CN113308374A - System and method for detecting pollen storage and in-vitro culture viability of cerasus campanulata - Google Patents
System and method for detecting pollen storage and in-vitro culture viability of cerasus campanulata Download PDFInfo
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- 238000000338 in vitro Methods 0.000 title claims abstract description 20
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- 230000035899 viability Effects 0.000 title claims description 14
- 235000003476 Prunus cerasoides var campanulata Nutrition 0.000 title claims description 11
- 240000008245 Prunus cerasoides var. campanulata Species 0.000 title claims description 11
- 238000001514 detection method Methods 0.000 claims abstract description 74
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Abstract
The invention relates to a primula sikkmensis flower pollen storage and in-vitro culture activity detection system and a method thereof, and the primula sikkmensis flower pollen storage and in-vitro culture activity detection system comprises a bearing rack, a bearing table, a test bed, a storage box, a refrigerated cabinet, a constant temperature box, a base liquid configuration mechanism, a culture dish, an electron microscope and an infrared analyzer. The detection method comprises pollen collection, culture solution preparation, pollen microscopic detection, pollen optical detection and detection verification. On one hand, the invention can effectively meet the requirements of pollen collection, storage, culture and detection operation; on the other hand, the working efficiency and quality of detection operation can be improved, and the labor intensity of the detection operation is effectively reduced.
Description
Technical Field
The invention relates to a system and a method for detecting the storage and in vitro culture viability of cerasus campanulata pollen, and belongs to the technical field of pollen storage and in vitro culture viability detection.
Background
In the breeding and corresponding plant cross breeding research and other works of cerasus campanulata, the core element of the cross breeding work is the biological activity of cerasus campanulata pollen, because of the influence of factors such as plant development factors, the biological activity of the pollen, the effective biological activity retention period of the pollen and the like, the pollen is required to be collected in advance and stored in a freezing way, and germination rate difference factors caused by the difference of the pollen during breeding are often required, so the activity of the pollen is inevitably influenced to a certain extent, therefore, the pollen activity detection operation is required in the cross breeding, and various detection technologies are developed during the pollen activity detection operation at present, for example, the patent publication number is CN105567783B, the publication number is 20190419, the patent application number is 2016101363209, and the patent name is a method for detecting the storage and in-vitro culture activity of the actinidia chinensis pollen. Although the requirements for pollen activity detection can be met to a certain extent, the detection operation is relatively complicated, and the equipment integration and modularization degree of the detection operation is low, so that the detection operation efficiency and convenience are poor; on the other hand, in the detection process, the current detection means are relatively single, so that the precision of the detection operation is relatively poor.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides the primula sikkmensis pollen storage and in-vitro culture viability detection system and the detection method thereof, the detection operation efficiency is high, the detection precision is high, the detection operation integration and the modularization degree are high, the detection operation working efficiency and the detection operation quality can be improved, and meanwhile, the labor intensity of the detection operation is effectively reduced.
The utility model provides a clockflower cherry pollen is preserved and separation culture vigor detecting system, including bearing the frame, the plummer, the test bench, the storage tank, the freezer, the thermostated container, base liquid configuration mechanism, the culture dish, electron microscope, infrared analyzer, wherein bear the frame construction that the frame is axis and horizontal plane vertical distribution, its up end is connected and coaxial distribution with the test bench, the plummer is at least one, with bear the frame side surface and be connected and with horizontal plane parallel distribution, the storage tank, the freezer, the thermostated container is all at least one, encircle and bear frame axis equipartition and be connected with bearing frame lateral surface, base liquid configuration mechanism, the culture dish, electron microscope, infrared analyzer all is connected with the test bench up end, and encircle and bear frame axis equipartition.
Furthermore, a waste liquid recovery tank which is coaxially distributed with the bearing rack is arranged in the bearing rack, a liquid collecting tank is arranged on the lower end face of the test bed, a backflow port is arranged on the upper end face of the test bed corresponding to the liquid collecting tank, and the liquid collecting tank is communicated with the waste liquid recovery tank through a flow guide pipe.
Furthermore, an infrared irradiation light source is arranged in the constant temperature box, the spectrum of the infrared irradiation light source is 1000-2500 nm, and the wavelength interval is 2-5 nm.
Further, base liquid configuration mechanism including bearing base, compounding jar, ultrasonic oscillation structure, wherein the compounding jar inlay in bearing the base, with bear the base coaxial distribution and with bear between the base through spout sliding connection, bear the base for axis and test bench up end vertical distribution's frame construction, ultrasonic oscillation structure is at least two, inlays in the compounding jar and encircles the equipartition of compounding jar axis.
