CN109566416B - Method for inducing adventitious buds to carry out rapid propagation of ginger plant seedlings - Google Patents
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Abstract
The invention discloses a method for inducing adventitious buds to carry out rapid propagation of ginger plant seedlings, which comprises the following steps: selecting strong tissue culture seedlings of Zingiberaceae, and removing peripheral roots; removing main stem, retaining root and expanded stem, and retaining basal leaf sheath part of leaf; respectively crosscutting the expanded stem and leaf sheath parts, inoculating the cross-cut expanded stem and leaf sheath parts on an induction culture medium, and culturing by illumination; transferring the adventitious bud to a proliferation culture medium, and culturing by illumination. The invention establishes a novel rapid seedling propagation method for Zingiberaceae, the seedlings can be directly grown, the callus stage is not passed, the risk of variation is avoided, the period is short, and the efficiency is high.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to a method for inducing adventitious buds to perform rapid propagation of ginger plant seedlings.
Background
In tropical and subtropical areas of the world where the zingiberaceae plants are spread, in China, 22 zingiberaceae plants belong to 209 species, which are mainly distributed in southern and southwest provinces, wherein the Guangdong, Guangxi and Yunnan provinces are most abundant. The Zingiberaceae plants are a treasury of plant resources, and the knowledge and the utilization of the Zingiberaceae plants have a long history in China. The Zingiberaceae plants have many functions and are indispensable in life. For example, ginger is called as one of four spicy ingredients of food kingdom, contains multiple ingredients, is rich in nutrition, is an important seasoning vegetable, is also used as seasoning spice, has pungent ginger taste and mild nature, has the functions of sweating, relieving exterior syndrome, warming the middle and dissipating cold and the like, has very high medicinal value, can be used as pickles, ginger soup, cakes, beverages and the like, is wide in cultivation region of China, is more and more popular in markets at home and abroad, becomes an important export commodity of China, and brings huge economic benefit to China every year; the turmeric has the medicinal values of promoting qi and blood circulation, and stimulating the menstrual flow and relieving pain, the contained curcumin has various effects of resisting tumor, reducing blood fat, resisting radiation and the like, is also used as a main raw material of the curry powder, can be used as a yellow dye, and has very important economic value; fructus Amomi can be used for warming spleen and invigorating stomach, promoting qi circulation, regulating middle warmer, and treating dysentery, and can be made into confection, fructus Amomi sugar, etc.; xianghe and YangHe are eaten as vegetables from ancient times. Besides the above functions of eating and medicinal use, the ginger plants have excellent ornamental property, are mostly wet or aquatic plants, have special negative resistance and aromaticity, and have great development prospect in special plant gardens, such as the white ginger flowers and the flower-leaf alpinia japonica are widely applied to landscaping in south China. In addition, part of the plants in the family Zingiberaceae, such as the flowers of white ginger, the flowers of Molbilus zingiberis, the flowers of Emeira glabra and the like, have strong adaptability to sewage environment, can play a purifying effect on sewage, and are suitable for being applied to remediation engineering of polluted water.
The method has the advantages that the propagation coefficient is low, the cost is high, the ginger plants are easy to suffer from natural disasters and plant diseases and insect pests, the production requirement is difficult to meet, the sexual degeneration and the disease are extremely easy to occur due to long-term asexual propagation, the quality and the yield are rapidly reduced, and the production of the ginger plants is seriously threatened. Such as tobacco mosaic virus, cucumber mosaic virus, bacterial wilt and rot (also known as ginger blast), root rot, ginger anthracnose and ginger pythium rot, which are the most serious diseases to the ginger; the amomum fruit is seriously harmed by the amomum fruit leaf blight, the amomum fruit anthracnose, the leaf spot, the fruit rot and the like. These diseases cause significant economic losses to the production of plants of the Zingiberaceae family, and have become one of the major factors limiting the development of the Zingiberaceae family. Therefore, the rapid propagation by using plant tissue culture technology is a key measure and important guarantee for obtaining healthy seedlings on a large scale in a short time.
