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CROP BREEDING & GENETICS

An Accelerated Postharvest Seed-Coat Darkening Protocol for Pinto Beans Grown across Different Environments

Dep. of Plant Sciences, Univ. of Saskatchewan, Saskatoon, SK, Canada S7N 5A8

^* Corresponding author (k.bett{at}usask.ca).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Pinto beans (Phaseolus vulgaris L.) that darken more slowly than conventional pinto beans would be more desirable in the market place and have been identified in the bean breeding program at the University of Saskatchewan. To incorporate the slow-darkening trait into new cultivars there is a need for a quick, reliable, and inexpensive method to accelerate darkening without affecting seed germination. Three different accelerated darkening protocols were compared. The greenhouse protocol was conducted in the greenhouse by placing the bean seeds in plastic bags with a 1-cm² piece of moistened felt. For the ultraviolet C (UVC) light protocol, bean seeds were placed 10 cm below a 254-nm UVC lamp. For the third protocol, bean seeds were placed in a cabinet set at 30°C, 80% relative humidity, and full fluorescent light. All three protocols examined could be used to distinguish darkening beans from slow-darkening beans, however the UVC protocol was considered superior as it was quick, consistent over years, and economical and, unlike the greenhouse and the cabinet protocols, had no effect on seed germination. A genotype by environment (g x e) study was conducted to validate the UVC light protocol. After accelerated darkening, line and environment effects were found to be significant (P < 0.0001) but the g x e interaction was not significant (P = 0.29), which indicated that the UVC protocol could be used to distinguish slow-darkening pinto beans from darkening pinto beans, regardless of where the beans were grown.

Abbreviations: CDC, Crop Development Centre • g x e, genotype by environment • HTC, hard to cook • UVC, ultraviolet C.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
IN MANY MARKET classes of dry bean, including pinto, the seed coat color of the bean slowly changes to a darker brown color after the seed is physiologically mature. Postharvest seed coat darkening occurs more rapidly in environments that have high temperature, humidity, and light (Park and Maga, 1999). These same conditions cause beans to develop the hard-to-cook (HTC) phenomenon (Barrón et al., 1996; Hincks and Stanley, 1986; Richardson and Stanley, 1991; Sievwright and Shipe, 1986; Srisuma et al., 1989; Stanley, 1992; Stanley et al., 1989). Thus, consumers presume that beans with dark seed coats are HTC. Hard-to-cook beans are undesirable as they require longer soaking and cooking time compared to fresh beans and are less palatable.

Currently, few inexpensive, practical cultural techniques are available to successfully prevent seed coat darkening and the HTC effect. One cultural technique to prevent seed coat darkening is to store the beans in a controlled, N-enriched atmosphere, as is done for horticulture crops such as apples (Malus spp.). Sartori (1982) found that after 6 mo of storage at 24°C and 75% relative humidity, pinto beans that were stored in an enriched N atmosphere showed no change in color while pinto beans stored in a natural atmosphere began to significantly darken after 2 mo of storage. The undarkened pinto beans stored under forced N still developed long cooking times, became hard, and produced poor flavor, all of which was similar to the natural atmosphere pinto beans. Park and Maga (1999) found that low moisture content reduced seed coat darkening but would not prevent it entirely.

Merchants have expressed interest in beans that maintain the same seed coat color present at harvest. At the Crop Development Centre (CDC), University of Saskatchewan, several pinto bean lines were identified that maintain their seed coat color better than currently grown cultivars. These new lines are referred to as slow-darkening lines and traditional cultivars are referred to as darkening lines. The differences in the darkening rates of these lines may be due to differing amounts of proanthocyanidins (syn. condensed tannins) in the seed coats. A recent study showed that slow-darkening line 1533-15 had lower levels of proanthocyanidins in the seed coat than darkening cultivar CDC Pintium (Beninger et al., 2005).