Furthermore, the outer side surface of the test bed is additionally provided with a control circuit based on an industrial single chip microcomputer, the control circuit is respectively and electrically connected with the refrigerated cabinet, the thermostat, the base liquid configuration mechanism, the electron microscope and the infrared analyzer, the control circuit is additionally provided with a display and an operation key, and the display and the operation key are embedded on the outer surface of the test bed.
A detection method of a system for detecting pollen storage and in-vitro culture viability of cerasus campanulata comprises the following steps:
s1, collecting pollen, selecting healthy and strong Bell primrose buds which are full and have no defect and are in a large bud period, tearing off the tips of petals, cutting off the buds from the lower part of a pollen sac, placing the buds into a culture dish, adding a silica gel desiccant into the culture dish, placing the culture dish into a thermostat, freeze-drying at the constant temperature of-5-4 ℃ until the pollen sac leaves, transferring the pollen sac into centrifugal equipment, carrying out centrifugal separation on the pollen and the pollen sac, collecting the pollen, packaging the collected pollen in at least three parts, placing the packaged pollen into a refrigerated cabinet, and preserving at the constant temperature of-85-60 ℃;
s2, preparing a culture solution, preparing a culture mother solution through a base solution preparation mechanism while the step S1 is carried out, and storing the prepared culture mother solution at the constant temperature of 10-25 ℃ for later use;
s3, performing pollen microscopic detection, after steps S1 and S2 are completed, firstly, taking at least two parts of pollen, uniformly heating to the same temperature with a culture mother solution at a speed of 5-15 ℃/min, simultaneously taking the culture mother solution into a culture dish, enabling the culture mother solution amount in the culture dish to be 5-20 times of the mass of each part of pollen, then uniformly spraying one part of pollen on the surface of the culture mother solution in one culture dish, placing the pollen and the culture dish together in a thermostat, culturing for 1-5 hours at a constant temperature of 20-30 ℃, finally, sampling the pollen and the culture mother solution together from the culture dish after culture, preparing at least ten glass slides, sequentially observing the pollen in each glass slide by an electron microscope, recording the number of the observed pollen and the length of a pollen tube, and performing rate statistics on each glass slide sample according to the condition that the length of the pollen tube is larger than the particle size of the pollen as a pollen germination judgment standard, finally, summarizing and averaging the germination rates of all glass slide samples, and preliminarily obtaining the pollen activity detection result;
s4, optically detecting pollen, namely, filling one part of the pollen which is heated in the step S3 into a transparent container sample cup, sealing the transparent container sample cup to obtain a detection sample, preparing more than 10 parts of the detection sample, sequentially scanning each detection sample by an infrared analyzer in sequence with an infrared radiation light source spectrum of 1000-2500 nm and a wavelength interval of 2-5 nm, and counting pollen spectral data to obtain pollen activity judgment;
and S5, detection and verification, wherein the detection results of the step S4 and the step S5 are summarized and compared to obtain the accurate detection result of the cherry Bellis pollen storage and in-vitro culture activity.
Further, in the step S1, the storage time of the pollen in the refrigerated cabinet is not more than 72 hours; the preservation time of the culture mother liquor prepared in the step of S2 is not more than 24 hours.
Further, in the step S4, relative analysis error RPD of the detected spectrum is determined according to the detected pollen spectrum data of each detected sample by AACC39-00CVPerforming verification, establishing a standard model of pollen germination rate, and determining by RPDCVAnd 2.5 or more is a standard for verifying the data of the step S4.
On one hand, the system has simple structure and flexible and convenient use, and relative analysis error RPD of the detection spectrum is determined by taking AACC39-00 as a standard according to pollen spectrum data detected by each detection sampleCVThe standard model for measuring the pollen germination rate is established by verification, so that the requirements of pollen collection, storage, culture and detection operation can be effectively met; on the other hand, the detection efficiency is high, the detection precision is high, the detection operation is integrated and the modularization degree is high, the working efficiency and the quality of the detection operation can be improved, and meanwhile, the labor intensity of the detection operation is effectively reduced.
Drawings
The invention is described in detail below with reference to the drawings and the detailed description;
FIG. 1 is a schematic diagram of the system of the present invention;
FIG. 2 is a schematic structural view of a base liquid preparation mechanism;
FIG. 3 is a schematic flow chart of the method of the present invention.
