CN112106665A - Culture medium for one-step seedling formation of amorphophallus rivieri and application and tissue culture method - Google Patents
Culture medium for one-step seedling formation of amorphophallus rivieri and application and tissue culture method Download PDFInfo
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- CN112106665A CN112106665A CN202011175007.9A CN202011175007A CN112106665A CN 112106665 A CN112106665 A CN 112106665A CN 202011175007 A CN202011175007 A CN 202011175007A CN 112106665 A CN112106665 A CN 112106665A
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- culture medium
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention provides a culture medium for one-step seedling formation of konjac as well as application and a tissue culture method thereof, and relates to the technical field of plant tissue culture. The culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.5-1.5 mg/L, NAA 0.2.2-1.0 mg/L of 6-BA, 30g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8, transplanting the explant to the culture medium for culture, starting to induce the swelling callus on one surface close to the culture medium after 2 weeks, continuously increasing the volume of the callus, and inducing a large amount of white callus after 2 months. Moreover, most white callus is converted into red tumor-shaped and green tumor-shaped bulges along with the lapse of time, and the callus induction rate is 100 percent; the culture is continued on the culture medium, the culture can be differentiated into buds and roots, the proliferation is continued, the proliferation coefficient can reach 3.0, the differentiation rate reaches 21.7 percent, and the rooting rate reaches 80 percent.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a one-step seedling culture medium for konjac as well as application and a tissue culture method thereof.
Background
The konjac (Amorphophalus konjac) is a perennial herb of Araceae konjac, and is especially produced in forest margin or bush at the altitude of 200-3000 m in Yunnan of China. The konjac glucomannan is a main effective component of konjac, has the characteristics of high molecular weight, high viscosity, high expansion, high moisture content and the like, can be used as a gelling agent, a thickening agent, an emulsifying agent, a stabilizing agent, a film forming agent and the like, and has important and wide application value in a plurality of fields. At present, the problems of good variety degradation, low variety propagation coefficient and the like are faced in the large-scale production of the konjac. The rapid propagation technology of plant tissue culture is an effective way for solving the problems of large seed consumption, low natural propagation rate and the like in the rapid propagation and development of the konjac milling production. The one-time seedling formation means that the four stages of induction, differentiation, seedling strengthening and rooting are realized in one or two culture media in production at one time, and the seedlings which can be transplanted can be obtained without cutting and bottle rotating, so that the production procedure is simplified, the efficiency is improved, and the cost is saved.
Disclosure of Invention
In view of the above, the invention aims to provide a culture medium for one-step seedling formation of konjac as well as an application and a tissue culture method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a culture medium for one-step seedling formation of konjac, which takes an MS culture medium as a basic culture medium and further comprises the following components: 0.5-1.5 mg/L, NAA 0.2.2-1.0 mg/L of 6-BA, 30g/L of cane sugar and 5.6g/L of agar, and the pH value is 5.8.
Preferably, the culture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.0mg/L, NAA 0.25.25 mg/L of 6-BA, 30g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8.
The invention also provides application of the culture medium in tissue culture of the konjac.
The invention also provides a tissue culture method of the one-step seedling of the konjac based on the culture medium, which comprises the following steps: taking the leaf of the dioscorea opposita as an explant, inoculating the explant on the culture medium, and culturing in an environment with the temperature of 25 +/-1 ℃ and the illumination intensity of 2000 lx.
Preferably, the explant comprises the basal leaf, petiole or upper middle leaf of konjac.
Preferably, when the leaf base is taken as the explant, taking the position where three small leaves grow together as a base point, and cutting a part which is in a three-fork shape within the range of 0.5-1.0 cm as the explant;
taking a part of the petiole with the length of 1.0-2.0 cm as an explant when the petiole is taken as the explant;
when the middle upper part of the leaf is taken as an explant, the part with the width of 0.8-1.2 cm at the middle upper part of the leaf is taken as the explant.
Preferably, the explant comprises the petiole or leaf base of konjac.
Preferably, the illumination period of the culture is 12 h/d.
The invention provides a culture medium for one-step seedling formation of konjac, which is characterized in that an explant is transplanted to the culture medium for culture, and after 2 weeks, the callus close to one surface of the culture medium is induced to expand, the volume of the callus is continuously increased, and a large amount of white callus can be induced after 2 months. Moreover, most white callus is converted into red tumor-shaped and green tumor-shaped bulges along with the lapse of time, and the callus induction rate is 100 percent; the culture is continued on the culture medium, the culture can be differentiated into buds and roots, the proliferation is continued, the proliferation coefficient can reach 3.0, the differentiation rate reaches 21.7 percent, and the rooting rate reaches 80 percent.
