Evaluation of Seedling Size Following Germination Using Computer-aided Analysis of Digital Images from a Flat-bed Scanner

Early seedling growth rate can be used to estimate seed vigor for small-seeded vegetable and flower seeds. However, hand measurement of small seedlings is tedious and difficult to reproduce among analysts. Computer-aided analysis digital images of seedlings should improve accuracy and reproducibility. A flat-bed scanner fitted with base and top lighting provided high resolution images of even small-seeded species like petunia [Petunia ×hybrida ‘Blue Picotee’ (Hort) Vilm.] and lisianthus [Eustoma grandiflorum ‘Mariachi Pure White’ (Raf.) Shinn]. Uniform lighting was provided and images were captured and analyzed in less than 2 minutes. A clear, cellulose film was used as the germination substrate in petri dish germination assays to facilitate capturing images with a flat-bed scanner. The transparent medium permitted seedlings to be imaged without removal from the petri dish and also allowed for repeated measures of the same seedlings in order to calculate growth rate. Six species evaluated in this study included cauliflower (Brassica oleracea L., var. Botrytis), tomato (Lycopersicon esculentum Mill. ‘New Yorker’), pepper (Capsicum annuum L. ‘North Star’), impatiens [Impatiens walleriana Hook. f. ‘Impact Lavender’], vinca [Catharanthus roseus (L.) G. Don. ‘Little Bright Eye’], and marigold (Tagetes patula L. ‘Little Devil Flame’). For germination and early seedling growth, the cellulose film compared favorably with other standard germination media (blue blotter and germination paper) for five of the six species tested. Computer analysis of seedling length was possible for all six species and was statistically similar to hand measurements averaged for three analysts. Seed vigor is an important aspect of seed quality. Unlike standard germination tests, which are a required for each commercial seed lot, seed vigor tests attempt to provide an assessment of germination performance under field (Association of Official Seed Analysts, 1983) or greenhouse conditions. Although numerous seed vigor tests have been developed, five major ones are used by most seed analysts. These include accelerated aging, cold test, cool test, electrolyte leakage and seedling growth rate (Hartmann et al., 1997). Of these, accelerated aging and cold test account for the majority of vigor tests for agronomic crops (Ferguson Spears, 1995). Seed vigor tests used to evaluate largeseeded agronomic crops are generally not useful for evaluating smaller-seeded vegetable and flower species. In a standard accelerated aging test, rapid water uptake by small seeds may result in high mortality unrelated to the vigor of the seed. The accelerated aging test has been adapted to evaluate flower seeds by replacing water with saturated salt solutions (KCl or NaCl) to keep moisture content in the seeds below 25% (Jianhua and McDonald, 1996). Termed the saturated salt accelerated aging test, it has been used successfully to evaluate seed vigor in impatiens. This is an important development for vigor assessment in flower seeds and with further research could be adapted for other flower species. The cold test, as currently conducted for agronomic crops, involves imbibing seeds at low temperatures (10 to 15 °C) in a sterile or nonsterile field soil, or in other media for several days prior to a standard germination test. Studies on small-seeded crops are limited, but the nonsterile conditions of this test were not tolerated by black-eyed Susan (Rudbeckia fulgida L.) seeds (Fay et al., 1993). A cold test without soil may prove a useful alternative, but could be confounded in some species where dormant seeds could benefit from the cold treatment, leading to higher germination percentages than for control seeds. Cool temperature germination (<15 °C) as a stress test may be a useful alternative for selected small-seeded crops. Seedling growth rate tests can also be used to assess vigor in seeds. Ball Seed (Chicago) developed a vigor index by using a video camera to capture digital images of emerged seedlings in a plug tray to determine cotyledon area (Conrad, 1999). This has proven to be very successful in a limited number of bedding plant species. However, it relies on seedling emergence under greenhouse or growth chamber conditions. This limits its usefulness as a general vigor test for the industry, where greenhouse conditions vary with location or time of the year. The significant amount of space required to evaluate seedlings in plug flats would be limiting for evaluation of many species and seed lots. One alternative is to measure seedling or radicle length in plants grown under controlled environments as an indicator of seed vigor. Evaluating seedling growth rate, especially radicle growth under controlled conditions, has been used successfully to test vigor in a number of small-seeded vegetable crops, including carrot (Daucus carota L.), lettuce (Lactuca sativa L.) (McCormac et al., 1990; Smith et al., 1973), radish (Raphanus sativus L.), sugar beet (Beta vulgaris L.) (Perry 1981), cauliflower (Brassica oleracea L. Botrytis Group), onion (Allium cepa L.) and leek (Allium ampeloprasum Tausch.) (Finch-Savage, 1986). Radicle length or growth rate, measured by using a slant-board test, was correlated with field emergence in these crops. These studies and others have established a strong correlation between radicle growth and the vigor level of a seed lot (Bingham et al., 1994). One problem with the slant-board test is that it is time consuming for the analyst to evaluate radicle length by hand and it can introduce analyst error in measuring radicle length. McCormac et al. (1990) attempted to use digital image analysis of radicle length of small-seeded vegetable crops using the slantboard test. Although this was an accurate measure of radicle length, problems with lighting and background color made it difficult to evaluate the small roots of these crops. Paradigm Research Corp. (South Haven, Minn.) developed an improved digital camera system that has