The reference numbers in the figures: the device comprises a bearing frame 1, a bearing table 2, a test bed 3, a storage box 4, a refrigerated cabinet 5, a constant temperature box 6, a base liquid configuration mechanism 7, a culture dish 8, an electron microscope 9, an infrared analyzer 10, a waste liquid recovery tank 11, a liquid collecting tank 12, a return port 13, a guide pipe 14, an infrared irradiation light source 15, a control circuit 16, a bearing base 71, a mixing tank 72, an ultrasonic oscillation structure 73, a sliding groove 74, a display 161 and an operation key 162.
Detailed Description
In order to facilitate the implementation of the technical means, creation features, achievement of the purpose and the efficacy of the invention, the invention is further described below with reference to specific embodiments.
As shown in figure 1, a primula sikkmensis pollen storage and in-vitro culture viability detection system comprises a bearing frame 1, a bearing table 2, a test bed 3, a storage box 4, a refrigerated cabinet 5, a constant temperature box 6, a base liquid configuration mechanism 7, a culture dish 8, an electron microscope 9 and an infrared analyzer 10, wherein the bearing frame 1 is a frame structure with an axis vertically distributed with a horizontal plane, the upper end face of the frame structure is connected with the test bed 3 and coaxially distributed, at least one bearing table 2 is connected with the side surface of the bearing frame 1 and is parallelly distributed with the horizontal plane, at least one storage box 4, the refrigerated cabinet 5 and the constant temperature box 6 are uniformly distributed around the axis of the bearing frame 1 and are connected with the outer side face of the bearing frame 1, the base liquid configuration mechanism 7, the culture dish 8, the electron microscope 9 and the infrared analyzer 10 are uniformly connected with the upper end face of the test bed 3 and uniformly distributed around the axis of the bearing frame 1. The waste liquid recovery tank 11 which is coaxially distributed with the bearing frame 1 is arranged in the bearing frame 1, the liquid collecting tank 12 is arranged on the lower end face of the test bed 3, the upper end face of the test bed 3 corresponding to the liquid collecting tank 12 is provided with a backflow port 13, and the liquid collecting tank 12 is communicated with the waste liquid recovery tank 11 through a diversion pipe 14. The outer side surface of the test bed 3 is additionally provided with a control circuit 16 based on an industrial single chip microcomputer, the control circuit 16 is respectively and electrically connected with the refrigerated cabinet 5, the thermostat 6, the base liquid configuration mechanism 7, the electron microscope 9 and the infrared analyzer 10, the control circuit 16 is additionally provided with a display 161 and an operation key 162, and the display 161 and the operation key 162 are embedded on the outer surface of the test bed 3. An infrared irradiation light source 15 is arranged in the constant temperature box 6, the spectrum of the infrared irradiation light source 15 is 1000-2500 nm, and the wavelength interval is 2-5 nm.
Referring to fig. 2, the base liquid configuration mechanism 7 includes a bearing base 71, mixing tanks 72, and ultrasonic oscillation structures 73, wherein the mixing tanks 72 are embedded in the bearing base 71, coaxially distributed with the bearing base 71, and slidably connected with the bearing base 71 through sliding grooves 74, the bearing base 71 is a frame structure with an axis vertical to the upper end face of the test bed 3, and at least two of the ultrasonic oscillation structures 73 are embedded in the mixing tanks 72 and uniformly distributed around the axis of the mixing tanks 72.
