the foxtail blue as an explant, and is cultured with the bud-inducing medium of the foxtail blue of the present invention, and has a short induction time and brown The advantages of low chemistry rate and high survival rate are achieved, and the rapid propagation of foxtail orchids is realized.

A foxtail blue side bud induction medium which is based on an improved MS medium and is supplemented with isopentenyl adenine, indolebutyric acid, polyvinylpyrrolidone, agar powder and white granulated sugar.

In one embodiment, the organic component in the modified MS medium is 100 mg/L of inositol, 1 mg/L of niacin, 1 mg/L of vitamin B6, and 10 mg/L of vitamin B1.

In one embodiment, the concentration of isoprene adenine is 2-8 mg/L, the concentration of indolebutyric acid is 0.1-0.5 mg/L, and the concentration of polyvinylpyrrolidone is 0.5-1.0 g/L, agar powder. The concentration is 6-7 g/L, and the concentration of white sugar is 25-30 g/L.

In one embodiment, the concentration of isoprenoid adenine is 6 mg/L, the concentration of indolebutyric acid is 0.3 mg/L, the concentration of polyvinylpyrrolidone is 0.8 g/L, and the concentration of agar powder is 6 g/L. The concentration of white sugar is 30 g/L.

In one embodiment, the pH of the foxtail blue side bud induction medium is between 5.5 and 6.0.

A method for cultivating lateral buds of foxtail blue, comprising the steps of: taking a robust foxtail blue bud as an explant, and sterilizing, inoculation into the lateral bud induction medium of the foxtail orchid of the present invention; It is cultured under dark culture conditions for 7-10 days at 25-28 °C, and then cultured for 40-50 days under the conditions of 12h light/12h dark and light intensity of 1900～2200lux to obtain foxtail orchids. bud.

In one embodiment, the sterilization treatment comprises the steps of: taking the explant, soaking it with alcohol with a concentration of 70-75% for 25-30 seconds, rinsing with sterile water, and placing it in a mass concentration. Sterilize for 0.1 to 0.12% of mercury chloride solution for 5 to 7 minutes, then rinse with sterile water.

In one embodiment, the side bud of the foxtail blue is a lateral bud that is germinated at the leaf stalk of the original plant after cutting and removing the top bud.

The invention improves the existing tissue culture method of Foxtail blue, and uses the lateral bud of Foxtail blue as an explant to develop an induction medium capable of promoting the growth and development of the lateral bud of Foxtail blue, which solves the difficulty in germination and differentiation of the lateral bud of Foxtail. The problem of bud emergence has the advantages of short induction time, low browning rate and high induction survival rate. It can realize the rapid propagation of foxtail blue and maintain the consistency of foxtail blue characteristics, overcoming the existing foxtail orchid. Defects in tissue culture methods.

The bud-inducing medium of the foxtail blue of the present invention adopts a modified MS medium, the organic component does not contain glycine, and the content of niacin, vitamin B6 and vitamin B1 is increased, wherein the vitamin is produced. The content of the element B1 was greatly increased from 0.1 mg/L to 10 mg/L. The addition of glycine in MS medium can avoid the adverse effects of organic amines on the lateral bud induction of foxtail; by increasing the concentrations of niacin, vitamin B6 and vitamin B1, it can provide the necessary growth of cells and tissues of the lateral buds of foxtail Vitamins can also speed up metabolism, promote lateral bud sprouting, promote bud growth and development, and make the sprouts induced by lateral buds grow stronger. At the same time, the concentration of niacin, vitamin B6 and vitamin B1 should be controlled within the appropriate dosage limit. If the concentration is too high, the number of induced shoots will be small, and the sprouts will be prolonged and the induction success rate will be reduced.

In the bud-inducing medium of Foxtail blue, the isoprenoid adenine (2-IP) has a regulating effect on protein synthesis, enzyme activity and cell metabolic balance, and can promote cell division and differentiation, and promote active growth sites. Growth and development, shortening the induction time, can also increase the activity of antioxidant enzymes, reduce browning and increase the survival rate of lateral buds; 吲哚butyric acid (IBA) is an endogenous auxin that promotes cell growth and accelerates reproduction. Polyvinylpyrrolidone (PVP) is a specific adsorbent for phenolic substances, which can effectively adsorb phenolic oxidative substrates produced by lateral buds, thereby inhibiting browning and increasing lateral bud-induced survival rate; agar is a solid culture platform for plants. The role of nutrients, water and gas permeability; while white sugar is a carbon source, the preparation cost of the medium can be reduced under the premise that the induction time, the induction rate and the small shoot robustness are not affected.