To determine the genetics of slow-darkening and to efficiently introgress the trait into new bean cultivars, a quick method to identify slow-darkening individuals would be useful. Chemical analysis of proanthocyanidin is destructive, expensive, and time consuming. Allowing the seed to naturally darken under room conditions is a lengthy process and could possibly produce variable results from year to year. The objective of this study was to develop and validate a nondestructive darkening protocol that can reliably differentiate slow-darkening from darkening lines in a way that is quick, inexpensive, and independent of growing and environmental effects and that will not affect seed germination.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Eighteen lines, three slow-darkening and 15 darkening, were grown in the field in 2003 and 2004 to produce seeds for this study. Of the 15 darkening lines, CDC Pintium, ‘CDC Minto’, and ‘CDC Camino’ were commercially available cultivars and the remaining 12 lines were entries in the 2003 Canadian Prairie Dry Bean Cooperative Registration Trials. The beans were grown at Saskatoon, SK, as a randomized complete block design with the first replicate grown in 2003 and the second and third replicates grown in 2004. The 2003 growing season was long, warm, and dry whereas the 2004 growing season was short, cool, and wet, with an early frost in August. After harvest seeds were kept in cold (4°C), dark storage before darkening to limit natural darkening. Three darkening protocols were tested using seed from the three replicates. The amount of seed tested for each line was equal to the volume of seed that could cover a 100-mm petri dish in a single layer.

Greenhouse Darkening Protocol
The first protocol was based on the results of Park and Maga (1999) who found that darkening accelerates when beans have high moisture contents and/or are stored under high relative humidity and high temperatures. To achieve this in an economically feasible way, seeds were placed in plastic bags (16.5 by 9.5 cm; Nasco Whirl-Pak, Fort Atkinson, WI) along with a cut piece of felt (1 cm²) and the plastic bags were placed on benches in a greenhouse for 57 d. For the 2003 darkening season, average daily accumulated light levels were approximately 2.8 x 10^–2 µmol m^–2 s^–1 compared to the 2004 darkening season which had roughly 3.2 x 10^–2 µmol m^–2 s^–1. The average air temperature inside the greenhouse was comparable in both years with temperatures ranging between 16°C (night) and 26°C (day).

UVC Light Darkening Protocol
This protocol was designed to maximize the effect of light, which was partially blocked in the greenhouse protocol by the greenhouse glass and the polybag plastic. Beans were placed in open 100-mm petri dishes directly under a germicidal UVC light bulb ( = 254 nm; model G40T10, Ushio America, Inc., Cypress, CA). In the light standards, UVC light bulbs alternated with fluorescent light bulbs every 12 cm. The two UVC and two fluorescent light bulbs were raised 10 cm above the beans and emitted a light intensity of 4.06 mW cm^–2 at the 254 nm light spectrum.

Cabinet Darkening Protocol
The third protocol was based on protocols used to simulate the HTC effect (Barrón et al., 1996; Hincks and Stanley, 1986; Richardson and Stanley, 1991; Sievwright and Shipe, 1986; Srisuma et al., 1989; Stanley, 1992; Stanley et al., 1989). The seeds were placed in open 100-mm petri dishes in a seed germination cabinet set at 30°C, 80% relative humidity, and full fluorescent light. In 2003, a Conviron model 630 cabinet (Controlled Environments Ltd., Winnipeg, MB) was used but due to its malfunction in 2004, a Conviron model PGR15 cabinet was used instead in 2004.