As shown in FIG. 3, a detection method of a primula sikkmensis pollen storage and in vitro culture viability detection system comprises the following steps:
s1, collecting pollen, selecting healthy and strong Bell primrose buds which are full and have no defect and are in a large bud period, tearing off the tips of petals, cutting off the buds from the lower part of a pollen sac, placing the buds into a culture dish 8, adding a silica gel desiccant into the culture dish 8, placing the culture dish 8 into a constant temperature box 6, freeze-drying the flowers at the constant temperature of-5-4 ℃ until the pollen sac leaves, transferring the pollen sac into a centrifugal device, carrying out centrifugal separation on the pollen and the pollen sac, collecting the pollen, packaging the collected pollen in at least three parts, placing the packaged pollen into a refrigeration cabinet 5, and preserving the pollen at the constant temperature of-85-60 ℃;
s2, preparing a culture solution, preparing a culture mother solution through a base solution preparation mechanism 7 while the step S1 is carried out, and storing the prepared culture mother solution at the constant temperature of 10-25 ℃ for later use;
s3, performing pollen microscopic detection, after steps S1 and S2 are completed, firstly, taking at least two parts of pollen, uniformly heating to the same temperature with the culture mother liquor at the speed of 5-15 ℃/min, simultaneously taking the culture mother liquor to the culture dish 8, enabling the culture mother liquor amount in the culture dish 8 to be 5-20 times of the mass of each part of pollen, then uniformly spraying one part of pollen to the surface of the culture mother liquor in one culture dish 8, placing the pollen and the culture dish 8 into the incubator 6 together, culturing for 1-5 hours at the constant temperature of 20-30 ℃, finally sampling the pollen and the culture mother liquor together from the culture dish 8 after culture and preparing at least ten glass slides, observing the pollen in each glass slide in sequence through an electron microscope 9, recording the observed number and the pollen tube length, and taking the pollen tube length larger than the pollen particle size as the pollen germination judgment standard, counting the germination rate of each glass slide sample, and finally summarizing and averaging the germination rate of each glass slide sample to obtain a pollen activity detection result preliminarily;
s4, optically detecting pollen, namely, filling one part of the pollen which is heated in the step S3 into a transparent container sample cup, sealing the transparent container sample cup to obtain a detection sample, preparing more than 10 parts of the detection sample, sequentially scanning each detection sample by an infrared analyzer 10 in sequence, scanning the detection sample with an infrared radiation light source (15) spectrum of 1000-2500 nm and wavelength intervals of 2-5 nm, and counting pollen spectral data to obtain pollen activity judgment;
and S5, detection and verification, wherein the detection results of the step S4 and the step S5 are summarized and compared to obtain the accurate detection result of the cherry Bellis pollen storage and in-vitro culture activity.
It should be noted that the storage time of the pollen in the refrigerated cabinet 5 in the step S1 is not more than 72 hours; the preservation time of the culture mother liquor prepared in the step of S2 is not more than 24 hours.
Specifically, in the step S4, the relative analysis error RPD of the detected spectrum is determined according to the pollen spectrum data detected by each detected sample by using AACC39-00 as standardCVPerforming verification, establishing a standard model of pollen germination rate, and determining by RPDCVAnd 2.5 or more is a standard for verifying the data of the step S4.
On one hand, the system has simple structure and flexible and convenient use, and can effectively meet the requirements of pollen collection, storage, culture and detection operation; on the other hand, the detection efficiency is high, the detection precision is high, the detection operation is integrated and the modularization degree is high, the working efficiency and the quality of the detection operation can be improved, and meanwhile, the labor intensity of the detection operation is effectively reduced.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. The utility model provides a cloaca sakura pollen storage and isolated culture vigor detecting system which characterized in that: the cerasus campanulata pollen storage and in-vitro culture viability detection system comprises a bearing frame (1), a bearing table (2), a test table (3), a storage box (4), a refrigerated cabinet (5), a thermostat (6), a base liquid configuration mechanism (7), a culture dish (8), an electron microscope (9) and an infrared analyzer (10), wherein the bearing frame (1) is of a frame structure with an axis and a horizontal plane which are vertically distributed, the upper end face of the bearing frame is connected with the test table (3) and coaxially distributed, at least one bearing table (2) is connected with the side surface of the bearing frame (1) and is distributed in parallel with the horizontal plane, at least one of the storage box (4), the refrigerated cabinet (5) and the thermostat (6) is uniformly distributed around the axis of the bearing frame (1) and is connected with the outer side surface of the bearing frame (1), and the base liquid configuration mechanism (7), the culture dish (8), The electron microscope (9) and the infrared analyzer (10) are connected with the upper end face of the test bed (3) and are uniformly distributed around the axis of the bearing rack (1), and the thermostat (6) is internally provided with an infrared irradiation light source (15).
2. The system for detecting the storage and in vitro culture viability of cerasus campanulata pollen according to claim 1, wherein: bear frame (1) in establish with the waste liquid recovery jar (11) that bear frame (1) coaxial distribution, collecting tank (12) are established to test bench (3) lower terminal surface, return opening (13) are established to test bench (3) up end that collecting tank (12) correspond, collecting tank (12) are in addition through honeycomb duct (14) and waste liquid recovery jar (11) intercommunication.
3. The system for detecting the storage and in vitro culture viability of cerasus campanulata pollen according to claim 1, wherein: the spectrum of the infrared radiation light source (15) is 1000-2500 nm, and the wavelength interval is 2-5 nm.
