CN104488709A - Method for culturing bulb tissues of tulbaghia violacea floral leaf - Google Patents

Abstract

The invention relates to a method for culturing bulb tissue of Zijiao japonica, belonging to the field of plant tissue culture, comprising the following steps: medium preparation, explant selection and disinfection, bulb germination, adventitious bud induction and proliferation, rooting induction, tissue culture seedlings Domestication and transplanting. The basic medium is MS medium, sucrose 25-30g/L, agar powder 8-9g/L, medium pH 5.6-5.8; bulb germination medium is MS+6-BA 0.1-0.5mg/L+NAA 0.1 ～0.2mg/L; adventitious bud induction medium is MS+6-BA 3.0～6.0mg/L+NAA 0.05～0.5mg/L; adventitious bud proliferation medium is MS+6-BA 0.5～2.0mg/L+ NAA 0.05～0.2mg/L; rooting medium is MS+IBA 0.01～0.5mg/L or 1/2MS+IBA 0.01～0.5mg/L; culture conditions at each stage are: culture temperature 25±2℃, light time 12~16h/d, the light intensity is 40~60μ·mol·m ^-2 ·s ^-1 . The advantages of the present invention are that the established rapid multiplication system of Zijiao japonica has fast propagation speed, robustness and uniformity, less variation, high transplanting survival rate, and is suitable for large-scale production of Zijiao japonica seedlings.

Description

A kind of method for culturing the bulb tissue of Zijiao japonica

technical field

The invention relates to plant tissue culture technology, in particular to a method for culturing the bulb tissue of Zijiao japonica.

Background technique

Tulbaghia violacea, also known as wild garlic and African lily, is a perennial herb of the family Amaryllidaceae. It is a bulbous flower with a plant height of 30-50cm and small cylindrical bulbs. The height of the flower stem is about 40-60cm. The terminal cyme has small pink and purple flowers, and the flowering period is long. The actual cultivated species are often cultivated in patches. They are excellent ground cover landscape flowers. At the same time, the whole plant has a leek flavor and is edible. Generally, the leaves of Zijiao are all green, but the whole edge of the leaves of Zijiao used in the present invention has white lace, which can enrich the planting resource bank of Zijiao. Full green purple flowers.

The traditional propagation of Zijiao mosaic is the same as that of Zijiao mosaic, mainly based on scale cuttings and ramet proliferation, but the propagation efficiency of this method is low. According to Sun Lei et al. Technology, the progeny of Zijiao mosaic only doubles in 2 years, and when there are few germplasm resources, it is difficult to obtain a large number of seedlings in a short period of time. Utilize the plant tissue culture technology to carry out the existing report of the rapid propagation of the Zijiao flower seedling of the all-green variety of conventional leaves, Zhu Zhiyong et al. The regenerated plants of Zijiaohua were obtained; Chen Jianhua (CN101869068B) also obtained the patent of tissue culture and rapid propagation of Zijiaohua; in addition, He Yueqiu et al. Regenerated plants of delicate flowers. However, there is no report on the technology of directly obtaining the proliferation adventitious buds of Zijiao japonica through plant tissue culture and rapid propagation.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for culturing the bulb tissue of Zijiao japonica. Zijiao Hua uses sterile materials obtained from bulb induction to establish a good tissue culture regeneration system of Zijiao Huaye through direct induction of adventitious buds, providing technical support for the planting and propagation of Zijiao Huaye and the cultivation of new varieties.

The purpose of the present invention is accomplished through the following technical solutions. The method for culturing the bulb tissue of the flower leaf Zijiao flower, the method comprises the following steps:

1) Selection and treatment of explants: cut off the bulb from the root of Zijiao mosaic, cut off the upper leaves and lower roots, keep the length of the bulb 2-3cm, and peel off a layer of epidermis on the outer surface;

2) Germination of bulbs: On the ultra-clean workbench, transfer the surface-sterilized bulbs of Zijiao japonica to a sterile inoculation tray, use sterile filter paper to absorb the water on the surface of the seeds, and cut off some of the tissues at both ends and place The bulbs are inoculated in the germination medium MS+6-BA 0.1~0.5mg/L+NAA 0.1~0.2mg/L for cultivation, the cultivation temperature is 25±2℃, the light time is 12~16h/d, and the light intensity is 40~ 60μmol m ^-2 s ^-1 ;

