CN101199269B - Method of sterilized monitoring growing dynamic states of tissue cultured seedling root - Google Patents
Method of sterilized monitoring growing dynamic states of tissue cultured seedling root Download PDFInfo
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- CN101199269B CN101199269B CN 200710134021 CN200710134021A CN101199269B CN 101199269 B CN101199269 B CN 101199269B CN 200710134021 CN200710134021 CN 200710134021 CN 200710134021 A CN200710134021 A CN 200710134021A CN 101199269 B CN101199269 B CN 101199269B
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- tissue culture
- bottle
- length
- cultivating seedling
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Abstract
Disclosed is a method for aseptically monitoring the growth dynamics of plant tissue culture seedling root system, which relates to the biotechnology field. The invention selects a linear object with known length to be positioned in a tissue culture container containing a rooting medium to serve as a reference bottle, the reference bottle and a tissue culture container to be detected grown with a tissue culture root seedling are positioned on the same horizontal position, a camera is utilized to shoot the two bottles in the same picture from the bottom parts, each shooting keeps the same object distance and the same focal length and angle, the image analysis software can analyze and acquire the product pixel space of the linear object in the reference bottle and the root to be surveyed, and image analysis lengths of the tissue culture seedling root can be acquired by conversion; the image analysis lengths of the root to be surveyed can be acquired at regular intervals so as to draw a variation curve chart over the culture time, a fitting curve is gotten by the Logistic equation, and the growth dynamics of the tissue culture seedling root to be surveyed can be acquired according to the fitted Logistic equation and the curve chart. The invention can realize the aseptic and harmless online detection to the growth of the tissue culture seedling root.
Description
Technical field
The present invention relates to biological technical field, refer in particular to the dynamic method of a kind of aseptic monitoring plant tissue cultivating seedling root growth.
Background technology
Utilizing tissue culture to breed fast is a kind of effective modes of reproduction of at present a lot of flowers, vegetables and rare plant resource.The growth and development state of dynamically describing tissue cultivating seedling has great importance in production and scientific research.The production of the screening of medium, the regulation and control of environmental condition, order flowers all needs to be grasped the growth-development law of tissue cultivating seedling, understands the influence of growing of external factor to tissue cultivating seedling.Rely on perusal or the detection of artificial destruction property can't dynamically describe the growth and development state of tissue cultivating seedling.Therefore, must seek a kind of method of harmless, aseptic monitoring tissue cultivating seedling growth.The present invention utilizes image processing techniques to come the growth of monitoring plant tissue cultivating seedling root dynamic, and this does not at home and abroad appear in the newspapers.
Summary of the invention
Basic principle of the present invention is to utilize the image of image processing techniques analysis bank training shoot root, and the accumulation pel spacing of the root that obtains compares the chromosome that obtains as analysis length with the accumulation pel spacing of object of reference.Chromosome has extremely significant dependency relation as analysis length and manual measurement length, like Fig. 1.Dynamically obtain the graphical analysis length of same root continuously, the growth that can aseptic monitor out the plant tissue culture shoot root is dynamic.
Specific embodiments is: the thread of choosing the class rooty of known length is positioned in the tissue culture bottle that fills root media in advance; With this reference bottle as the graphical analysis of root; To there be the tissue culture bottle of the tissue cultivating seedling of taking root to be positioned on the same horizontal level with reference to bottle and length to be checked; Utilize digital camera to take pictures from the bottom of tissue culture bottle; To there be the tissue culture bottle of the tissue cultivating seedling of taking root to take in same image with reference to bottle and length to be checked; Take pictures at every turn and keep identical object distance and identical focal length and identical angle; Utilize image analysis software to image analysis obtain with reference to thread in the bottle and by the long-pending pel spacing of the root investigated, obtain the graphical analysis length of tissue cultivating seedling root through conversion, conversion method is: the accumulation pel spacing of the root that the graphical analysis length of the root of being investigated equals to be investigated and the ratio with reference to the accumulation pel spacing of thread in the bottle multiply by the length of known object of reference; Obtain the graphical analysis length of the root of being investigated at regular intervals; Make the change curve of graphical analysis length with incubation time; Come this curve of match with logistic (Logistic) equation; According to the Logistic equation and the curve map of match, the growth of root that draws the tissue cultivating seedling of being investigated is dynamic.
Advantage of the present invention:
(1) this method can realize aseptic, harmless, the online detection to the growth of tissue cultivating seedling root; Can obtain the growth information of more root according to the logistic after the match (Logistic) equation; Like the growth rate of exponential phase, this is that manual measurement can't be realized.
(2) efficient is high, and is easy to operate.
(3) range of application is wider.Can be used for the screening of medium, the regulation and control of environmental condition, the production of order flowers and the research of growing of tissue cultivating seedling root.
