CN117063840A - A cutting-free propagation method for potato tissue culture seedlings suitable for mechanized production - Google Patents
A cutting-free propagation method for potato tissue culture seedlings suitable for mechanized production Download PDFInfo
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- CN117063840A CN117063840A CN202311032560.0A CN202311032560A CN117063840A CN 117063840 A CN117063840 A CN 117063840A CN 202311032560 A CN202311032560 A CN 202311032560A CN 117063840 A CN117063840 A CN 117063840A
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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
- A01H4/002—Culture media for tissue culture
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Abstract
本发明公开了一种适宜机械化生产的马铃薯组培苗免扦插扩繁方法，包括以下步骤：步骤1：将经过组织培养后的马铃薯幼苗切段免扦插接种在MS固体培养基上，进行光照培养；步骤2：将步骤1中马铃薯组培苗光照培养3周左右，对组培苗进行切割扩繁。本发明通过使用倒置组培盒，利用激光切割组培苗免扦插扩繁，是机械扩繁马铃薯组培苗的新方式；显著提高了人工扩繁马铃薯组培苗的效率，同时可以减少扩繁操作步骤，有利于机械的设计和操作，具有较高的实际应用价值，适合马铃薯组培苗的工厂化生产。
The invention discloses a cutting-free propagation method of potato tissue culture seedlings suitable for mechanized production, which includes the following steps: Step 1: cut the tissue-cultured potato seedlings into sections and inoculate them on MS solid medium without cutting, and conduct light culture ; Step 2: Cultivate the potato tissue culture seedlings in step 1 under light for about 3 weeks, then cut and propagate the tissue culture seedlings. The present invention uses an inverted tissue culture box and uses laser cutting to propagate tissue culture seedlings without cutting. It is a new way to mechanically propagate potato tissue culture seedlings; it significantly improves the efficiency of artificial propagation of potato tissue culture seedlings and can reduce the number of propagations at the same time. The operating steps are conducive to the design and operation of machinery, have high practical application value, and are suitable for factory production of potato tissue culture seedlings.
Description
Technical Field
The invention relates to the technical field of potato planting, in particular to a cutting-free propagation method for potato tissue culture seedlings suitable for mechanized production.
Background
Researches show that the yield of the seed potato can be increased by 30% -50% when the seed potato is planted with the seed potato without the virus. Therefore, the application rate of the high-quality detoxified seed potato in China can be improved, and the single yield of potato planting in China can be effectively and greatly improved.
The shortening of the breeding time of the potato seeds requires enough original seed quantity for initial breeding, so that the seed requirement of potato planting in China every year can be met. The potato is planted mainly by tuber asexual propagation, so that the cultivated potato is easy to be affected by viruses, and the yield and quality of the potato are affected by variety degeneration. The detoxicated miniature potato produced by the potato tissue culture seedling replaces the three-generation propagation seed potato, can effectively improve the single yield of potato planting by 1.3-1.5 times of the original yield, and can even reach 3-4 times of the original yield.
The labor cost in the potato virus-free seed potato breeding system is mainly reflected in that a great deal of propagation is needed to be manually carried out after the potato stem tip is detoxified to obtain virus-free tissue culture seedlings, so as to obtain a certain basic quantity of virus-free tissue culture seedlings. The market demand of China on potato virus-free tissue culture seedlings is large each year, but the degree of mechanization of expanding propagation of virus-free tissue culture seedlings is low and mainly depends on manpower, so that the efficiency of expanding propagation of tissue culture seedlings is low, the cost is high, and the expanding propagation and mechanization of potato tissue culture seedlings are urgently needed in production.
However, she Yeya is required to be identified in the traditional tissue culture seedling propagation machine design, and is sheared into single sections, clamped by a mechanical arm and cut on a culture medium, and the traditional tissue culture seedling propagation machine is complex in design, high in operation difficulty and low in efficiency.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to solve the problems of complex design, high operation difficulty and low efficiency of the traditional tissue culture seedling propagation machinery in the prior art, and provides a cutting-free propagation method for potato tissue culture seedlings suitable for mechanized production.
2. Technical proposal
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a cutting-free propagation method of potato tissue culture seedlings suitable for mechanized production comprises the following steps:
step 1: cutting the potato seedlings subjected to tissue culture into sections, inoculating the sections onto an MS solid culture medium without cutting, and carrying out illumination culture;
step 2: and (3) culturing the potato tissue culture Miao Guangzhao in the step (1) for about 3 weeks, and cutting and expanding propagation of tissue culture seedlings.
Preferably, the culture inoculation density in step 1 is 25-50 segments/cassette.
Preferably, in the MS culture medium in the step 1, the mass ratio of sucrose is 4% or no sugar, and the mass ratio of agar is 0.8%.
