CN110583485A - Method for inducing and rapidly propagating axillary buds of persimmon - Google Patents
Method for inducing and rapidly propagating axillary buds of persimmon Download PDFInfo
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- CN110583485A CN110583485A CN201910994772.4A CN201910994772A CN110583485A CN 110583485 A CN110583485 A CN 110583485A CN 201910994772 A CN201910994772 A CN 201910994772A CN 110583485 A CN110583485 A CN 110583485A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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 method for inducing and rapidly propagating axillary buds of persimmons, which comprises the steps of inoculating scales of retained bud with bud short branches into a culture medium for culturing aseptic seedlings, cutting off the aseptic seedlings and inoculating the cut aseptic seedlings into the culture medium to be connected with axillary bud induction, cutting off the induced axillary buds and inoculating the cut aseptic seedlings again to finish propagation. The method provided by the invention can efficiently promote the rapid growth of the axillary buds of the persimmons, has no browning phenomenon, and achieves the purpose of rapid propagation. Is suitable for the tissue culture method for rapid propagation of the persimmon.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing and rapidly proliferating axillary buds of persimmons.
Background
Yueying persimmon belongs to Diospyros kaki (Diospyros kaki) of Ebenaceae, and arbor deciduous fruit trees are mainly produced in Yao autonomous county and Pingle county of May City of Zhuang nationality autonomous region of Guangxi province in China, are internationally famous and have been cultivated and processed for over 400 years. The Yueyu persimmon has beautiful shape, bright color, no pit and excellent quality, and is the top grade of persimmon. The fresh persimmon is sweet and delicious, the frozen persimmon is fragrant and sweet, and the dried persimmon is most suitable for being prepared, which is one of the traditional export-earning high-quality products. The good social and economic benefits of the persimmon industry put higher requirements on the cultivation and the fine variety cultivation work.
However, persimmons are very easy to be infected with brown spot disease during cultivation and breeding, especially in rainy season, and the virus is easy to remain in pedicel disease to cause next-year disease, thereby causing great serious wound to the industry; in addition, the conventional seedling raising method of the persimmons is long in period and low in survival rate. Therefore, the detoxification treatment of persimmon seedlings and the rapid and efficient propagation of good persimmon seedlings by adopting a plant tissue culture technology become very important fine variety breeding means. The plant tissue culture technology can rapidly obtain a large number of healthy plants with stable characters and no plant diseases and insect pests by utilizing the totipotency of cells. Therefore, by exploring the optimal culture conditions for the growth and development of the persimmons and establishing a persimmons tissue culture system, the method can provide technical support for the rapid, efficient and healthy propagation of the persimmons, provide experimental materials for subsequent genetic transformation and lay a foundation for the genetic transformation and the character directional genetic improvement of the persimmons.
At present, tissue culture research aiming at a small part of persimmon varieties is carried out, but the tissue culture work of the persimmons is not reported yet. Previous researches prove that the genotypes of different persimmon varieties have great difference in adventitious bud induction and rooting culture, and show that the tissue culture of the persimmons has variety difference, so that the different persimmon varieties are pertinently used for establishing an optimal culture system.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, a rapid persimmon tissue culture proliferation method is provided. In order to realize the purpose of the invention, the following technical scheme is adopted for realizing the purpose: a method for inducing and rapidly proliferating axillary buds of persimmon sequentially comprises the following steps.
(1) Selection and sterilization of explants: taking 12-1 month-after-year-old persimmon budding branches from a net room, cutting into short branches with 4-6 full budding branches, putting the short branches into a beaker, wrapping the opening of the beaker with experimental gauze, putting the beaker under flowing water, and washing for about 2 hours to clean dirt carried on the surfaces of the branches and reduce the microbial pollution degree on the surfaces of explants. Then cutting into 2-4 cm short branches with sprouts, transferring the short branches to an ultra-clean workbench, sterilizing the surfaces of the short branches with 75% ethanol for 30 seconds under an aseptic condition, soaking the short branches with 2% NaClO for 5 minutes, and then soaking the short branches with 0.1% HgCl2Immersing for 5 minutes, shaking occasionally in each disinfection process, and finally washing for 5 times by using sterile distilled water;
(2) and (3) sterile seedling cultivation: shearing the disinfected short branches with buds into about 1 cm under the aseptic condition, directly inoculating the short branches with buds to a culture medium of MS (1/2N), 0.1-1.0 mg/L of indoleacetic acid (IBA), 0.05g/L of polyvinylpyrrolidone (PVP-40), 20g/L of sucrose and 3g/L of phytogel (Sigma) without removing scales, and culturing at constant temperature (25 +/-1) DEG C for 16/8 hours in a dark period and under the condition of illumination intensity of 30-40 umol/m 2/s;
(3) and (3) inducing axillary buds: cutting off the generated 0.5-1 cm sterile seedlings, inoculating the cut sterile seedlings to MS (1/2N), IBA 0.1-1.0 mg/L, zeatin 4.0mg/L, PVP-400.05g/L, sucrose 20g/L, Phytogel (Sigma)3g/L culture medium, keeping the temperature at (25 +/-1) DEG C, 16/8 hours in light-dark period and illumination intensity at 30-40 umol/m2Culturing under the condition of/s. After 50 days, the growth height of the aseptic seedlings reaches 4.5 cm, the average axillary bud number is 12.7, and the aseptic seedlings grow vigorously;
(4) and (3) rapid propagation: and (4) cutting the axillary buds with the height of 0.5-1 cm, then continuously inoculating the cut axillary buds to the axillary bud induction culture medium in the step (3), and inducing the axillary buds again to achieve the aim of rapid propagation.