In the bud-inducing medium of Foxtail blue, the isoprenoid adenine and the indolebutyric acid have a synergistic effect, and the two are used together to promote plant tissue growth in cell division, differentiation and growth, and can be obviously The shortening of the induced germination time and the success rate of lateral bud induction; in addition, isoprenoid adenine can increase the activity of antioxidant enzymes in the lateral buds and inhibit the oxidation of phenolic substances, and it can reduce the oxidation substrate while using it in combination with polyvinylpyrrolidone. Improve plant antioxidant capacity, thereby effectively inhibiting browning, reducing browning rate, and improving the success rate of lateral bud induction.

The concentration of isoprenoid adenine in the bud-inducing medium of the foxtail blue of the present invention is preferably 2 to 8 mg/L. If the concentration is too low, the lateral bud induction rate is low or even difficult to induce budding, and if the concentration is too high, The induced buds are prone to variability; the concentration of succinic acid is preferably 0.1-0.5 mg/L. If the concentration is too low, the lateral buds have a long germination time and a low germination rate. If the concentration is too high, the buds are inhibited. Induction and growth; the concentration of polyvinylpyrrolidone is preferably 0.5-1.0 g/L. If the concentration is too low, the phenolic substance cannot be sufficiently adsorbed, and the browning inhibition effect is poor; when the concentration is higher than 1.0 g/L, browning The inhibition effect is not significantly improved.

The method for inducing and cultivating the lateral bud of Foxtail blue according to the present invention, using the lateral bud of Foxtail blue as an explant, using the bud-inducing medium of foxtail blue according to the present invention, can shorten the induction time, increase the induction survival rate, and realize The rapid propagation of the foxtail orchid.

In the method for inducing lateral bud induction of the foxtail orchid according to the present invention, the lateral bud which is germinated in the leaf stalk of the original plant after cutting and removing the top bud is used as an explant, which can reduce damage to the mother plant and not damage the ornamental of the mother plant. The value, and direct induction of clustering buds by lateral buds, can also make the superior traits of the mother plants stably inherited.

detailed description

Example 1: Preparation of the foxtail blue bud induction medium of the present invention

According to the formulas shown in Tables 1 and 2, the modified MS medium and the components of the foxtail blue side bud induction medium of the present invention were weighed and placed in a triangular flask, and an appropriate amount of distilled water was added thereto, stirred and dissolved, and the volume was adjusted to a volume. 1L, adjust the pH to 6.0, then dispense into tissue culture flask, seal it, place it in autoclave, sterilize at 121 °C for 20 minutes, cool and solidify, and obtain the foxtail blue bud of the present invention. Induction medium.

Table 1 Compositions and amounts of the modified MS medium of the present invention

Table 2 Composition and amount of the bud-inducing medium of the foxtail blue of the present invention

成分ingredient	终浓度Final concentration
成分ingredient	终浓度Final concentration	改良MS培养基Modified MS medium	--
异戊烯腺嘌呤(2-IP)Isopentenyl adenine (2-IP)	6mg/L6mg/L	改良MS培养基Modified MS medium	--
异戊烯腺嘌呤(2-IP)Isopentenyl adenine (2-IP)	6mg/L6mg/L	吲哚丁酸(IBA)Butyric acid (IBA)	0.3mg/L0.3mg/L
聚乙烯吡咯烷酮(PVP)Polyvinylpyrrolidone (PVP)	0.8g/L0.8g/L	吲哚丁酸(IBA)Butyric acid (IBA)	0.3mg/L0.3mg/L
聚乙烯吡咯烷酮(PVP)Polyvinylpyrrolidone (PVP)	0.8g/L0.8g/L	琼脂粉Agar powder	6g/L6g/L
白砂糖White sugar	30g/L30g/L	琼脂粉Agar powder	6g/L6g/L

Example 2: Method for inducing lateral bud induction of foxtail

After the foxtail orchid plant has been cut and removed, the robust lateral bud germinated in the original plant leaf stalk is used as an explant, soaked in alcohol with a concentration of 70-75% for 25-30 seconds, rinsed with sterile water. Sterilize in a mercury chloride solution with a mass concentration of 0.10 to 0.12% for 5 to 7 minutes, then rinse with sterile water.

In the ultra-clean workbench, the sterilized foxtail buds were inoculated into the bud-inducing medium of Foxtail blue prepared in Example 1, and cultured under dark culture conditions for 7 days at a temperature of 25-28 °C. Then, the cells were cultured for 45 days under the conditions of a light dark cycle of 12 h light/12 h darkness and an illumination intensity of 1900 to 2200 lux, to obtain a bud of the foxtail.

Example 3: Effect test of lateral bud induction medium of Foxtail

Referring to the bud-inducing culture method of the foxtail blue bud, the foxtail blue explants were inoculated into the medium shown in Table 3, and 45 days after the induction culture, the brown color of the foxtail blue explants was recorded. The rate, induction rate, induction time and small shoot robustness are shown in Table 4.

