Assessment of dormancy and sprouting behavior of elite and advanced clones
C. Carli, E. Mihovilovich, and M. Bonierbale
In potato breeding and selection, storability or keeping quality of potatoes should be regarded as equally important as yield, disease resistance, and quality. It is one of the considerations that need to be evaluated before releasing any variety so that farmers are able to store their produce for a desired period of time at their farm under traditional storing conditions or in refrigerated storage infrastructure, depending whether the end-use is for fresh consumption, processing, or planting as seed. For estimating keeping quality of a particular potato clone, its dormancy period, sprouting behavior, and weight loss are major criteria that should be documented before any promising clone is released.
The present protocol is formulated to support the assessment and documentation of the dormancy period, sprout growth and weight loss of CIP’s elite and advanced potato clones. This information will guide farmers to manipulate sprouting to occur only when it is desirable.
Review of literature
Dormancy is a physiological state characterized by a period during which autonomous sprout growth will not occur even under optimal sprouting conditions, i.e., darkness, 15 to 20 °C, relative humidity about 90% (Wiersema, 1985). Dormancy should be regarded as the period in the tuber life cycle from initiation to the time when sprouting starts (Burton 1989). However, since this period is difficult to determine, post harvest dormancy is used for practical purposes and defined as the period from dehaulming to the time 80% of tubers show sprouts at least 2 mm long.
Dormancy is considered to be a varietal character yet influenced by environmental and management conditions. Since dormancy is not related to earliness of varieties, it is possible to breed late varieties with relatively short dormancy and early varieties with relatively long dormancy (Beukema and van der Zaag, 1979). Dormancy period depends on soil and weather conditions during growth, tuber maturity at harvest, storage conditions, and whether the tuber is injured or not (Ezequiel and Singh 2003). High temperatures, low soil moisture and fertility during tuber growth accelerate physiological development and reduce the dormant period. On the other hand, tubers harvested at an immature stage have a longer post-harvest dormancy than tubers harvested at maturity. Regarding storage, fluctuating storage temperatures shorten dormancy more than constant high temperatures. Therefore, storage temperatures should remain as consistent as possible when retarding sprout development is desired. Finally, tuber injuries caused by harvest or by diseases and pests, can result in earlier sprouting.
Sprouting is a physiological stage that commence when dormancy is broken. It is the major visible milestone in determining tuber physiological age. The earliest observable stage of sprouting is characterized by visible small white buds, often termed “pipping” or “peeping” (Daniels-Lake and Prangel, 2007). The physiological age of the tuber has a great effect on the pattern of sprout growth but the basis is genetic. In turn, the physiological age of the tuber is greatly influenced by growing conditions, storage conditions, and length of storage period.
Patterns of sprout growth:
Apical dominance: This is a physiological phenomenon characterized by the exhibition of a dominant bud over the others, that is suppressing the sprouting of other buds (Pavlista, 2004). The suppressing bud is at the apical end of the tuber, which is the furthest bud from where the tuber was attached to the vine. Physiological young tubers exhibit apical dominance and thus the apical sprout will need to be removed (de-sprouted) for the other buds to develop sprouts.
Multiple sprouting: This pattern develops gradually in time as apical dominance diminishes, and is characterized by the appearance of several buds sprouting along the tuber. The duration of apical dominance as well as the number of sprouts per tuber is a varietal characteristic (Sunoschi 1981, van Es and Hartmans 1987).
Middle aged tubers exhibit multiple sprouts, and are at the optimum stage for planting. However, as mentioned before, this pattern can be induced in young tubers by removing the apical sprout, although apical dominance may be reinstated by growing of the next bud closest to the apical end (Pavlista, 2004). In such a situation, a second de-sprouting will be necessary for inducing more sprouts (Beukema and van der Zaag, 1979).
Branching This pattern appears as middle aged tubers age further. Since sprouts are comprised of multiple nodes with meristematic tissue and leaf primordia at each node, branching occurs when apical dominance within the sprouts is overcome, either after the sprouts are sufficiently large because of tuber senility, or following damage to the apex. These branches are referred to as “hairy” because they tend to be weak. Even more, old tubers may also show a proliferation of small stolons (Daniels-Lake and Range, 2007).
Effect of tuber size on sprout growth: Since the sprout depends on the tuber for the materials for growth, if there are several sprouts on the tuber, an inter-sprout competition for growth factors will be imposed by the size of the tuber (Burton, 1989). With fairly large tubers, no effect of size will be noticeable, but with decreasing size, a point can clearly be reached at which growth will be impaired. This competition has been shown to be independent of the distance between the competing sprouts, suggesting that it is not a local matter but of growth factors distributed throughout the tuber (Morris, 1966; cited by Burton, 1989).