As a preferable scheme: the medium in step (2) was MS (1/2N) + IBA 0.75mg/L + PVP-400.05g/L + sucrose 20g/L + phytogel (Sigma)3g/L.
As a preferable scheme: the medium in step (3) was MS (1/2N) + IBA 0.1mg/L + ZR 4.0mg/L + PVP-400.05g/L + sucrose 20g/L + phytogel (Sigma)3g/L.
The other purpose of the invention is to provide the application of the method in the rapid propagation of the persimmon tissue culture seedlings, and the method can rapidly obtain a large amount of persimmon tissue culture seedlings so as to realize the purpose of rapid propagation.
After other reported persimmon plant tissue culture systems are not suitable for persimmon tissue culture, the invention discloses a method suitable for cultivating and rapidly propagating aseptic seedlings of persimmons based on a large amount of experimental data. The method can promote rapid growth of axillary buds of persimmon efficiently, and has no browning phenomenon, thereby achieving the purpose of rapid propagation.
Drawings
FIG. 1 shows the stage of cultivating aseptic seedlings of axillary buds of Yueykaki.
FIG. 2 shows the axillary bud induction stage of Yuezhiki.
FIG. 3 shows the rapid propagation stage of axillary buds of Yuezhiki.
Detailed Description
The invention is further explained by the accompanying drawings and examples.
Example 1
A method for inducing and rapidly proliferating axillary buds of persimmon sequentially comprises the following steps.
(1) Selection and sterilization of explants: taking 12-1 month-after-year-old persimmon budding branches from a net room, cutting into short branches with 4-6 full budding branches, putting the short branches into a beaker, wrapping the opening of the beaker with experimental gauze, putting the beaker under flowing water, and washing for about 2 hours to clean dirt carried on the surfaces of the branches and reduce the microbial pollution degree on the surfaces of explants. Then cutting into 2-4 cm short branches with sprouts, transferring the short branches to an ultra-clean workbench, sterilizing the surfaces of the short branches with 75% ethanol for 30 seconds under an aseptic condition, soaking the short branches with 2% NaClO for 5 minutes, and then soaking the short branches with 0.1% HgCl2Immersing for 5 minutes, shaking occasionally in each disinfection process, and finally washing for 5 times by using sterile distilled water;
(2) and (3) sterile seedling cultivation: shearing the disinfected short branches with buds into about 1 cm under the aseptic condition, directly inoculating the short branches with buds to a MS (1/2N) + IBA 0.1-1.0 mg/L + PVP-400.05g/L + sucrose 20g/L + Phytogel (Sigma)3g/L culture medium without peeling, and culturing at constant temperature (25 +/-1) DEG C, under the conditions of 16/8 hours light-dark period and illumination intensity of 30-40 umol/m 2/s;
(3) and (3) inducing axillary buds: cutting off aseptic seedlings with the height of 0.5-1 cm, inoculating the aseptic seedlings to MS (1/2N) + IBA 0.1-1.0 mg/L + ZR 0-6.0 mg/L + PVP-400.05g/L + sucrose 20g/L + Phytogel (Sigma)3g/L culture medium, keeping the temperature at (25 +/-1) DEG C, carrying out 16/8 hours of light-dark period and illumination intensity at 30-40 umol/m2Culturing under the condition of/s. After 50 days, the growth height of the aseptic seedlings reaches 4.5 cm, the average axillary bud number is 12.7, and the aseptic seedlings grow vigorously;
(4) and (3) rapid propagation: and (3) cutting the axillary buds with the height of 0.5-1 cm, then continuously inoculating the cut axillary buds into an axillary bud induction culture medium, and inducing the axillary buds again to achieve the aim of rapid propagation.