Preferably, the potato tissue culture seedling in the step 1 is cut into single segments or double segments, wherein each single segment is provided with one axillary bud or one leaf, and each double segment is provided with two axillary buds or two leaves.
Preferably, the tissue culture Miao Jieduan which is not cut manually or by laser in the step 1 is not cut into the culture medium; manual shearing tissue culture Miao Jieduan is randomly dropped on the culture medium, and laser cutting tissue culture Miao Jieduan falls on the culture medium.
Preferably, the culture mode in the step 1 can be sugar-free culture, so that the requirement on the sterile environment during propagation of tissue culture seedlings is reduced, and the mechanical design and operation are facilitated.
Preferably, the propagation of the potato tissue culture seedlings in the step 2 is performed in a culture container, and the culture container comprises an inverted tissue culture box and a traditional upright tissue culture box; the culture container is a square transparent PVE plastic culture box, the size of the culture box is 76mm multiplied by 90mm (length multiplied by width multiplied by height), and the external size of the culture medium box is 76mm multiplied by 20mm (length multiplied by width multiplied by height).
Preferably, the cutting mode of the tissue culture seedling in the step 2 is to cut the tissue culture seedling manually by using a laser sent by a scissors or a laser cutting machine.
Preferably, the laser cutting tissue culture seedling specifically comprises the following steps:
step 1: the laser emitted by the laser cutting machine starts cutting from the position 1.5cm upwards from the top end of each box of tissue culture seedlings in the horizontal direction; the laser cutting machine is fixed in position, and tissue culture seedlings growing on the base of the inverted tissue culture box and the box body with the culture medium arranged right below move at a uniform speed in the same direction in the horizontal direction, so that the tissue culture Miao Jieduan cut by the laser uniformly falls on the culture medium;
step 2: after the tissue culture seedlings are cut once by laser, the base of the inverted tissue culture box moves downwards by 1.5cm, and the tissue culture seedlings are cut once again in the horizontal direction by the laser; the tissue culture seedlings growing for about three weeks can be cut for 4-5 times by laser in the horizontal direction, namely, each box of mother seedlings can be propagated for 4-5 boxes of tissue culture seedlings.
Preferably, the photoperiod of the daily light culture in the step 2 is 16/8h, the light intensity is 2500lx-3000lx, the culture temperature is 19-21 ℃, and the air humidity is 70-80%.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
in the invention, the inverted tissue culture box is used, and the cutting propagation is avoided by utilizing laser to cut the tissue culture seedlings, so that the method is a novel mode of mechanically propagating the potato tissue culture seedlings; the efficiency of artificially propagating the potato tissue culture seedlings is remarkably improved, the propagation operation steps can be reduced, the mechanical design and operation are facilitated, the practical application value is high, and the method is suitable for industrial production of the potato tissue culture seedlings.
Drawings
Fig. 1 is a schematic diagram of artificial cutting and cutting-free propagation of a potato tissue culture seedling in embodiment 2 of the present invention;
FIG. 2 shows the artificial cutting-free propagation culture of different segments for 21 days of the potato tissue culture seedling in example 2 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Example 1:
a cutting-free propagation method of potato tissue culture seedlings suitable for mechanized production comprises the following steps:
step 1: cutting potato seedlings subjected to tissue culture into sections, inoculating the sections to an MS solid culture medium without cutting, and carrying out illumination culture, wherein the culture inoculation density is 25-50 sections/box, and the mass ratio of sucrose in the MS culture medium is 4% and the mass ratio of agar is 0.8%;
the potato tissue culture seedling is cut into single segments or double segments, wherein each single segment is provided with one axillary bud or one leaf, and each double segment is provided with two axillary buds or two leaves;
the tissue culture Miao Jieduan without cutting is manually or by laser cutting is not cut into a culture medium; the artificial shearing tissue culture Miao Jieduan is randomly dropped on a culture medium, and the culture mode can be selected to be sugar-free or sugar-free culture;
step 2: culturing the potato tissue culture Miao Guangzhao in the step 1 for about 3 weeks, and cutting and expanding propagation the tissue culture seedlings; propagating potato tissue culture seedlings in a culture container, wherein the culture container comprises an inverted tissue culture box and a traditional upright tissue culture box; the photoperiod of the daily illumination culture is 16/8h, the illumination intensity is 2500lx-3000lx, the culture temperature is 19-21 ℃, and the air humidity is 70-80%;
the culture vessel is a square transparent PVE plastic culture box, the size of the culture box is 76mm multiplied by 90mm (length multiplied by width multiplied by height), and the external size of the culture medium box is 76mm multiplied by 20mm (length multiplied by width multiplied by height).