Color Analysis
Color L*, a*, and b* values were measured using a Hunter Lab colorimeter (model No 45/0-L MiniScan XE, Hunter Associates Lab Inc., Reston, VA). The Commission Internationale de L'Eclairage (CIE) recommended the use of the L*a*b* color system as it is similar to how the ganglion cells in the human eye sense the amount of green or red, the amount of blue or yellow, and the amount of lightness or darkness (Marcus, 1998). The L*a*b* color system is advantageous as it has an approximately uniform color scale and provides a way to compare color values between different samples (Marcus, 1998). The L*a*b* color system uses three axes to describe color: the L* values run on the z axis with 100 being perfect white and 0 being perfect black, the a* values run on the x axis with positive values being more red and negative values being more green, and the b* values run on the y axis with positive values being more yellow and negative values being more blue. Color measurements were taken every 7 d for bean seeds from the greenhouse and cabinet protocol and every 8 h for bean seeds from the UVC protocol. During each color measurement, the beans were randomly mixed and reoriented in the petri dish or plastic bag.

The greenhouse darkening protocol was terminated after 57 d in 2003 as the seeds had become infected with fungi and would have been destroyed had the darkening procedure continued. At this time point, there was a color difference of roughly 10 L* values and 4 a* values between the slow-darkening lines and the darkening lines. Ten L* values and 4 a* values were then used as a guideline for ending the other two darkening protocols.

Statistics were completed using the SAS System 8.2 (SAS Institute Inc., Cary, NC) using Proc GLM. Analyses of variance were conducted on the L* color values according to the outline by Baker and Briggs (1982). The basic analysis was a split-block with the factors being time and line. The combined analyses of data from 2 yr were as described in Section 14.31 of Cochran and Cox (1957) for combining experiments of unequal size. The pooled error mean squares were calculated by weighting the error mean squares from each year by their degrees of freedom.

When time was significant, it was partitioned to determine the linear and quadratic significance. When the linear and quadratic components were found to be significant, time by line was subdivided into linear and quadratic components to determine if the lines differed at any of these components. This allowed for linear equations to be determined (y = a + bx + cx²) and the regression coefficients—intercept, linear, and quadratic—to be compared using Fisher's Protected Least Significant Difference (LSD) test with a P 0.05 significance level. For easement of terminology, the intercept represents the color at harvest, the linear component is the slope or rate of change of color, and the quadratic component is the diminishing rate of color change. Color after harvest was also compared using Fisher's Protected LSD test with a P 0.05 significance level.

Seed Germination
Following darkening, seed germination tests were conducted on undarkened seed that had been stored in a cold room and on darkened seed from the three protocols. Seed germination tests were conducted in accordance with Canadian Food Inspection Agency Methods and Procedures for Testing Seed (Anonymous, 1997). Before germination, seeds were surface sterilized with 70% ethanol for 1 min and with 1.25% sodium hypochlorite for 10 min followed by three rinses with distilled water. Seeds were placed between two 38-lb rolled towels (Anchor Paper Company, St. Paul, MN) and the rolled towels were placed inside a pail in a seed germination cabinet (model 2015, VWR Signature Diurnal Growth Chamber, Sheldon Manufacturing, Inc., Cornelius, OR) set at 20°C with 8 h of light every 24 h. Three subsamples of 10 seeds were taken from each of the protocols and each of the replications and germinated consecutively. Replicate one and its three subsamples were germinated in 2004 and the three subsamples of each of replicates two and three were germinated in 2005. Data analysis was conducted using the SAS System 8.2 (SAS Institute Inc.) using Proc GLM with data subjected to analysis of variance (ANOVA) and protocol and treatments means compared using standard errors.

Environmental Effects
Beans are grown in indoor (greenhouse and phytotron) and outdoor (field and polyhouse) environments at different stages of the breeding program at the University of Saskatchewan. The objective of this experiment was to determine if there are significant interactions between different lines, growing environments, and darkening protocols. Nine known darkening pinto bean cultivars and one slow-darkening pinto bean breeding line, SC11743-3, were grown in the phytotron, greenhouse, polyhouse, and field at Saskatoon in 2003. Before darkening, the seed was stored in a dark, cold room set at 8°C to allow the moisture content of the beans to equilibrate. Seed was darkened via the greenhouse, UVC, and cabinet protocols as described previously. Data analysis was conducted using SAS System 8.2 (SAS Institute Inc.) using Proc GLM with data subjected to analysis of variance and means compared using standard errors.