3) Adventitious bud induction: the aseptic seedlings with new leaves growing and 3-5 cm high were cut in half from the middle of the bulb on a sterile workbench and inoculated into adventitious bud induction medium MS+6-BA 3.0～6.0mg/L+NAA 0.05～0.5mg/L, to induce adventitious buds, the culture temperature is 25±2℃, the light time is 12～16h/d, and the light intensity is 40～60μ·mol·m ^-2 s ^-1 ;

4) Proliferation of adventitious buds: On a sterile workbench, separate the induced clusters of adventitious buds with a height of 2 to 3 cm from the base, using 2 to 3 conjoined adventitious buds as the inoculation unit, and inoculate them in the adventitious bud proliferation medium Adventitious bud proliferation medium: MS+6-BA 0.5～2.0mg/L+NAA 0.05～0.2mg/L, culture temperature 25±2℃, light time 12～16h/d, light intensity 40～ 60μmol m ^-2 s ^-1 ;

5) Rooting induction of adventitious buds: on a sterile workbench, cut the clustered adventitious buds growing to 3-4 cm high from the base into individual ones, and insert them into the rooting medium to induce adventitious roots. The rooting medium is MS+ IBA 0.01～0.5mg/L or 1/2MS+IBA 0.01～0.5mg/L, culture temperature 25±2℃, light time 12～16h/d, light intensity 40～60μmolm- ² s ^-1 ;

6), domestication and transplanting of tissue cultured seedlings: After 2 to 3 adventitious roots of 2 to 3 cm grow out from the base of the adventitious buds, move the rooted seedlings to the greenhouse, cultivate for 5 days with scattered light, open the bottle cap, and cultivate for 1 to 2 days. Take out the rooted plant of Zijiao mosaic flower carefully from the culture bottle, wash the medium on the surface of the tissue culture seedling with tap water, plant the tissue culture seedling of Zijiao mosaic flower into the substrate, keep the moisture content above 90% and cultivate it for 15 days, and then culture it normally in the greenhouse ;

The basic medium is MS medium, the amount of sucrose is 25-30g/L, the coagulant is agar powder, the amount is 8-9g/L, the pH of the medium is adjusted to 5.6-5.8 before subpackaging; the bulb germination medium is MS+ 6-BA 0.1～0.5mg/L+NAA 0.1～0.2mg/L; adventitious bud induction medium is MS+6-BA 3.0～6.0mg/L+NAA 0.05～0.5mg/L; adventitious bud proliferation medium is MS+6-BA 0.5～2.0mg/L+NAA 0.05～0.2mg/L; rooting medium is MS+IBA 0.01～0.5mg/L or 1/2MS+IBA 0.01～0.5mg/L; bulb germination, adventitious The culture conditions for bud induction, multiplication and adventitious shoot rooting are: culture temperature 25±2°C, light time 12-16h/d, and light intensity 40-60μ·mol·m ^-2 ·s ^-1 .

Selection and treatment of initial explants, bulb germination, induction of adventitious buds, proliferation of adventitious buds, induction of rooting, acclimatization before transplanting, and culturing with more than 90% moisture after transplanting for 15 days.

The beneficial effects of the present invention are:

1. Rapid propagation of Zijiao japonica seedlings through tissue culture technology. The production is not restricted by region, season, climate and the growth period of the mother plant. It is convenient for industrial seedling cultivation. It can be produced according to the order, and the production time and scale are controllable. The popularization and application of Mosaic Zijiao provides sufficient high-quality seedling protection.

2. The reproduction coefficient reaches 2.5-3.0, the rooting rate is 100%, and the transplanting survival rate is 100%, which meets the requirements of factory seedling production.

3. The present invention adopts the multiplication method of directly inducing adventitious buds from the bulbs of Mosaic Zijiao, without callus injury induction and callus differentiation induction, which can greatly reduce the occurrence of offspring variation in the process of plant tissue culture, and the seedlings can keep the mother to the maximum extent. Ben's excellent properties.

4. It is conducive to the establishment, reference and promotion of excellent individual plants and new varieties of Tissue Culture Rapid Propagation System.

5. Establish a good tissue culture and rapid propagation system to lay the foundation for the future research work on the cultivation of new varieties and genetic improvement of Zijiao japonica using plant biotechnology.

Detailed ways

The present invention will be further elaborated below through specific embodiments. Examples will help to better understand the present invention, but the present invention is not limited only to the following examples.