In the embodiment, the cutting mode of the tissue culture seedlings is to manually cut the tissue culture seedlings by using a pair of scissors or a laser cutter; the laser cutting tissue culture seedling specifically comprises the following steps:
step 1: the laser emitted by the laser cutting machine starts cutting from the position 1.5cm upwards from the top end of each box of tissue culture seedlings in the horizontal direction; the laser cutting machine is fixed in position, and tissue culture seedlings growing on the base of the inverted tissue culture box and the box body with the culture medium arranged right below move at a uniform speed in the same direction in the horizontal direction, so that the tissue culture Miao Jieduan cut by the laser uniformly falls on the culture medium;
step 2: after the tissue culture seedlings are cut once by laser, the base of the inverted tissue culture box moves downwards by 1.5cm, and the tissue culture seedlings are cut once again in the horizontal direction by the laser; the tissue culture seedlings growing for about three weeks can be cut for 4-5 times by laser in the horizontal direction, namely, each box of mother seedlings can be propagated for 4-5 boxes of tissue culture seedlings.
In the embodiment, the inverted tissue culture box is used, and the cutting propagation is avoided by utilizing laser to cut the tissue culture seedlings, so that the method is a novel mode of mechanically propagating the potato tissue culture seedlings; the efficiency of artificially propagating the potato tissue culture seedlings is remarkably improved, the propagation operation steps can be reduced, the mechanical design and operation are facilitated, the practical application value is high, and the method is suitable for industrial production of the potato tissue culture seedlings.
Example 2:
in the embodiment, the seedling adopted by the method is 'Hua potato No. 1' (Hua 1 for short) detoxification tissue culture seedling, the seedling is sheared into single segments, and the single segments are inoculated into an MS culture medium containing 4% of sucrose and 0.8% of agar, and the pH is 5.8.
The method comprises the following steps:
each box of the artificial cuttage-free plant is inoculated with 25, 30, 40 and 50 plants respectively, 10 boxes are inoculated as one repetition per treatment, 5 times are repeated per treatment, 25 plants are inoculated by controlling the artificial cuttage per box of the cuttage, 5 times are repeated, 10 boxes are inoculated per repetition, the artificial cuttage-free plant is cultivated in an illumination cultivation room, the illumination intensity is 2500-3000 lx every day, the temperature in the cultivation room is 19-21 ℃, and the air humidity is 70-80%. Fig. 1 is a schematic diagram of artificial cutting and cutting-free inoculation.
After 21d of culture, the growth condition of each treated and control potato tissue culture seedling is measured, and the measurement indexes are the seedling rate, plant height, stem thickness (about 1cm above the basal part) and internode number of each box of the tissue culture seedling. And performing analysis of variance on the measured data, and performing multiple comparison by using the Duncan type new complex polar difference. The comparison result of the growth condition of the tissue culture seedling of 'Hua potato No. 1' under the artificial cuttage-free inoculation condition with the comparison result is shown in fig. 2 and table 1;
table 1 'Hua potato No. 1' tissue culture seedling propagation cutting-free influence on growth
In this example, the results show that: from the index of strong seedlings, the average plant height, the internode number and the stem thickness of the test tube seedlings growing in the cutting-free 40 sections are obviously superior to those of the cutting-free 25 and 50 sections, and have no obvious difference in the growth indexes of the cutting-free 25 sections and the cutting-free 30 sections, but the seedling rate per box is 91.57 percent obviously lower than that of the cutting-free 25 sections and has no obvious difference with that of the cutting-free 30 sections.
In the embodiment, the efficiency of analyzing the cutting and the propagation of each treatment without cutting of the test tube plantlet is shown in table 2, and the propagation time of each box of 25 sections without cutting is obviously shorter than that of other treatments, and the propagation efficiency and the effective plantlet number per hour are obviously higher than those of the modes of 25 sections without cutting; the cutting-free 40-segment processing per hour propagation efficiency and the effective seedling number are obviously higher than those of other processing, and are 1.42 times of the actual propagation efficiency of the 25-segment artificial cutting. Therefore, each box is considered to be free of 40 sections, and the artificial propagation efficiency can be remarkably improved.
Table 2 comparative analysis of artificial propagation cutting and cutting-free efficiency of test tube plantlet of Hua potato No. 1
Example 3:
in this example, the cutting-free method provided in example 2 uses laser cutting to propagate potato tissue culture seedlings, except that an inverted tissue culture box is used to culture potato tissue culture seedlings, the laser cutting is used to cut the tissue culture seedlings into 1.5cm sections, and other culture conditions are the same as in example 2, and the culture mode is sugar-free culture. The adopted seedling is 'Hua potato No. 1' (Hua 1 for short) detoxification tissue culture seedling, the seedling is sheared into single segments, and the single segments are inoculated into an MS culture medium added with 0.4% agar by MS, and the pH value is 5.8.