Genotype by Environment Effects
The validity of the UVC light protocol was tested on seed grown under different field conditions in 2004. Nine known darkening cultivars and three slow-darkening breeding lines were grown in a randomized complete block design at four locations in Saskatchewan: Saskatoon, Davidson, Oxbow, and Outlook. The CDC's bean breeding program tests beans at all of these locations and these sites vary in their location coordinates, soil type, date of seeding, and growing degree days (Table 1). The Outlook site was irrigated while the other sites were dryland. The site at Saskatoon had a hail storm on 12 July. Saskatoon and Oxbow had an early frost on 20 August, and Davidson had a 10 October snowfall which delayed harvest until after the snow had melted (Table 1).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The color change of beans as they darken has never been studied before. This study not only looked at three different darkening protocols, but it also revealed how the beans darken over time. The best model to describe the color change over time is a linear model in the form of y = a + bx + cx², where a is equal to the intercept or color at harvest, b is equal to the slope or color change over time, and c is equal to the quadratic component of the equation or the leveling off of the curve in this case.

Figures 1 and 2 are representative of the three darkening protocols for the L* and a* color value response over time with the results of the darkening and slow-darkening lines pooled together, respectively. At harvest, the darkening and slow-darkening lines had significantly different L* values but did not significantly differ in their a* values. The nonsignificant difference for the a* value indicates that it is not possible to distinguish darkening from slow-darkening beans at harvest. However, the significant difference in the L* values most likely is due to the pooling of the darkening and slow-darkening lines together, respectively. When the lines are not pooled based on their darkening aspects, the slow-darkening lines cannot always be distinguished from the darkening lines as shown later. Over time the darkening and slow-darkening lines decreased in L* value but increased in a* value indicating that the beans were becoming darker and redder respectively, in a linear fashion. The darkening lines changed color at a much faster rate than the slow-darkening lines for both L* and a* values. Eventually, the rate of darkening began to diminish with the darkening lines diminishing more than the slow-darkening lines as indicative of the x² or quadratic component. This indicated that the darkening lines had reached their maximum color darkening sooner than the slow-darkening lines. At the end, the slow-darkening lines had significantly higher L* values and lower a* values than the darkening lines indicating the slow-darkening lines were lighter and less red.

View larger version (18K): [in this window] [in a new window]   Figure 1. The L* color value response (mean ± SE) of 15 darkening and three slow-darkening pinto bean lines during exposure to ultraviolet C light. LSD values: intercept = 7.88, slope = 0.06, quadratic coefficient = 0.0003, end point = 7.80.

View larger version (17K): [in this window] [in a new window]   Figure 2. The a* color value response (mean ± SE) of 15 darkening and three slow-darkening pinto bean lines during exposure to ultraviolet C light. LSD values: intercept = 0.59, slope = 0.06, quadratic coefficient = 0.0004, end point = 2.60.

Greenhouse Darkening Protocol
There was a significant year effect for the L* color values (P < 0.0001) which could be due to differences in the environmental conditions experienced during both the growing and the postharvest seed coat darkening seasons. In 2003, the growing season was warm, dry, and relatively long, whereas in 2004, the growing season was wet, cool, and relatively short due to an early frost on 20 August. For the 2003 darkening season, average daily accumulated light levels were approximately 2.8 x 10^–2 µmol m^–2 s^–1 compared to the 2004 darkening season which had roughly 3.2 x 10^–2 µmol m^–2 s^–1. The average air temperature inside the greenhouse was comparable in both years with temperatures ranging between 16°C (night) and 26°C (day). The time by year interaction was highly significant for the L* color values (P < 0.0001). Again, this could be a result of the differences in the conditions during the growing and darkening seasons. The L* color values changed significantly with time (P < 0.0001) and the lines had significantly different slopes (P < 0.0001).