At present, the tissue culture explants of the Zingiberaceae plants usually adopt buds, stem tips, leaves, seeds and the like, and most of the explants mainly comprise buds. For example, the method usually adopts a germination accelerating mode to induce the ginger to sprout, and after the stem tip is cut, a sterile system is established through surface disinfection, and the method has the defects that: firstly, the time is consumed, the germination acceleration usually needs to wait for 1 to 2 weeks, and the time from the germination acceleration to the establishment of an explant usually needs 6 to 8 months; secondly, the cost is high, an environment with the temperature of more than 20 ℃ is usually required to be provided when germination is performed in winter, and the room temperature needs to be improved in production by burning a boiler, burning electricity and the like; thirdly, the pollution rate is high, the ginger blocks are uneven, the surface disinfection is not easy to be thorough, and the pollution rate is very high. In addition to stem tip stripping for ginger germination acceleration, stem tip stripping by tissue culture seedlings is also studied, but the stem tip of ginger is semitransparent and water-soaked, and has high stripping difficulty and low survival rate. Therefore, large-scale propagation and detoxification by using the stem tip are difficult. Inducing callus by leaf to induce adventitious bud is a common method in Zingiberaceae, such as placing basal leaf of Zingiber officinale Roscoe in a solution containing 0.9-10mg.L-1Dense callus was generated on 2, 4-D medium and transferred to 0.9mg.L-12, 4-D can induce the formation of adventitious buds. However, this method has problems that: firstly, as the number of subcultures is increased, the regeneration capacity of the callus is obviously degraded; ② the variation rate is high through callus stage. The aseptic seeding of seeds is a commonly used technique for rapid propagation, but this method is not suitable for plants of Zingiberaceae, such as ginger, which are not fruity in nature. Therefore, at present, a safe, simple and rapid propagation method is extremely lacking in the production of the Zingiberaceae plants.
Disclosure of Invention
In view of the above, the invention provides a method for rapidly propagating zingiberaceae plant seedlings, which aims at the defects of long time consumption, easy pollution, easy variation caused by callus stages and the like in the conventional rapid propagation technology of zingiberaceae plants, takes simple and effective thin-layer and thin-sheet tissue induced adventitious buds as a technical core.
The invention is realized by the following technical scheme:
a method for inducing adventitious buds to carry out rapid propagation of seedlings of Zingiberaceae plants comprises the following steps:
1) selecting strong tissue culture seedlings of Zingiberaceae, and removing peripheral roots under the condition of an ultra-clean workbench;
2) preparing materials: removing main stem, retaining root and expanded stem, and retaining basal leaf sheath part of leaf;
3) transversely cutting the expanded stem and leaf sheath part, inoculating on an induction culture medium, and culturing under illumination;
4) transferring the adventitious bud to a proliferation culture medium, and culturing by illumination.
The step (1) is specifically as follows: selecting healthy ginger tissue culture seedlings with the age of 5-6 weeks, reserving 1.0-2.0cm of main roots, and removing the rest peripheral roots.
The step 2 specifically comprises the following steps: retaining the main root and 1.0-1.5cm above the main root, removing the rest stem and leaf, and cutting the leaf to obtain 1.0-1.5cm base leaf sheath.
The step 3 specifically comprises the following steps: transecting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating on an induction culture medium of MS + TDZ 1.0-4.0mg/L + NAA 0-2.0mg/L, and culturing in a light culture room at 25 + -2 deg.C for 10-15 days.
The step 4 is specifically as follows: transferring the induced adventitious bud to proliferation culture medium of 6-BA 2.0-3.0mg/L + NAA 0.2-0.5mg/L, and culturing in 25 + -2 deg.C illumination culture room for 4-5 weeks.
Compared with the prior art, the invention can obtain the following technical effects:
1) the method establishes a novel rapid propagation method of the seedling of the Zingiberaceae plant, the seedling is directly grown, the callus stage is not passed, and the risk of variation is avoided.
2) The period is short, adventitious buds are directly induced through a thin layer and a thin tissue, and then the propagation and rooting stages are carried out, wherein the total period is 43-50 days.
3) The propagation efficiency is high, taking 1 ginger seedling as an example, the ginger seedling has 1 stem and 4 leaf sheaths, the stem can be cut into 5-6 thin layers, each thin layer can induce 1-2 adventitious buds, the leaf sheaths are cut into 8 sections of thin sheets, each thin sheet induces 1-2 adventitious buds, namely 13-26 adventitious buds are shared, the propagation coefficient is 4-6, therefore, the number of the seedlings propagated in the first generation is as follows based on 1 ginger seedling: the number of seedlings grown in the second generation and the later generations is increased in geometric grade from 52 to 156.