The method for culturing the bulb tissue of the flower leaf Zijiao flower, the method comprises the following steps:

1. Culture medium and culture conditions: The basic medium for bulb germination, adventitious bud induction, adventitious bud proliferation and adventitious bud rooting induction is MS medium, the amount of sucrose is 25-30g/L, and the coagulant is agar powder. 8～9g/L, the pH of the medium is adjusted to 5.6～5.8 before subpackaging; the bulb germination medium is MS+6-BA 0.1～0.5mg/L+NAA 0.1～0.2mg/L; the adventitious bud induction medium is MS +6-BA 3.0～6.0mg/L+NAA 0.05～0.5mg/L; adventitious bud proliferation medium is MS+6-BA 0.5～2.0mg/L+NAA 0.05～0.2mg/L; rooting medium is MS +IBA 0.01～0.5mg/L or 1/2MS+IBA 0.01～0.5mg/L; the culture conditions for bulb germination, adventitious bud induction and proliferation, adventitious bud rooting are: culture temperature 25±2℃, light time 12～16h /d, the light intensity is 40-60 μ·mol·m ^-2 ·s ^-1 .

2. Treatment and disinfection of explants: select the bulb of Zijiao japonica as the starting material, cut off the bulb from the root of Zijiao huaye, cut off the upper leaves and lower roots, keep the length of the bulb 2-3cm, and peel off the outer surface 1 layer of epidermis, the treated explants were cleaned with detergent solution for 30 minutes, during which they were constantly stirred gently, and then the detergent solution remaining on the surface of the clean bulb was rinsed with running water, and placed on a sterilized ultra-clean workbench Transfer the cleaned flower-leaf Zijiao flower bulbs to a sterile conical flask, soak in 70-75% alcohol for 60 seconds, and gently shake the conical flask during this period to remove air bubbles attached to the surface of the flower-leaf Zijiao flower seeds, pour out the 75% alcohol, Then soak the seeds with 0.1% HgCl ₂ solution for 10-15 minutes, or soak the seeds with 1% NaClO solution with available chlorine concentration for 20-30 minutes. Prepare 0.1% HgCl ₂ solution or 1% NaClO solution, wash the surface-sterilized bulbs with sterile water for 4 to 5 times, soak the treated seeds with a small amount of sterile water, and set aside.

3. Seed germination: On the ultra-clean workbench, transfer the surface-sterilized Zijiao japonica seeds to a sterile inoculation tray, blot the water on the surface of the seeds with sterile filter paper, and inoculate them in the seed germination medium MS+6-BA 0.1～0.5mg/L+NAA 0.1～0.2mg/L for seed germination, culture temperature 25±2℃, light time 12～16h/d, light intensity 40～60μmol m ^-2 s ^{- 1} .

4. Induction of adventitious buds: the aseptic seedlings with a height of 3 to 5 cm, which have grown new leaves, are cut in half from the middle of the bulb on a sterile workbench, and then inoculated into the adventitious bud induction medium MS+6-BA 3.0～6.0mg/L+NAA 0.05～0.5mg/L, to induce adventitious buds, the culture temperature is 25±2℃, the light time is 12～16h/d, and the light intensity is 40～60μ·mol·m ^-2 · s ^-1 .

5. Proliferation of adventitious buds: On a sterile workbench, separate the induced clusters of adventitious buds with a height of 2 to 3 cm from the base, using 2 to 3 conjoined adventitious buds as the inoculation unit, and inoculate them in the adventitious bud proliferation medium Adventitious bud proliferation medium: MS+6-BA 0.5～2.0mg/L+NAA 0.05～0.2mg/L, culture temperature 25±2℃, light time 12～16h/d, light intensity 40～ 60 μ·mol·m ^-2 ·s ^-1 .

6. Rooting induction of adventitious buds: On a sterile workbench, cut the clustered adventitious buds that grow to a height of 3 to 4 cm from the base into individual pieces, and insert them into the rooting medium to induce adventitious roots. The rooting medium is MS+IBA 0.01～0.5mg/L or 1/2MS+IBA 0.01～0.5mg/L, culture temperature 25±2℃, light time 12～16h/d, light intensity 40～60μmol m ^-2 s ^{- 1} .