Detailed Description
The invention provides a culture medium for one-step seedling formation of konjac, which takes an MS culture medium as a basic culture medium and further comprises the following components: 0.5-1.5 mg/L, NAA 0.2.2-1.0 mg/L of 6-BA, 30g/L of cane sugar and 5.6g/L of agar, and the pH value is 5.8.
The culture medium comprises 6-BA, the concentration of the 6-BA is preferably 1.0mg/L, and the 6-BA can promote the proliferation of buds after differentiation.
The culture medium comprises NAA, the concentration of the NAA is preferably 0.5mg/L, and the NAA can induce the growth and rooting of newly differentiated buds. In the invention, the 6-BA and the NAA with the concentrations are used in a matching way, the growth of the konjac plant and the formation of roots can be simultaneously promoted, and the combined use effect is better than that of single use. The sources of the components in the medium are not particularly limited in the present invention, and are preferably conventional commercial products in the art.
The invention also provides application of the culture medium in tissue culture of the konjac.
When the culture medium is used for one-step seedling tissue culture of the konjac, the leaf is preferably used as an explant, the leaf base, the petiole or the middle upper part of the leaf of the konjac is more preferably selected as the explant, and the leaf or the leaf base is most preferably used as the explant. Before the explant is cultured, the explant preferably further comprises the following treatment steps: when the leaf base is taken as an explant, taking the position where three small leaves grow together as a base point, and cutting a part which is in a three-fork shape within the range of 0.5-1.0 cm as the explant;
taking a part of the petiole with the length of 1.0-2.0 cm as an explant when the petiole is taken as the explant;
when the middle upper part of the leaf is taken as an explant, the part with the width of 0.8-1.2 cm at the middle upper part of the leaf is taken as the explant.
The invention also provides a tissue culture method of the one-step seedling of the konjac based on the culture medium, which comprises the following steps: taking the leaf of the dioscorea opposita as an explant, inoculating the explant on the culture medium, and culturing in an environment with the temperature of 25 +/-1 ℃ and the illumination intensity of 2000 lx. The selection and treatment method of the explant according to the present invention is preferably the same as described above and will not be described herein. When the explant is cultured, the illumination period of the culture is preferably 12 h/d.
The culture medium for one-step seedling formation of amorphophallus konjac as well as the application and the tissue culture method thereof provided by the invention are described in detail below with reference to the examples, but the invention is not to be construed as being limited by the scope of the invention.
Example 1
1, material: the seeds of the konjac are aseptically germinated to obtain the leaves of the seedlings, including small leaves and petioles.
2, the method comprises the following steps:
2.1 screening of the most suitable explant for tissue culture and rapid propagation
Taking the leaf base, the petiole and the upper part of the leaf as explants, and the specific interception method comprises the following steps:
1) leaf base: taking the position where the three lobules are grown together as a base point, and cutting a part which is in a three-fork shape within the range of 0.5-1.0 cm to be used as an explant.
2) Leaf stalks: taking the part of the petiole with the length of about 1.0-2.0 cm as an explant.
3) Upper part of leaf: the explant was taken from the upper middle part of the leaf about 1.0 cm.
Respectively adding 6-BA and NAA with different mass concentrations by taking MS as a basic culture medium; wherein the NAA mass concentration is 0.2, 0.5 and 1.0mg/L, the 6-BA mass concentration is 0.5, 1.0 and 1.5mg/L, and the reference is set, the treatment numbers are respectively M1-M7, and the table 1 shows. And respectively inoculating the three explants in 7 culture media for culture, inoculating 5 bottles for each treatment, inoculating 5 explants for each bottle, observing every week, and timely removing the polluted culture media. Counting the number of the callus in the culture medium twice a month, carrying out subculture after primary culture for two months, cutting off all leaves and roots growing in the later period during inoculation, and counting the proliferation number of buds. 30g/L of sucrose and 5.6g/L of agar are added to the culture medium, and the pH value is adjusted to 5.8. The temperature of the culture room is 25 +/-1 ℃, the illumination intensity is 2000lx, and the illumination time is 12h/d (the same below).
TABLE 1 Effect of different hormone combinations on explant callus induction
Note: the data in the table are the statistical results of 2 months of culture
2.2 screening of optimal Medium for Single seedling
The optimum explant of the dioscorea opposita is inoculated in four culture mediums (M4-M7 medium) for culture, each treatment is inoculated in 12 bottles, and each bottle is inoculated with 5 explants. The number of calluses, the rooting rate and the proliferation number of buds in the culture medium are counted twice per month.