With the greenhouse protocol, the L* color values at harvest could not be used to distinguish the slow-darkening lines from the darkening lines (Table 2). There were significant differences among the lines, but the slow-darkening lines could not be clearly distinguished from the darkening lines before darkening. The L* color value slopes could be used to distinguish the slow-darkening lines from the darkening lines as the slopes of the slow-darkening lines were larger for the L* values (Table 2). There were also differences among the slopes of the darkening lines. Similarly, the L* color following darkening could be used to distinguish the slow-darkening lines from the darkening lines, with the darkening lines having significantly lower L* values than the slow-darkening lines. However there were no differences among the darkening lines or among the slow-darkening lines following darkening (Table 2).

With the UVC light protocol, the L* color values at harvest could not be used to distinguish the slow-darkening lines from the darkening lines (Table 3). The L* value slopes could be used to clearly distinguish the slow-darkening lines from the darkening lines (Table 3). The slopes of the L* values were significantly larger for the slow-darkening lines than the darkening lines. The L* color values following darkening were clearly different between the darkening lines and the slow-darkening lines with the darkening lines having significantly lower L* values than the slow-darkening lines (Table 3). There were no significant differences among the darkening lines or among the slow-darkening lines.

For the cabinet protocol in 2003, the slow-darkening lines had slightly higher L* color values at harvest when compared with the darkening lines (Table 4). The slopes of the L* color values were larger for the slow-darkening lines compared to those of the darkening lines. After the cabinet protocol was completed, color was darker for the darkening lines than the slow-darkening lines (Table 4).

In summary, all darkening protocols could be used to distinguish slow-darkening from darkening lines on the basis of the rate of darkening as indicate by the slopes of the L* color value as well as by the final L* color value following darkening. The color at harvest was not indicative as to whether or not a line was darkening or slow-darkening.

Seed Germination
The results of the seed germination study indicated that the lines did not differ in percentage of germination (P = 0.82). This was interesting since the seed coats of the slow-darkening lines have less condensed tannins than the darkening lines (Beninger et al., 2005) and one might expect the slow-darkening lines to have lower germination than the darkening lines, based on the results in other crops. Tannin-free faba beans (Vicia faba L.) and zero-tannin lentils (Lens culinaris Medik.) have poorer germination in the field than tannin-containing lines, although this may be due to their increased susceptiblity to soil-borne pathogens (Kantar et al., 1996; Matus Munoz, 1991). However, the seeds in this germination study were surface sterilized before germination indoors in clean, sterile conditions. Gesto and Vazquez (1976) found that fresh bean seeds with lower levels of phenolic compounds germinated readily indoors while 5-yr-old seeds with considerably higher levels of phenolic compounds had a low rate of germination indoors. Caryopsis tannin content in shattercane [Sorghum bicolor (L.) Moench] was shown to be negatively correlated with seed germination indoors but positively correlated with seed germination in the field (Fellows and Roeth, 1992).

The protocol used to darken the seed coats significantly affected seed germination (P < 0.0001). Seed darkened using the UVC protocol had a high percentage of seed germination (94% ± 0.02) that was not significantly different than the percentage of seed germination for the nondarkened seed (93% ± 0.02). Both the cabinet and the greenhouse protocol seed had low germination (53% ± 0.03 and 52% ± 0.07, respectively) and they were not significantly different from one another.

A possible explanation for the difference in percentage of germination between the protocols was that the conditions experienced during the cabinet and the greenhouse protocols not only darken the seed coats of the beans, but also age the cotyledons and embryos of the seed while the UVC protocol only darken the seed coats of the beans and does not age the cotyledons or embryos of the seeds. It has been shown that when beans have been stored under high humidity, high temperature, and light, the germination declines (Barrón et al., 1996; Gesto and Vazquez, 1976) whereas beans that are subjected to ultraviolet or cool-white light, without high temperatures or high humidity, have little loss in germination (Hughes and Sandsted, 1975). As well, a previous study showed that the cooking time for UVC light darkened beans was not significantly different from that of untreated, fresh beans which again suggests that the UVC light protocol was only affecting the bean seed coat and not the cotyledons (unpublished data, 2004).