4. The system for detecting the storage and in vitro culture viability of cerasus campanulata pollen according to claim 1, wherein: base liquid configuration mechanism (7) including bearing base (71), compounding jar (72), ultrasonic oscillation structure (73), wherein compounding jar (72) inlay in bearing base (71), with bear base (71) coaxial distribution and with bear base (71) between through spout (74) sliding connection, bear base (71) for axis and test bench (3) up end vertical distribution's frame construction, ultrasonic oscillation structure (73) are two at least, inlay in compounding jar (72) and encircle compounding jar (72) axis equipartition.
5. The system for detecting the storage and in vitro culture viability of cerasus campanulata pollen according to claim 1, wherein: the test bench (3) lateral surface establish in addition based on industrial singlechip control circuit (16), control circuit (16) respectively with freezer (5), thermostated container (6), base liquid configuration mechanism (7), electron microscope (9), infrared analyzer (10) electrical connection, and control circuit (16) establish display (161), control key (162) in addition, display (161), control key (162) all inlay in test bench (3) surface.
6. The method for detecting primula sikkmensis franch pollen storage and in vitro culture viability detecting system according to claim 1, comprising the steps of:
s1, collecting pollen, selecting healthy and strong Bell cherry buds which are full and have no defect and are in a large bud period, tearing off the tips of petals, cutting off the buds from the lower part of a pollen sac, placing the buds into a culture dish (8), adding a silica gel desiccant into the culture dish (8), placing the culture dish (8) into a constant temperature box (6) to freeze-dry in a constant temperature environment of-5 ℃ to 4 ℃ until the pollen sac leaves, transferring the pollen sac into a centrifugal device, performing centrifugal separation on the pollen and the pollen sac, collecting the pollen, packaging the collected pollen in at least three parts, placing the packaged pollen into a refrigerated cabinet (5) and storing the packaged pollen at a constant temperature of-85 ℃ to 60 ℃;
s2, preparing a culture solution, preparing a culture mother solution through a base solution preparation mechanism (7) while the step S1 is carried out, and storing the prepared culture mother solution at the constant temperature of 10-25 ℃ for later use;
s3, performing pollen microscopic detection, after steps S1 and S2 are completed, firstly, taking at least two parts of pollen, uniformly heating to the same temperature of the pollen with a culture mother solution at a speed of 5-15 ℃/min, simultaneously taking the culture mother solution into a culture dish (8), enabling the culture mother solution amount in the culture dish (8) to be 5-20 times of the mass of each part of pollen, then uniformly spraying one part of pollen on the surface of the culture mother solution in one culture dish (8), placing the pollen and the culture dish (8) into a constant temperature box (6) to culture for 1-5 hours at a constant temperature of 20-30 ℃, finally sampling the pollen and the culture mother solution together from the cultured culture dish (8) and preparing at least ten glass slides, sequentially observing each glass slide by an electron microscope (9), recording the number of the observed pollen and the length of a pollen tube, and taking the pollen tube length larger than the pollen particle size as a germination judgment standard, counting the germination rate of each glass slide sample, and finally summarizing and averaging the germination rate of each glass slide sample to obtain a pollen activity detection result preliminarily;
s4, optically detecting pollen, namely, filling one part of the pollen which is heated in the step S3 into a transparent container sample cup, sealing the transparent container sample cup to obtain a detection sample, preparing more than 10 parts of the detection sample, sequentially scanning each detection sample by an infrared analyzer (10) through an infrared radiation light source (15) with the spectrum of 1000-2500 nm and the wavelength interval of 2-5 nm, and counting the pollen spectral data to obtain the pollen activity judgment;
and S5, detection and verification, wherein the detection results of the step S4 and the step S5 are summarized and compared to obtain the accurate detection result of the cherry Bellis pollen storage and in-vitro culture activity.
7. The method of claim 6, wherein the system comprises: in the step S1, the storage time of the pollen in the refrigerated cabinet (5) is not more than 72 hours; the preservation time of the culture mother liquor prepared in the step of S2 is not more than 24 hours.
8. The method of claim 6, wherein the system comprises: in the step S4, according to the pollen spectrum data detected by each detection sample, the relative analysis error RPD of the detection spectrum is determined by taking AACC39-00 as a standardCVPerforming verification, establishing a standard model of pollen germination rate, and determining by RPDCVAnd 2.5 or more is a standard for verifying the data of the step S4.
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