7. Domestication and transplanting of tissue-cultured seedlings: After 2-3 adventitious roots of 2-3 cm grow out of the base of the adventitious buds, move the rooted seedlings to the greenhouse, and open the bottle cap after cultivating with scattered light for 5 days, and then cultivate for 1-2 days. The rooted plants of Yezijiao peanut were carefully taken out from the culture bottle, the culture medium on the surface of the tissue culture seedlings was washed with tap water, and the tissue culture seedlings of Mosaicia japonica were planted in the substrate, and the moisture content was more than 90% and cultivated for 15 days, and then cultivated normally in the greenhouse.

Claims (2)

1. a method for floral leaf tulbaghia violacea bulb tissue cultures, is characterized in that: the method comprises the steps:

1), the choosing and processing of explant: from floral leaf purple tender root, bulb is connected root and cut, cut off upper blade and bottom root, retain bulb length 2 ~ 3cm, peel off outer surface 1 layer of epidermis;

2), bulb is sprouted: on superclean bench, floral leaf tulbaghia violacea bulb through surface sterilization is being transferred in aseptic inoculation dish, the surface of the seed moisture is sucked with aseptic filter paper, after slightly cutting two end portions tissue, bulb is inoculated in germination medium MS+6-BA 0.1 ~ 0.5mg/L+NAA 0.1 ~ 0.2mg/L and cultivates, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

3) adventitious bud inducing: grow young leaves by what obtain, the aseptic seedling that 3 ~ 5cm is high is inoculated into adventitious bud induction culture base MS+6-BA 3.0 ~ 6.0mg/L+NAA 0.05 ~ 0.5mg/L after being divided into two in the middle of bulb on aseptic working platform and cutting open, carry out the induction of indefinite bud, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

4) adventitious bud proliferation: on aseptic working platform, induction is obtained cluster, indefinite bud that 2 ~ 3cm is high from base portion with 2 ~ 3 disjunctor indefinite buds for inoculation unit is separated, be inoculated in adventitious bud proliferation medium and breed, adventitious bud proliferation medium is MS+6-BA 0.5 ~ 2.0mg/L+NAA 0.05 ~ 0.2mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

5), adventitious bud rooting induction: on aseptic working platform, Seedling height is cut into single to the indefinite bud of growing thickly of 3 ~ 4cm from base portion, the induction of adventive root is carried out in access root media, root media is MS+IBA 0.01 ~ 0.5mg/L or 1/2MS+IBA 0.01 ~ 0.5mg/L, cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1;

6), plantlet in vitro domestication and transplanting: after indefinite bud base portion grows the adventive root of 2 ~ 32 ~ 3cm, seedling of taking root moves to greenhouse, scattered light opens bottle cap after cultivating 5d, cultivate 1 ~ 2d again, the plant that taken root by floral leaf tulbaghia violacea is carefully taken out from blake bottle, cleans the medium on plantlet in vitro surface with running water, by floral leaf tulbaghia violacea plantlet in vitro, plant in matrix, 15d is cultivated in moisturizing more than 90%, then normally cultivates in greenhouse;

Minimal medium selects MS medium, and dosage of sucrose is 25 ~ 30g/L, and coagulating agent is agar powder, and consumption is 8 ~ 9g/L, is adjusted to 5.6 ~ 5.8 before medium pH packing; Bulb germination medium is MS+6-BA 0.1 ~ 0.5mg/L+NAA 0.1 ~ 0.2mg/L; Adventitious bud induction culture base is MS+6-BA 3.0 ~ 6.0mg/L+NAA 0.05 ~ 0.5mg/L; Adventitious bud proliferation medium is MS+6-BA 0.5 ~ 2.0mg/L+NAA 0.05 ~ 0.2mg/L; Root media is MS+IBA 0.01 ~ 0.5mg/L or 1/2MS+IBA 0.01 ~ 0.5mg/L; Bulb is sprouted, adventitious bud inducing and propagation, adventitious bud rooting condition of culture are that cultivation temperature 25 ± 2 DEG C, light application time is 12 ~ 16h/d, and intensity of illumination is 40 ~ 60 μm of olm ^-2s ^-1.

2. the method for floral leaf tulbaghia violacea bulb tissue cultures according to claim 1, is characterized in that: before the selection of initial explant and process, bulb sprouting, adventitious bud inducing, adventitious bud proliferation, root induction, transplanting domestication process and after transplanting moisturizing more than 90% cultivate 15d.