Drawings
FIG. 1 shows the fast seedling propagation process of Zingiberaceae plant (taking Zingiber officinale as an example) with the thin-layer and thin-sheet tissue-induced adventitious bud technology as the core; wherein a is a ginger seedling to be tested; b is a schematic diagram of the preparation of lamina and lamina tissues, wherein b1 is the lamina tissue and b2 is the lamina tissue; c, the thin-layer tissue grows in an unsuitable induction culture medium, browning occurs, and no adventitious bud grows; d is the case of thin tissue growth in a more suitable induction medium, the adventitious bud starts to grow but is weaker; e is the condition that the thin-layer tissue grows in the most suitable induction culture medium, and the adventitious bud starts to grow and is very strong; f, growing the slices on a suitable induction medium, and starting to grow adventitious buds; g is adventitious bud induced by lamina and flake tissues; h is the growth state of the adventitious bud on an improper multiplication culture medium, the plant is yellowed, and the multiplication coefficient is extremely low; i is the state that the adventitious bud grows on a proper multiplication culture medium, the plant is green and strong, and the multiplication coefficient is 4-6.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
The invention discloses a method for rapidly propagating ginger plant seedlings by inducing adventitious buds through thin-layer and thin-sheet tissues, which comprises the following steps:
step 2, reserving the main root and the expanded stem with the length of 1.0-1.5cm above the main root, removing the rest stems and leaves, and reserving the leaf sheath part at the base part for 1.0-1.5cm after the leaves are cut;
and 3, transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each section of the leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaf sheath on an induction culture medium of MS + TDZ 1.0-4.0mg/L + NAA 0-2.0mg/L, and placing the cut leaf sheath on a light culture room at the temperature of 25 +/-2 ℃ for culturing for 10-15 days.
And 4, transferring the induced adventitious bud to a proliferation culture medium of 2.0-3.0 mg/L6-BA and 0.2-0.5mg/L NAA, and culturing in a light culture room at 25 +/-2 ℃ for 4-5 weeks.
The key points of the method are the preparation of thin layers and thin sheet materials, the hormone types and the concentration ratio. Preparation of thin layer material: keeping 1.0-1.5cm of expanded stem above the main root, and transversely cutting the expanded stem to obtain thin layers each with a thickness of 2.0-3.0 mm. Preparation of the sheet material: the leaf sheath part at the base part is kept 1.0-1.5cm after the leaf blade is cut, and each section of the leaf sheath is cut into 5.0-7.0 mm. The inducing hormones of the adventitious bud are TDZ and NAA, and the concentration ranges are 1.0-4.0mg/L of TDZ and 0-2.0mg/L of NAA; the proliferation hormone of adventitious bud is 6BA and NAA, and the concentration range is 6BA 2.0-3.0mg/L, and NAA 0.2-0.5 mg/L. In the experiment, the data show that the type and concentration of hormones have a significant effect on the induction and proliferation of adventitious buds (fig. 1).
TABLE 1 Effect of different hormone types and concentration ratios on the induction of adventitious buds by thin and thin tissue
TABLE 2 Effect of different hormone ratios on adventitious bud proliferation
Example 1
Selecting a healthy ginger variety 'Sichuan castrated ginger' with the age of 5 weeks as a tissue culture seedling of the yellow ginger, reserving a main root of 1.0-2.0cm under the condition of an ultra-clean workbench, and removing the rest peripheral roots; retaining the main root and 1-1.5cm of the expanded stem, removing the rest stem and leaf, and cutting the leaf to retain 1.0-2.0cm of the leaf sheath part of the base part; transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaves on an induction culture medium of MS + TDZ 2.0mg/L + NAA 1.0mg/L, and culturing in a light culture room at 25 +/-2 ℃ for 15 days; the obtained adventitious bud was transferred to MS medium of 2.0 mg/L6-BA + 0.5mg/L NAA, and cultured in a light culture room at 25. + -. 2 ℃ for 4 weeks with a proliferation rate of 6.
Example 2
Selecting a robust ginger variety Leshanxi dam ginger tissue culture seedling with the age of 5 weeks, reserving a main root of 1.0-2.0cm under the condition of an ultra-clean workbench, and removing the rest peripheral roots; retaining the main root and the 1-1.5cm enlarged stem above the main root, removing the rest stem and leaf, and retaining the 1.0-2.0cm basal leaf sheath part after the leaf blade is cut; transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaves on an induction culture medium of MS + TDZ 2.0mg/L + NAA 1.0mg/L, and culturing in a light culture room at 25 +/-2 ℃ for 15 days; transferring the obtained adventitious bud to 6-BA 2.0-3.0mg/L + NAA 0.2-0.5mg/L MS culture medium, and culturing in 25 + -2 deg.C illumination culture room for 4 weeks with adventitious bud proliferation rate of 4-6.
Example 3
Selecting a robust ginger variety 'Sichuan bamboo root ginger' tissue culture seedling with the age of 5 weeks, reserving a main root of 1.0-2.0cm under the condition of an ultra-clean workbench, and removing the rest peripheral roots; retaining the main root and the 1-1.5cm enlarged stem above the main root, removing the rest stem and leaf, and retaining the 1.0-2.0cm basal leaf sheath part after the leaf blade is cut; transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaves on an induction culture medium of MS + TDZ 3.0mg/L + NAA 0.5mg/L, and culturing in a light culture room at 25 +/-2 ℃ for 15 days; the obtained adventitious bud was transferred to MS medium of 2.0 mg/L6-BA + 0.5mg/L NAA, and cultured in a light culture room at 25. + -. 2 ℃ for 4 weeks with a proliferation rate of 4.