The method comprises the following steps:
cutting the tissue culture Miao Jieduan by laser without cutting, inoculating 25 strains and 40 strains respectively to each tissue culture box, wherein 10 boxes are inoculated as one repetition per treatment, 5 times per treatment are repeated, 25 strains are inoculated by cutting each box by contrast with artificial cutting, 5 times of repetition are set, and 10 boxes are inoculated per repetition; culturing in an illumination culture room, wherein illumination is carried out for 8 hours every day, illumination intensity is 2500-3000 lx, temperature in the culture room is 19-21 ℃, and air humidity in the culture room is 70-80%; the inoculated tissue culture seedlings are cultured for about 3 weeks, and the statistical related growth indexes are measured.
In this example, after the tissue culture seedlings were cut by laser and cultured for 3 weeks, the relevant growth indexes were measured and analyzed as shown in table 3;
table 3 'Hua potato No. 1' test tube seedling propagation machinery cutting-free influence on growth
In this example, the results show that: after the test-tube plantlet of 'Hua potato No. 1' is inverted and subjected to cutting-free mechanical propagation and cutting control growth for 3 weeks, counting the seedling rate, plant height, stem thickness and internode number of each box of test-tube plantlet, and obtaining that each index of the test-tube plantlet subjected to cutting-free 40-segment growth has no significant difference from the test-tube plantlet subjected to cutting-free 25-segment treatment except the stem thickness; the plant height, the internode number and the seedling rate of the 25-segment cutting are obviously higher than those of the 25-segment cutting and 40-segment cutting treatment; cutting-free 25 and 40-segment treatment averages 22.5 seedlings and 35.68 seedlings per box; and the average plant height of the cutting-free 40-segment treatment is 57.9mm, the internode number is 5.05, and the stem thickness is 0.66mm, so that the cutting-free method and the inverted tissue culture box for mechanically propagating the potato tissue culture seedlings can better meet the actual production requirements.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. A cutting-free propagation method of potato tissue culture seedlings suitable for mechanized production is characterized by comprising the following steps:
step 1: cutting the potato seedlings subjected to tissue culture into sections, inoculating the sections onto an MS solid culture medium without cutting, and carrying out illumination culture;
step 2: and (3) culturing the potato tissue culture Miao Guangzhao in the step (1) for about 3 weeks, and cutting and expanding propagation of tissue culture seedlings.
2. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanical production according to claim 1, wherein the culture inoculation density in the step 1 is 25-50 segments/box.
3. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanical production according to claim 1, wherein in the MS culture medium in the step 1, the mass ratio of sucrose is 4% or no sugar, and the mass ratio of agar is 0.8%.
4. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanical production according to claim 1, wherein the potato tissue culture seedlings in the step 1 are cut into single segments or double segments, each segment is provided with one axillary bud or one leaf, and each segment is provided with two axillary buds or two leaves.
5. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanical production according to claim 1, wherein the cutting-free tissue culture Miao Jieduan in the step 1 is artificial or laser cutting and does not cut into a culture medium; the artificial shearing tissue culture Miao Jieduan is randomly dropped on a culture medium, and the culture mode can be selected from sugar or sugar-free culture.
6. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanical production according to claim 1, wherein the propagation of potato tissue culture seedlings in the step 2 is performed in a culture container, and the culture container comprises an inverted tissue culture box and a traditional normal tissue culture box; the culture container is a square transparent PVE plastic culture box, the size of the culture box is 76mm multiplied by 90mm (length multiplied by width multiplied by height), and the external size of the culture medium box is 76mm multiplied by 20mm (length multiplied by width multiplied by height).
7. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanical production according to claim 1, wherein the cutting mode of the tissue culture seedlings in the step 2 is to manually cut the tissue culture seedlings by using a laser emitted by a scissors or a laser cutting machine.
8. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanized production as claimed in claim 7, wherein the laser cutting tissue culture seedlings specifically comprise the following steps:
step 1: the laser emitted by the laser cutting machine starts cutting from the position 1.5cm upwards from the top end of each box of tissue culture seedlings in the horizontal direction; the laser cutting machine is fixed in position, and tissue culture seedlings growing on the base of the inverted tissue culture box and the box body with the culture medium arranged right below move at a uniform speed in the same direction in the horizontal direction, so that the tissue culture Miao Jieduan cut by the laser uniformly falls on the culture medium;
step 2: after the tissue culture seedlings are cut once by laser, the base of the inverted tissue culture box moves downwards by 1.5cm, and the tissue culture seedlings are cut once again in the horizontal direction by the laser; the tissue culture seedlings growing for about three weeks can be cut for 4-5 times by laser in the horizontal direction, namely, each box of mother seedlings can be propagated for 4-5 boxes of tissue culture seedlings.
9. The cutting-free propagation method of potato tissue culture seedlings suitable for mechanized production according to claim 1, wherein the photoperiod of daily illumination culture in the step 2 is 16/8h, the illumination intensity is 2500lx-3000lx, the culture temperature is 19-21 ℃, and the air humidity is 70% -80%.
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