Table 3 foxtail blue bud induction medium

Table 4 Test results of lateral bud induction medium of Foxtail

Remarks: (1) Comprehensive score y=a*0.8-b*0.3-c*0.2.

(2) indicators of small bud robustness: "-" means no small buds; "+" means small number of small buds, weak buds, slow growth; "++" means less buds, smaller buds, growth Slower; "+++" means that the number of small buds is large, the buds are strong, and the leaves are green.

From the comparison between the results of the control group 1 and the test group 8, it can be seen that the improved MS medium of the present invention is more favorable for the induction of new shoots by the lateral buds of the foxtail, which can shorten the induction time and increase the induction rate.

Comparing the results of the control group 2～4 with the test group 8, it can be seen that the isopentenyl adenine, indolebutyric acid and polyvinylpyrrolidone all significantly promoted the induction of lateral buds of the foxtail blue; among them, isoprenoid adenine The role of infusion is indispensable. In the control group 2, due to the lack of isoprenoid adenine, the lateral buds can not induce new shoots at all, and the browning rate is the highest; polyvinylpyrrolidone has an indispensable effect on inhibiting browning. Due to the lack of polyvinylpyrrolidone, a large amount of phenolic substances accumulated, and all the test samples were browned severely, and only a small amount of samples germinated, but all died due to severe browning.

The above test results show that there is a mutual synergistic effect between isoprenoid adenine, indolebutyric acid and polyvinylpyrrolidone. When the medium contains only indole butyrin and no isopentenyl adenine, the induction rate is 0; when the medium contains only isopentenyl adenine and does not contain indolebutyric acid, the induction rate is only 59%; When the medium contained both isopentenyl adenine and indolebutyric acid, the induction rate increased significantly to 86%, and the induction time was also significantly shortened. When the medium contains only isopentenyl adenine and does not contain polyvinylpyrrolidone, the browning rate is 100%; when the medium contains only polyvinylpyrrolidone and does not contain isopentenyl adenine, the browning rate is as high as 29%. When the medium contains both isopentenyl adenine and polyvinylpyrrolidone, the browning rate can be significantly reduced. To 8%.

From the results of the test groups 1 to 12, it can be seen that within a certain concentration range, as the amount of isoprenoid adenine, indolebutyric acid and polyvinylpyrrolidone increases, the induction time of the lateral buds of the foxtail is shortened, the induction rate is increased, The browning rate was lowered, and when the concentration of isoprene adenine was 6 mg/L, the concentration of indolebutyric acid was 0.3 mg/L, and the concentration of polyvinylpyrrolidone was 0.8 g/L, the optimal induction effect was obtained.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A foxtail blue side bud induction medium characterized by: modified MS medium-based medium supplemented with isopentenyl adenine, indolebutyric acid, polyvinylpyrrolidone, agar powder and white granulated sugar.
The foxtail blue side bud induction medium according to claim 1, wherein the organic component in the modified MS medium is inositol 100 mg/L, niacin 1 mg/L, vitamin B6 1 mg/L, vitamin B1. 10mg/L.
The foxtail blue side bud induction medium according to claim 1 or 2, wherein the concentration of isoprene adenine is 2 to 8 mg/L, and the concentration of indolebutyric acid is 0.1 to 0.5 mg/L. The concentration of vinylpyrrolidone is 0.5 to 1.0 g/L, the concentration of agar powder is 6 to 7 g/L, and the concentration of white sugar is 25 to 30 g/L.
The foxtail blue bud induction medium according to claim 3, wherein the concentration of isoprene adenine is 6 mg/L, the concentration of succinic acid is 0.3 mg/L, and the concentration of polyvinylpyrrolidone is 0.8. g/L, the concentration of agar powder was 6 g/L, and the concentration of white sugar was 30 g/L.
The foxtail blue side bud induction medium according to claim 1, wherein the foxtail blue side bud induction medium has a pH of 5.5 to 6.0.
A method for cultivating lateral buds of foxtail blue, comprising the steps of: taking a robust foxtail blue bud as an explant, and sterilizing, inoculation into the foxtail blue bud induction medium according to claim 1; The temperature is 25-28 ° C, cultured in dark culture for 7 to 10 days, and then cultured for 40 to 50 days under the conditions of 12 h light/12 h dark and light intensity of 1900-2200 lux. Bud buds.
The method according to claim 6, wherein the sterilization treatment comprises the steps of: taking the explants and soaking for 25 to 30 seconds with an alcohol having a mass concentration of 70 to 75%, After rinsing with sterile water, sterilize it in a mercury chloride solution at a concentration of 0.10 to 0.12% for 5 to 7 minutes, then rinse off with sterile water.
The foxtail blue bud induction culture method according to claim 6, wherein: The side buds of the foxtail blue bud are lateral buds that have been germinated in the original plant leaf stalk after cutting and removing the top bud.