The abbreviations referred to herein have the following meanings:
IBA indoleacetic acid;
PVP-40 polyvinylpyrrolidone;
MS(1/2N)(KNO3、NH4NO3halving the content) modified MS culture medium;
ZR zeatin.
Example 2
The influence of different sterilized treatment modes of the sprouting on the survival rate and the pollution rate of the explant is analyzed, namely, the sterilized short branches with the sprouts are cut into about 1 cm under the aseptic condition, and the scales on the outer layers of the sprouting are reserved or stripped and then inoculated into a culture medium. After 20 days of culture, the bacterial contamination rate and survival rate of the explant are counted, and the survival rate of the bud of the non-peeled scale reaches 92 percent, and the pollution rate is only 6 percent; whereas the survival rate of the sprouting of the peeled scales and the contamination rate were both 0% (table 1).
TABLE 1 influence of different ways of germinating treatment on survival and contamination rates
| Different measures for treating sprouts | Survival rate | Rate of contamination |
| Non-peeled scale | 92％ | 6％ |
| Peeling off the scale | 0％ | 0％ |
Example 3
The effect of different IBA concentrations on the growth of aseptic seedlings was analyzed, in this example after the sterilization treatmentThe shoots with buds were inoculated on MS (1/2N) + PVP-400.05g/L + sucrose 20g/L + phytogel (Sigma)3g/L medium containing 0.1, 0.25, 0.5, 0.75 and 1.0mg/L IBA, respectively, to induce sprouting. Keeping the temperature at 25 +/-1 ℃ for 16/8 hours, the light-dark period and the illumination intensity at 30-40 umol/m2And (5) culturing under the condition of s. Statistics shows that the germination rate of the sprouts treated after 20 days is 0-92%, wherein the germination rate of the sprouts in a culture medium added with 0.75mg/L of IBA is the highest and 92%, the sprouts grow well, leaves are dark green, and the browning phenomenon does not occur basically (Table 2 and attached figure 1).
TABLE 2 analysis of the Effect of different IBA concentrations on the cultivation of aseptic seedlings
| IBA concentration (mg/L) | Rate of germination of sprouts | Growth conditions |
| 0.1 | 12％ | Light green leaves and short stem |
| 0.25 | 27％ | Light green leaves and short stem |
| 0.5 | 78％ | Dark green leaves, high and medium stem |
| 0.75 | 92％ | The leaves are dark green and the stem height is high |
| 1.0 | 75％ | Leaf curl and higher stem height |
Example 4
Analyzing the influence effect of different concentrations of ZR on the axillary bud induction of the persimmon tissue culture seedlings, cutting aseptic seedlings with the height of 0.5-1 cm, and respectively inoculating the aseptic seedlings into MS (1/2N) + IBA 0.1mg/L + PVP-400.05g/L + sucrose 20g/L + phytogel (Sigma)3g/L culture medium containing 1, 2, 3, 4, 5 and 6 mg/L. After 50 days, the growth height of the original tissue culture seedlings is observed to reach 0-57 mm, the average axillary bud number is 0-12.7, and the plant cultured by the culture medium added with ZR 4.0mg/L has the best performance: after 50 days, the growth height of the original tissue culture seedlings reaches 57 millimeters, the leaves are dark green, the average axillary bud number is 12.7, and the growth vigor is vigorous (table 3, attached figure 2). And (3) cutting the axillary buds with the height of 0.5-1 cm, then continuously inoculating the cut axillary buds into an axillary bud induction culture medium (shown in figure 3), and inducing the axillary buds again to achieve the aim of rapid propagation.