better control of lighting and contrast between the seedling and background and uses standard petri dish germination test conditions (D. McNertney, personal communication). Several other researchers have tried to automate this type of vigor test using machine vision (Howarth and Stanwood, 1993), timesequence photography (Tomas et al., 1992), and computerized automated seed analysis with a hand potentiometric caliper (Keys et al., 1984). The results of tests have correlated well with those of hand measurements of radicle length, but have failed to become routine tests used by commercial analysts for a variety of reasons related to standardized conditions for collected images used by the computer. A good vigor test should be: 1) reproducible from lab to lab and analyst to analyst; 2) Received for publication 16 Aug. 2000. Accepted for publication 2 Nov. 2000. Univ. of Kentucky, College of Agriculture journal number 00-11-77. We thank Patchara Wonprasaid and Kay Oakley for their technical assistance. Seeds for this study were generously supplied by Ball Seed, Sakata Seed America, and Goldsmith Seeds. This research was partially funded through a grant from the Gloeckner Foundation. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. To whom requests for reprints should be addressed. E-mail address: [email protected] correlated with field or greenhouse emergence tests; 3) be rapid, objective and simple; and 4) inexpensive. The objective of this research was to develop a computer-aided evaluation system using digital images of radicle emergence that would be suitable for use as a vigor test. Key aspects developed for this research include the use of a flat-bed scanner to obtain digital images and evaluation of a transparent germination medium to facilitate scanning. Materials and Methods Plant material. Six small-seeded species (cauliflower, tomato, pepper, impatiens, vinca, and marigold) were evaluated for germination on three different germination media. Petunia and lisianthus were included to evaluate the ability to image and measure very small-seeded species. Germination conditions. Twelve seeds per species were sown in 8.5-cm-diameter plastic petri dishes containing either one piece of blue blotter (Anchor Paper Co., St. Paul, Minn.), two pieces of germination paper (Hoffman Manufacturing Co., Albany, Ore.) or one piece of a clear, uncoated cellulose film (CeloreyPUT, Cydsa Monterrey, Mexico). This film is transparent, absorbs water readily, and is 31.3 mm thick with a tensile strength of 1268 kg·cm. The cellulose film was cut to fit the petri dish, soaked in distilled water for 30 min to remove surface contamination, and autoclaved for 15 min to eliminate microbes from the film. Water (1.5 mL) was added to the dish prior to placing seeds on the film. For the blue blotter and germination paper, 5.25 or 4 mL of water was added, respectively. These quantities of water saturated the medium, leaving a thin layer of free water on the surface. Characteristics of water-holding ability were evaluated for each medium by measuring initial dry weight and subsequent water-saturated weight. Water uptake for a 60-h imbibition period was also evaluated gravimetrically for tomato seeds on all three media. Petri dishes were sealed with Parafilm (American National Can, Menasha, Wis.) and placed in a single germination chamber held at a constant 23 °C at 40 μmol·s·m from cool-white fluorescent lamps. Evaluation of seedling growth. Seedling growth was evaluated either by hand using a ruler, or using computer-aided analysis of digital images. Three analysts separately evaluated the same set of seeds. Petri dishes were refrigerated (5 °C) between hand and digital image acquisition. Each species was evaluated 2 d after initial radicle emergence. Digital images were acquired using a flat-bed scanner (Hewlett Packard Scanjet 4c/t; Palo Alto, Calif.) that included both base and top lighting. Scans were made for seeds on the cellulose film through the petri dish with the lid removed. Each scan created a 300 dpi (118 dots per cm) resolution, black and white image. Seedling length was measured automatically using MacRhizo software (Regent Instruments, Quebec) set with a threshold between 180 and 205 depending on the size of each species. Data presented for seedling growth on each germination medium was from a single analyst, while data presented for analysis method used seedlings on the film medium. A total of 60 seeds (five petri dishes) were evaluated for each treatment and analyzed using Tukey’s test with petri dish as the experimental unit. Results and Discussion Cellulose film was an effective medium for growth of seedlings of all species except pepper (Table 1, Fig. 1). For tomato, impatiens, vinca, and marigold, there was no statistical difference between the blotter and cellulose film. Cauliflower seedling length was greater on paper and cellulose film compared to the blotter. In all species except pepper, growth on the cellulose film was statistically the same as or greater than that on the germination paper. Pepper seeds germinated poorly and growth was reduced on the cellulose film. Pepper seeds were the only seeds coated with a fungicide and this may have interacted with the film to reduce seedling growth. However, attempts to wash the fungicide from the seeds prior to germination on the cellulose film did not affect seedling growth. The relative water-holding ability of the three germination media were similar, holding 66%, 74%, and 77% water at saturation for the blotter, paper and cellulose film, based on dry weight. The trend for water uptake by tomato seeds from the various media was similar over a 60-h period (Fig. 2); however, seeds tended to take up more water from the blotter. These data and the germination results observed on the three media indicate that the cellulose medium could be a suitable substitute for germination paper and blue blotter when imTable 1. Main effects of medium and method of measurement on data obtained for seedling length (mm) measured 2 d after initial radicle emergence in six small-seeded crops.