Environmental Effects
ANOVA was conducted on the L* values of the slow-darkening and darkening lines grown in the field, the polyhouse, the phytotron, and the greenhouse. Analysis of the data indicated that time, protocol, line, environment, g x e, protocol by line, and protocol by environment were all highly significant (all P < 0.0001). It was suspected that the differences among the darkening lines had created significant g x e interaction so the analysis was repeated again but with the lines classified as either slow-darkening or darkening. In this case, the g x e interaction was found to be nonsignificant (P = 0.7702). Thus while the darkening lines responded differently to environments, the slow-darkening trait was stable across indoor and outdoor environments. The significant protocol (P < 0.0001), protocol by line interaction (P = 0.0163), and protocol by environment interaction (P < 0.0001) were to be expected as each of the protocols darkened the beans to a different final color at the time of color scoring.

For the means of the L* values of the slow-darkening and darkening lines following darkening, the slow-darkening line was significantly lighter (L* = 34.34 ± 0.22) than those of the darkening beans (L* = 28.78 ± 0.11), as expected. The outdoor environments (polyhouse L* = 30.23 ± 0.27; field L* = 29.83 ± 0.22) produced significantly lighter beans than the indoor environments, with the greenhouse producing the darkest beans (phytotron L* = 28.96 ± 0.20; and greenhouse L* = 28.53 ± 0.19). Seed produced in the greenhouse and phytotron most likely had seed coats darker than the others as these seeds were not harvested in as timely a manner as was the case in the other environments. Thus, the seeds were physiologically mature in the pods and were beginning to darken naturally in the phytotron and the greenhouse whereas the seeds did not have as much time to darken in the field or polyhouse.

Genotype by Environment Effects
Analysis of the L* color values of the undarkened seed grown in four different locations revealed that line, environment, and the g x e interaction were all significant (all P < 0.0001). Differences among the L* value means at harvest at each location were minimal and the slow-darkening lines could not always be distinguished from the darkening lines. Thus, good seed coat color at harvest could be the result of adaptation and vigor as well as expressing the slow-darkening trait. As well, the L* value differences among the darkening lines was minimal and the relative ranking was different across the different environments. These results may explain why there is an ongoing debate in the pinto bean industry as to which of the currently sown varieties produces the lightest color seed coat.

Analysis of the L* values of the seed coats after accelerated darkening using UVC light, revealed that environment and line effects were significant (both P < 0.0001) but the g x e interaction was no longer significant (P = 0.29). Thus, regardless of the environment, the darkening lines always produce darker beans (lower L* value) and the UVC light protocol can be used to differentiate the slow-darkening and darkening lines (Table 5). It should be noted that lines such as CDC Pintium, Maverick, and Bill Z which are known to have good color do darken with age like other conventional lines. In this experiment, Bill Z most likely had a poor color relative to the other darkening pintos because it was poorly adapted to the short growing environment. This highlights the importance of harvest conditions when predicting color without darkening, particularly among conventional lines.

These results differ from the findings of a study involving seed coat darkening of lentil, where Vaillancourt and Slinkard (1985) found environment, line, and g x e to be significant following darkening. However, Vaillancourt and Slinkard (1985) suggested that the g x e interaction was significant for their study because one location received rain in August, causing those lentils that had already matured to darken while the late-maturing cultivar Laird, which had not fully matured at that time, did not darken. As a result, Laird had a much lower rate of darkening at their Hagen location compared to other cultivars at that location. These results also differ from a study by De Mejía et al. (2003) who studied trypsin inhibitor content and tannin content of beans from the Jalisco and Durango races and found the g x e interaction to be significant. However since genotype contributed three and a half times more in terms of the mean square value than the combined contribution of site and site by cultivar interaction they stated that it would be feasible to select against tannin content in beans (De Mejía et al., 2003).