Example 4
Selecting a robust ginger variety 'thick dam ginger' tissue culture seedling with the age of 5 weeks, reserving a main root of 1.0-2.0cm under the condition of an ultra-clean workbench, and removing the rest peripheral roots; retaining the main root and the 1-1.5cm enlarged stem above the main root, removing the rest stem and leaf, and retaining the 1.0-2.0cm basal leaf sheath part after the leaf blade is cut; transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaves on an induction culture medium of MS + TDZ 1.0mg/L + NAA 0.5mg/L, and culturing in a light culture room at 25 +/-2 ℃ for 15 days; the obtained adventitious bud was transferred to MS medium of 2.0 mg/L6-BA + 0.2mg/L NAA, and cultured in a light culture room at 25. + -. 2 ℃ for 4 weeks with a proliferation rate of 5.
Example 5
Selecting strong tissue culture seedlings of Zingiber mioga Rosc (hereinafter also referred to as Jerusalem artichoke) with the seedling age of 6 weeks, reserving 1.0-2.0cm of main roots under the condition of an ultraclean workbench, and removing the rest peripheral roots; retaining the main root and the 1.0-1.5cm enlarged stem above, removing the rest stem and leaf, and cutting the leaf to retain 1.0-1.5cm of leaf sheath part at the base part; transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaves on an induction culture medium of MS and TDZ 3.0mg/L, and culturing in a light culture room at 25 +/-2 ℃ for 10 days; the obtained adventitious bud was transferred to MS medium of 3.0 mg/L6-BA + 0.5mg/L NAA, and cultured in a light culture room at 25. + -. 2 ℃ for 4 weeks with a proliferation rate of 5.
Example 6
Selecting healthy and strong turmeric tissue culture seedlings with the seedling age of 6 weeks, reserving 1.0-2.0cm of main roots under the condition of an ultra-clean workbench, and removing the rest peripheral roots; retaining the main root and the 1.0-1.5cm enlarged stem above, removing the rest stem and leaf, and cutting the leaf to retain 1.0-1.5cm of leaf sheath part at the base part; transversely cutting the expanded stem, wherein the thickness of each thin layer is 2.0-3.0mm, each leaf sheath is cut into 5.0-7.0mm, inoculating the cut leaves on an induction culture medium of MS and TDZ 2.0mg/L, and culturing in a light culture room at 25 +/-2 ℃ for 10 days; the obtained adventitious bud was transferred to MS medium of 2.0 mg/L6-BA + 0.2mg/L NAA, and cultured in a light culture room at 25. + -. 2 ℃ for 4 weeks with a proliferation rate of 4.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (1)
1. A method for inducing adventitious buds to carry out rapid propagation of ginger plant seedlings is characterized by comprising the following steps:
step 1, selecting strong ginger tissue culture seedlings with the seedling age of 5-6 weeks, reserving 1.0-2.0cm of main roots under the condition of an ultra-clean workbench, and removing peripheral roots;
step 2, removing main stems, and reserving roots and 1.0-1.5cm of expanded stems; removing the rest stem and leaf, and reserving 1.0-1.5cm of leaf sheath part at the base part of the leaf;
step 3, transversely cutting the leaf sheath part, cutting each section of the leaf sheath into 5.0-7.0mm, inoculating the cut leaf sheath on an induction culture medium, and culturing in a light culture chamber at 25 +/-2 ℃ for 10-15 days, wherein the induction culture medium is MS + TDZ 1.0-4.0mg/L + NAA 0-2.0 mg/L;
step 4, transferring the adventitious bud to a proliferation culture medium, and culturing for 4-5 weeks in a light culture room at 25 +/-2 ℃, wherein the proliferation culture medium is MS +6-BA 2.0-3.0mg/L + NAA 0.2-0.5 mg/L;
the plant of Zingiberaceae is rhizoma Zingiberis recens, rhizoma Zingiberis recens or Curcuma rhizome.
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| CN112931228A (en) * | 2021-04-28 | 2021-06-11 | 青岛泽欣农业科技有限公司 | Detoxification subculture device for sterile tissue culture and rapid propagation of rhizomajaponicas and culture method thereof |
| CN116098063B (en) * | 2023-03-08 | 2024-02-13 | 南京林业大学 | Rapid propagation method and application of test-tube seedlings of lycoris radiata leaf sheath-induced test-tube bulblet |
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