Description of drawings
Fig. 1 chromosome is as the relation of analysis length and manual measurement length
The graphical analysis length of the root of the tissue cultivating seedling of Fig. 2 embodiment 1 over time
The graphical analysis length of the root of the tissue cultivating seedling of Fig. 3 embodiment 2 over time
The graphical analysis length of the root of the tissue cultivating seedling of Fig. 4 embodiment 3 over time
Embodiment
Embodiment 1
The unrooted tissue culture plant inoculation is carried out culture of rootage in the tissue culture bottle that is placed with root media, root media is the additional 0.5mgl of 1/2MS
-1IBA (indolebutyric acid)+0.05mgl
-1NAA (methyl)+0.1mgl
-1GA
3(gibberellin), condition of culture are that temperature is 25 ± 2 ℃, and light intensity is 500 μ molm
-2s
-1, the photoperiod is the dark 8h of light 16h/.Get one and do not inoculate in tissue culture bottle tissue cultivating seedling, that fill root media bottle as a reference, one section long white line of 7cm is put into this in advance with reference to bottle, with the object of reference of white line as the root system graphical analysis.Every subsequently at a distance from 1 day; Get tissue culture bottle to be checked and reference bottle, be positioned on the same horizontal level, keep object distance at 17.7cm; Utilize the Kodak digital camera of 5,000,000 pixels to take pictures from the bottom of tissue culture bottle; Obtain and object of reference tissue cultivating seedling root system image together, the root that utilizes image analysis software SigmaScan Pro to obtain to be investigated and the accumulation pel spacing (Distance parameter) of object of reference, and then be converted into graphical analysis length (table 1).With the incubation time is abscissa, and graphical analysis length is ordinate mapping (Fig. 2), and this figure has explained the growth course of root.Use logistic (Logistic) equation to come match again, the logistic of match (Logistic) equation is:
Y＝1/(1/6.50+125.811(0.681
X))(R
2＝0.997，n＝13，P＜0.001) (1)
Y is that the take root root of tissue cultivating seedling is long, and X is the time (fate) of tissue cultivating seedling growth, R
2For determination coefficient square, n for the statistics number, P is a significance.
Is 0.623cm/day. from Fig. 2 with the growth rate that (1) formula draws exponential phase
The graphical analysis length of the root of table 1 embodiment 1
| The incubation time of tissue cultivating seedling root (my god) | Object of reference accumulation pel spacing | The accumulation pel spacing of root | The graphical analysis length of root (centimetre) |
| 10 11 12 13 14 15 16 17 18 19 20 21 22 23 | 1113.762 1115.138 1064.576 1135.175 1161.948 1152.699 1115.141 1145.266 1078.388 1032.057 1114.446 1052.298 1009.656 932.768 | 0.000 86.315 99.114 142.289 224.049 322.030 415.009 514.254 536.478 603.774 691.460 786.560 799.406 780.008 | 0.000 0.542 0.652 0.877 1.350 1.956 2.605 3.143 3.482 4.095 4.343 5.232 5.542 5.854 |
Embodiment 2
The unrooted tissue culture plant inoculation is carried out culture of rootage in the tissue culture bottle that is placed with root media, root media is the additional 0.5mgl of 1/2MS
-1IBA (indolebutyric acid)+0.005mgl
-1NAA (methyl)+0.01mgl
-1GA
3(gibberellin), condition of culture are that temperature is 25 ± 2 ℃, and light intensity is 500 μ molm
-2s
-1, the photoperiod is the dark 8h of light 16h/.Get one and do not inoculate in tissue culture bottle tissue cultivating seedling, that fill root media bottle as a reference, one section long white line of 7cm is put into this in advance with reference to bottle, with the object of reference of white line as the root system graphical analysis.Every subsequently at a distance from 1 day; Get tissue culture bottle to be checked and reference bottle, be positioned on the same horizontal level, keep object distance at 17.7cm; Utilize the Kodak digital camera of 5,000,000 pixels to take pictures from the bottom of tissue culture bottle; Obtain and object of reference tissue cultivating seedling root system image together, the root that utilizes image analysis software SigmaScan Pro to obtain to be investigated and the accumulation pel spacing (Distance parameter) of object of reference, and then be converted into graphical analysis length (table 2).With the incubation time is abscissa, and graphical analysis length is ordinate mapping (Fig. 3), and this figure has enough explained the growth course of root.Use logistic (Logistic) equation to come match again, the logistic of match (Logistic) equation is:
Y＝1/(1/5.50+21.144(0.733
X))(R
2＝0.984，n＝10，P＜0.001)(2)
Y is that the take root root of tissue cultivating seedling is long, and X is the time (fate) of tissue cultivating seedling growth, R
2For determination coefficient square, n for the statistics number, P is a significance.
The growth rate that draws exponential phase from Fig. 3 and (2) formula is 0.420cm/day, and is littler than the growth rate of the growth rate of embodiment 1 exponential phase.
