Graduate Thesis Or Dissertation

 

Nondestructive evaluation of in vitro-stored plants : a comparison of visual and image analysis systems Public Deposited

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  • In-vitro plants in slow-growth storage must be routinely evaluated for repropagation and assessment of viability. It is often difficult to determine plant health by visual assessment due to variation in growth patterns and plant structure among genera. In order to evaluate cold-stored germplasm, laboratories need more effective approaches for viability testing. Current procedures in use for evaluation of stored plantlets are subjective and not easily replicated. In this study Pyrus (pear) and Humulus (hops) invitro stored germplasm were evaluated both by visual and digital-image analysis and the results compared. Pear shoot cultures stored at 4°C in tissue-culture bags were evaluated monthly by visual examination and by digital-image analysis. Digital images were evaluated for red, green, blue (RGB), MNDVI (modified normalized differences of vegetation index), Green/Red ratio, intensity, hue, and saturation at the first two nodes of each plantlet. The number of significant variations in MNDVI and R/G values from the image analysis was similar to those observed in the visual rating system. Significant differences in MNDVI and G/R values were more often observed in the 2nd node than in the 1st node. After 5 months the visual ratings declined steadily for quick deteriorating genotypes while the others did not begin to decline until 9 months and then reached a plateau. Regression analysis indicated that the MNDVI and G/R ratios changed significantly over the 15 month rating period for most cultivars. The average R2 color ratio verses the mean visual-rating values were significant for the fast declining cultivars 'Luscious' and 'Bartlett Swiss' as were the MNDVI and G/R values. All cultivars except 'Belle Lucrative' produced significant R² values for both MNDVI and G/R when compared to the visual ratings. For better understanding the pear RGB data was converted to HSB (hue, saturation and brightness) but no significant differences were observed during the storage period. Most of the hop cultivars showed positive correlations between MNDVI values of node one and whole-plant visual ratings. This indicates that node 1 MNDVI values can be used for evaluation of hop plants. Image analysis and the visual ratings correlated positively for most cultivars between the initial rating and the three or six month values. MNDVI values were significantly different for all hops cultivars between the initial reading and three or six months for those stored on EDTA chelated iron (MS formulation) alone compared with plants on medium with 100 mg /L. or 200 mg/L EDDHA chelated iron (sequestrene 138). In all Humulus genotypes, most of the plantlets grown on MS iron were in equal or better condition than those on either sequestrene iron concentration. The goal of cold storage is to keep plantlets stored as long as possible while still retaining viability. In 9 of the 12 genotypes the mean ratings for 9-month stored plantlets on MS iron remained well above the "2" rating given to plants in need of growth-room re-propagation. Only 7 of the 12 stored on sequestrene iron were rated > 2 at 6 months and all were < 2 by 9 months. Given the trend shown in this data it is likely that plantlets of most accessions stored on sequestrene iron will be dead at 12 months while those on MS will remain in storage. This research demonstrated that image analysis is a feasible approach to evaluate the health of in-vitro stored plantlets and has great potential for automated evaluation. Further evaluation of more diverse plants and an improvement in the image processing system are necessary to effectively assess the value of image analysis for routine in-vitro plant evaluation. The results of this research will assist in the further development of the digital-image system as an alternative evaluation technique for tissue culture plantlets.
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