3 results
3.1 selection of optimal explants
Three explants of petiole, leaf base and upper part of leaf were inoculated in culture medium M1-M7, and the statistical results of the number of calli are shown in Table 1. As can be seen from Table 1, different hormone treatments had an effect on the induction of explant callus, and the induction effect of both hormone treatments (M4-M7) was significantly better than that of the single hormone treatment and the control (M1-M3).
No enlarged callus was induced 2 weeks after explants were inoculated in M1-M3 medium, and minimal callus appeared at leaf bases over time. M2 induces callus at first, and the induction rate is 84.0%; the explants in the upper part of the leaves had not induced callus in M3 medium. The callus volume at the base of the leaf is the largest, followed by the petiole, and the callus volume at the upper part of the leaf is the smallest. The explants are inoculated into an M4-M7 culture medium, after 2 weeks, the callus induction and enlargement are started to be arranged on one surface close to the culture medium, the volume of the callus is continuously increased, and a large amount of white callus is induced after 2 months. Also, over time, most white callus turned into red nodules and green nodules.
3.2 screening of optimal Medium for Single seedling
Taking the leaf base as an explant, continuing to perform a one-time seedling experiment of the flower-grinding taro, and inoculating the explant into an M4-M7 culture medium for culture. As can be seen from Table 2, within 2 months, the callus was produced in all of the M4-M7 media, and the callus was produced in the M4 and M5 media more than in the other media, with an induction rate of 98.3%. The rooting block number of the culture medium M5 and M6 is the most, and the rooting rate is 80.0%. The M4 medium produced fibrous roots, the roots of M4 and M5 medium were dark green, and the roots of M6 and M7 medium were light green and white. The M4 and M5 medium had the largest number of buds and the differentiation rate was 21.7%. The proliferation fold of the M5 medium was the highest and was 3.1. In all, the induction rate, rooting rate, differentiation rate and proliferation fold of the M5 culture medium in the four culture media are highest. The culture medium MS + NAA 0.5mg/L +6-BA 1.0mg/L +30g/L sucrose +5.6g/L agar is the most suitable culture medium for one-time seedling formation of the flower milled taro, the leaf base of the flower milled taro is taken as an explant, and a regeneration plant can be obtained after 2 months.
TABLE 2 Effect of different hormone combinations on callus, root differentiation and shoot differentiation of primary shoot at leaf base
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The culture medium for one-step seedling formation of the konjac is characterized by taking an MS culture medium as a basic culture medium and further comprising the following components: 0.5-1.5 mg/L, NAA 0.2.2-1.0 mg/L of 6-BA, 30g/L of cane sugar and 5.6g/L of agar, and the pH value is 5.8.
2. The culture medium according to claim 1, wherein the culture medium is a MS culture medium as a minimal medium, and further comprises: 1.0mg/L, NAA 0.25.25 mg/L of 6-BA, 30g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8.
3. Use of the medium according to claim 1 or 2 for tissue culture of konjac.
4. A tissue culture method for one-step seedling formation of dioscorea opposita based on the culture medium of claim 1 or 2, which comprises the following steps: taking the leaf of the dioscorea opposita as an explant, inoculating the explant on the culture medium, and culturing in an environment with the temperature of 25 +/-1 ℃ and the illumination intensity of 2000 lx.
5. The tissue culture method of claim 4, wherein the explant comprises the basal leaf, petiole or upper middle leaf of Helianthus tuberosus.
6. The tissue culture method according to claim 5, wherein when the leaf base is used as the explant, the common growing position of three leaflets is used as a base point, and a part which is in a form of a three-fork shape within a range of 0.5-1.0 cm is cut out to be used as the explant;
taking a part of the petiole with the length of 1.0-2.0 cm as an explant when the petiole is taken as the explant;
when the middle upper part of the leaf is taken as an explant, the part with the width of 0.8-1.2 cm at the middle upper part of the leaf is taken as the explant.
7. The tissue culture method according to claim 5 or 6, wherein the explant comprises petiole or leaf base of Helianthus tuberosus.
8. The tissue culture method of claim 4, wherein the illumination period of the culture is 12 h/d.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113100063A (en) * | 2021-04-27 | 2021-07-13 | 安康市农业科学研究院 | Method for obtaining haploid plant by in vitro culture of konjac pollen |
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Cited By (2)
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| CN113100063A (en) * | 2021-04-27 | 2021-07-13 | 安康市农业科学研究院 | Method for obtaining haploid plant by in vitro culture of konjac pollen |
| CN113100063B (en) * | 2021-04-27 | 2022-03-22 | 安康市农业科学研究院 | Method for obtaining haploid plant by in vitro culture of konjac pollen |
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Application publication date: 20201222 |