The graphical analysis length of the root of table 2 embodiment 2
| The incubation time of tissue cultivating seedling root (my god) | Object of reference accumulation pel spacing | The accumulation pel spacing of root | The graphical analysis length of root (centimetre) |
| 10 11 12 13 14 15 16 17 18 19 20 | 1074.875 1122.643 1133.503 1122.542 1155.837 1095.223 1140.026 1159.813 1080.342 1039.434 1161.955 | 0.000 156.958 260.491 327.651 369.363 397.591 496.490 565.733 580.073 608.168 760.386 | 0.000 0.979 1.609 2.043 2.237 2.541 3.049 3.414 3.759 4.096 4.581 |
Embodiment 3
The unrooted tissue culture plant inoculation is carried out culture of rootage in the tissue culture bottle that is placed with root media, root media is the additional 5mgl of 1/2MS
-1IBA (indolebutyric acid)+0.005mgl
-1NAA (methyl)+0.01mgl
-1GA
3(gibberellin, condition of culture are that temperature is 25 ± 2 ℃, and light intensity is 500 μ molm
-2s
-1, the photoperiod is the dark 8h of light 16h/.Get one and do not inoculate in tissue culture bottle tissue cultivating seedling, that fill root media bottle as a reference, one section long white line of 7cm is put into this in advance with reference to bottle, with the object of reference of white line as the root system graphical analysis.Every subsequently at a distance from 1 day; Get tissue culture bottle to be checked and reference bottle, be positioned on the same horizontal level, keep object distance at 17.7cm; Utilize the Kodak digital camera of 5,000,000 pixels to take pictures from the bottom of tissue culture bottle; Obtain and object of reference tissue cultivating seedling root system image together, the root that utilizes image analysis software SigmaScan Pro to obtain to be investigated and the accumulation pel spacing (Distance parameter) of object of reference, and then be converted into graphical analysis length (table 3).With the incubation time is abscissa, and graphical analysis length is ordinate mapping (Fig. 4), and this figure has explained the growth course of root.Use logistic (Logistic) equation to come match again, the logistic of match (Logistic) equation is:
Y＝1/(1/4.20+426.690(0.623
X))(R
2＝0.985，n＝10，P＜0.001)(3)
Y is that the take root root of tissue cultivating seedling is long, and X is the time (fate) of tissue cultivating seedling growth, R
2For determination coefficient square, n for the statistics number, P is a significance.
The growth rate that draws exponential phase from Fig. 4 and (3) formula is 0.491cm/day, and is littler than the growth rate of the growth rate of embodiment 1 exponential phase, bigger than the growth rate of the growth rate of embodiment 2 exponential phases.
The graphical analysis length of the root of table 3 embodiment 3
| The incubation time of tissue cultivating seedling root (my god) | Object of reference accumulation pel spacing | The accumulation pel spacing of root | The graphical analysis length of root (centimetre) |
| 10 11 12 13 14 15 16 17 18 19 20 | 1109.237 1138.606 1104.218 1118.578 1124.146 1080.802 1102.240 1158.132 1054.454 1020.612 1095.134 | 0.000 62.214 92.209 150.255 183.206 258.686 319.383 445.242 506.912 510.238 554.182 | 0.000 0.383 0.585 0.940 1.141 1.675 2.028 2.691 3.365 3.500 3.542 |
Claims (1)
1. dynamic method of aseptic monitoring plant tissue cultivating seedling root growth; It is characterized in that: the thread of choosing the class rooty of known length is positioned in the tissue culture bottle that fills root media in advance; With this reference bottle as the graphical analysis of root; To there be the tissue culture bottle of the tissue cultivating seedling of taking root to be positioned on the same horizontal level with reference to bottle and length to be checked; Utilize digital camera from taking pictures with reference to the bottom of bottle and tissue culture bottle; To have the tissue culture bottle of the tissue cultivating seedling of taking root to take in same image with reference to bottle and length to be checked, take pictures keeps identical object distance and identical focal length and identical angle at every turn, utilizes image analysis software that image analysis is obtained the accumulation pel spacing with reference to root thread in the bottle and that investigated; Obtain the graphical analysis length of tissue cultivating seedling root through conversion, conversion method is: the accumulation pel spacing of the root that the graphical analysis length of the root of being investigated equals to be investigated and the ratio with reference to the accumulation pel spacing of thread in the bottle multiply by the length of known object of reference; Obtain the graphical analysis length of the root of being investigated at regular intervals; Make the change curve of graphical analysis length with incubation time; Come this curve of match with logistic (Logistic) equation; According to the Logistic equation and the curve map of match, the growth of root that draws the tissue cultivating seedling of being investigated is dynamic.
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| CN105210876B (en) * | 2015-10-13 | 2017-10-20 | 江苏大学 | A kind of large-scale planting method of Atractylis lancea high-quality strain |
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Non-Patent Citations (2)
| Title |
|---|
| 张云伟等.分形理论在植物根系图像放大中的应用.《昆明理工大学学报（理工版）》.2007,第32卷(第4期),80-84. * |
| 杨国梁等.计算机视觉技术在植物根系形态研究中的应用.《农业网络信息》.2005,(第11期),124-128. * |
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