Darkening Protocol for the Future
There are other factors that make the UVC light protocol more advantageous over the cabinet and greenhouse protocols. At the time of this study, the UVC light protocol was less expensive than the greenhouse and the cabinet protocols which cost 8 and 42 times more than the cost of the UVC light protocol, respectively. Setup time for the UVC light protocol is quick unlike the greenhouse protocol which requires long periods of time to insert the beans and moistened pieces of felt into plastic bags in such a manner to prevent the beans from touching.

The UVC light protocol could be used in many situations. Plant breeders could use the protocol to screen early generations for the slow-darkening trait. It could even be used to differentiate the lighter darkening beans and the lightest slow-darkening beans. Before releasing breeder seed to seed growers, sublines could be screened to ensure homogeneity. Likewise, growers, buyers, and sellers of the slow-darkening beans could use the UVC light protocol to ensure a homogenous product. Researchers interested in how seed coat darkening is related to other traits and agronomic practices would find this protocol useful. Although not scientifically tested, the UVC light protocol could probably be used for market classes of beans other than the pinto that are also known to darken as well as other pulse crops with darkening seed coats such as lentil and faba bean.

Based on the results from this study, the following darkening protocol is suggested as a means of differentiating between darkening and/or slow-darkening lines that darken at different rates. First, grow the beans to be tested so that the harvest date is the roughly the same for each line to prevent natural seed coat darkening from affecting the results. While harvesting the lines, store the harvested beans in cold, dark, dry conditions, so that they are subjected to the same temperature, humidity, and light conditions. Allow all of the beans to equilibrate in these conditions but do not prolong storage. Then, place the beans in open petri dishes, 10 centimeters below a 254-nm UVC lamp for 120 h or more without any disruption to the beans. Longer periods of darkening may be required to differentiate lines that have similar darkening rates.

For this study the color of the beans was always measured using a Hunter Lab colorimeter. However, in some of the above-mentioned scenarios Hunter Lab color readings would not be necessary as the difference between slow-darkening and darkening pinto beans is so great that they can be differentiated easily with the human eye.

If a Hunter Lab colorimeter is used to measure the color change in the beans, record the L* values of the beans before darkening. After darkening the beans, record the L* color value of the beans again with a Hunter Lab colorimeter. Best color measurements are obtained when the color of the sample is read three times and the color results are averaged. For data analysis, one can compare initial seed coat color and final seed coat color, and/or rate of seed coat color change depending on the user's needs.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
In summary, regardless of how the beans were darkened, the beans always became more red, as indicated by increasing a* values, and darker, as indicated by decreasing L* values, in a linear fashion with eventual diminishing of color change. Even though all darkening protocols could be used to distinguish slow-darkening lines from darkening lines, the UVC light protocol was the most useful as it was the most reliable over years, it was the fastest, and the seed had the least effect on germination following darkening. When compared to the UVC light protocol, the cabinet protocol was unfavorable as the period to darken the beans was long and depended on the cabinet model used, and the seed had a low percentage of germination following darkening. The greenhouse protocol was the most unfavorable protocol as the time required to darken the beans was greater than the UVC light protocol, and the seed had a low percentage of germination following darkening. The greenhouse protocol was also subject to seasonal darkening conditions, and the seed can become infected with fungi during darkening as occurred in 2003. Finally, the UVC light protocol could be used to distinguish slow-darkening pinto beans from darkening pinto beans, regardless of whether the beans were grown indoors or outdoors or even under different field environments.

	ACKNOWLEDGMENTS

 
We thank Dr. P.J. Hucl for allowing the use of his cabinet in 2003–2004. Funding for this project was provided by the Saskatchewan Pulse Growers and the Saskatchewan Agriculture and Food Agriculture Development Fund.

Received for publication May 17, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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