TABLE 3 Effect of different treatments on axillary bud Induction
| ZR concentration (mg/L) | Rate of axillary bud induction | Number of adventitious buds | Growth conditions |
| 1.0 | 0 | 0 | Original plant leaves are light green and short |
| 2.0 | 12％ | 1.4 | Small axillary bud and light green |
| 3.0 | 23％ | 2.4 | Small axillary bud and light green |
| 4.0 | 98％ | 12.7 | Axillary bud is dark green and large |
| 5.0 | 78％ | 8.3 | Axillary bud is dark green and bigger |
| 6.0 | 75％ | 7.2 | Axillary bud is dark green and bigger |
Claims (5)
1. A method for inducing and rapidly proliferating axillary buds of persimmons is characterized by comprising the following steps:
(1) selection and sterilization of explants: taking a persimmon budding branch of 12 months to 1 month every other year, shearing the persimmon budding branch into a short branch with 4-6 full budding buds, washing the short branch with flowing water for 2 hours, then shearing the persimmon budding branch into a short branch with 2-4 cm and 1 bud, and transferring the short branch into a clean bench; sterilizing the surface of the short branch with 75% ethanol for 30 s, soaking in 2% NaClO for 5 min, and soaking in waterWith 0.1% HgCl2Sterilizing for 5 min, and washing with sterile distilled water for 5 times;
(2) and (3) sterile seedling cultivation: shearing the disinfected short branch with buds into 1 cm under the aseptic condition, directly inoculating the short branch with buds to a culture medium of 1/2N MS + 0.1-1.0 mg/L indoleacetic acid + 0.05g/L polyvinylpyrrolidone + 20g/L sucrose + 3 Phytogel3g/L without removing scales, and culturing under the conditions of constant temperature, light-dark period of 16/8 hours and illumination intensity of 30-40 umol/m 2/s;
(3) and (3) inducing axillary buds: cutting off the generated aseptic seedlings of 0.5-1 cm, inoculating the aseptic seedlings to a culture medium of 1/2N MS + IBA 0.1-1.0 mg/L + zeatin 4.0mg/L + PVP-400.05g/L + sucrose 20g/L + Phytogel3g/L, keeping the temperature at 16/8 hours, and keeping the light-dark period and the illumination intensity at 30-40 umol/m2Culturing under the condition of/s, wherein the growth height of the aseptic seedlings reaches 4.5 cm after 50 days, the average axillary bud number is 12.7, and the aseptic seedlings grow vigorously;
(4) and (3) rapid propagation: and (4) cutting the generated 0.5-1 cm axillary bud, then continuing inoculating to the axillary bud induction culture medium in the step (3), and inducing the axillary bud again to achieve the aim of rapid propagation.
2. The method for inducing axillary buds of persimmon to rapidly proliferate as set forth in claim 1, wherein the step (1) comprises surface sterilization, soaking, immersion sterilization under aseptic conditions, and shaking at intervals during each sterilization.
3. The method for inducing axillary buds and rapidly proliferating persimmon according to claim 1, wherein the culture medium in the step (2) is replaced with 1/2N MS + IBA 0.75mg/L + PVP-400.05g/L + sucrose 20g/L + Phytogel 3g/L.
4. The method for inducing axillary buds and rapidly proliferating persimmon according to claim 1, wherein the culture medium in the step (3) is replaced with 1/2N MS + IBA 0.1mg/L + ZR 4.0mg/L + PVP-400.05g/L + sucrose 20g/L + Phytogel 3g/L.
5. The method of claim 1, which is applied to rapid propagation of persimmon tissue culture seedlings.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112106660A (en) * | 2020-10-09 | 2020-12-22 | 山东蓬勃生物科技有限公司 | Culture medium and culture method for improving potato propagation process efficiency |
| CN112616660A (en) * | 2020-12-15 | 2021-04-09 | 山东省烟台市农业科学研究院 | Method for establishing in-vitro regeneration system of persimmon |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103988776A (en) * | 2014-03-28 | 2014-08-20 | 南京农业大学 | Nantong xiaofang persimmon tissue culture rapid propagation method |
| CN104255499A (en) * | 2014-09-29 | 2015-01-07 | 中国计量学院 | Diospyros rhombifolia tissue culture and immediate propagation method |
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2019
- 2019-10-18 CN CN201910994772.4A patent/CN110583485A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103988776A (en) * | 2014-03-28 | 2014-08-20 | 南京农业大学 | Nantong xiaofang persimmon tissue culture rapid propagation method |
| CN104255499A (en) * | 2014-09-29 | 2015-01-07 | 中国计量学院 | Diospyros rhombifolia tissue culture and immediate propagation method |
Non-Patent Citations (4)
| Title |
|---|
| ZUZANA KOCHANOVÁ等: ""Adventitious shoot regeneration from dormant buds of persimmon (Diospyros kaki Thunb.) cv. Hachiya"", 《JOURNAL OF AGROBIOLOGY》 * |
| 刘恺等: ""甜柿品种‘富有’组织培养技术研究"", 《保定师范专科学校学报》 * |
| 刘月英等: ""磨盘柿离体繁殖技术研究"", 《江苏农业科学》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112106660A (en) * | 2020-10-09 | 2020-12-22 | 山东蓬勃生物科技有限公司 | Culture medium and culture method for improving potato propagation process efficiency |
| CN112616660A (en) * | 2020-12-15 | 2021-04-09 | 山东省烟台市农业科学研究院 | Method for establishing in-vitro regeneration system of persimmon |
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