Weight loss: This trait determines the longevity of tubers’ storability and hence their keeping quality. Variations in weight loss among cultivars are attributed to either their periderm characteristics and/or their sprouting behavior. Weight loss in unsprouted tubers occurs through the periderm and for a minimum proportion through the lenticels. Hence varieties with a thicker periderm (a greater number of cell layers in the periderm) and lesser number of lenticels on the tuber surface lose less weight than their counterparts (Ezekiel et al., 2004 and Pande et al., 2007). On the other hand, sprouted tubers loose much more weight than unsprouted tubers. After the onset of sprouting the rate of sprout growth and number of sprouts determine the weight loss in potatoes (van Es and Hartmans, 1987). Greater water loss with sprout growth occurs because of the high permeability of sprout wall to water vapor. A significant correlation between weight loss and both, the length of the longest sprout and number of sprouts per tuber was encountered by Pande et al. (2007).
Physiology of tuber dormancy and sprouting.
Dormancy and sprouting are controlled by the interactions of major plant growth regulators, predominantly the ratio of gibberellin (GA) and abscisic acid (ABA). ABA has been suggested as important to maintain dormancy, whereas the role of GA has been clearly determined in dormancy breakdown (Fernie and Willmitzer, 2001). On the other hand, some evidence has also implicated indole acetic acid (IAA), an auxin, in sprouting. IAA has been suggested to mediate the suppression of sprouting of lateral axillary buds by apical dominance (Pavlista, 2004).
Quantitative trait loci (QTL) analyses have indicated that tuber dormancy is controlled by at least nine distinct loci (van den Berg et al., 1996). The potential role of ABA in dormancy has also been supported by the observation of three of these QTL influencing ABA levels (Classens and Vreugdenhil, 2000).
Sprouting is associated with many physiological changes including the conversion of starch to sugars, respiration, water loss, and glycoalkaloid content (Burton, 1989). Although cool temperatures during storage can prolong the dormancy period, they generally result in an increase in reducing sugar content, primarily glucose, which is undesirable in the processing industry due to darkening of fried products. Low temperature storage is not appropriate for potatoes destined for the processing market. On the other hand, visible sprouts on potatoes are unacceptable to consumers.
Use at least 30 tubers of a calibre ranging from approximately 50 to 70 mm (Category 2) per test clone. Try to select tubers of uniform size within clones. Tubers with bruises or skinning damage must be avoided for storage
- Follow the practice of dehaulming i.e, cutting of haulms, by sickle or killing by chemicals (e.g. Gramoxone), 10-15 days before crop harvesting.
- Stop irrigation about two weeks before dehaulming.
- Select tubers without bruises or skinning damage to make them less susceptible to rot diseases during storage.
- Clean tubers from excess of soil or debris which can promote rot. It is recommended to clean them carefully by hand. Do not wash potatoes, dampness can cause decay.
Dry the harvested tubers and cure skin at 10 to 20oC and 85 to 95% relative humidity for 15 to 20 days. Avoid temperatures above 25oC. This period will allow wound healing and promote maturity of tubers.
Clean and repair your storage before potatoes are introduced. Equipment in the cold store should be examined for satisfactory storage operations.
Storability of the selected clones should be assessed under traditional storage (at ambient temperature) and cold storage conditions at 2-4°C and 95% relative humidity (RH). Maintaining this RH will minimize shrinkage of tubers.
Assessment of dormancy and sprouting pattern parameters of advanced and elite clones at CIP-Headquarters is performed under diffused light storage with natural ventilation and cold storage conditions.
The trial should last 4 months under diffused light storage and 6 under cold storage. However, this depends on the expected duration of the dormancy period of test clones and the location. A period of at least 45 days is required after dormancy is released[1|#_ftn1] to allow for assessment of sprout growth pattern. Under traditional storage, in areas where diffused light storage cannot be implemented due to the presence of strong winter as in the temperate geographic area, the trial may last up to 6 months depending on storage temperatures. Also in this case we should allow a period of 45 days to assess sprout growth for the clone(s) with the longest dormancy period before the end of the trial.
Experimental units should consist of at least 15 tubers that may be placed on trays. If available, you may use experimental units of up to 30 tubers. Tubers should be identified by enumerating them on the skin using a permanent marker. Treatments (test clones) are replicated at least twice (two experimental units/test clone). Replications of treatments should be randomized at the storage site following a completely randomized design (CRD).
The weight of tubers should be recorded before storage.
a) Tuber growing conditions
Since dormancy and sprouting of a given cultivar are greatly influenced by the geographic location and growing season, it is required to indicate the geographical growing area and weather conditions under which the tubers were grown. Data recommended for recording:
- Eco-geographic growing conditions, example Tropical lowlands, Temperate highlands or lowlands, etc..
- Photoperiod (daylight hours)
- Monthly average maximum and minimum air temperatures (oC)
- Monthly average relative air humidity (%)
- Monthly average precipitation (mm)
- Soil temperature (10 cm depth) (oC)
b) Storage conditions
- Storage system (cold store, diffused light store, cellars, etc.)
- Temperature (monthly average): Maximum, minimum and mean (oC)
- Relative humidity (RH) (monthly average)
c) Additional information
- Number of days from planting to haulm-cutting
- Number of days from haulm-cutting to harvest
- Number of days after harvest and before entering store (period of skin curing)
- Storage dates (beginning-end)
d) Evaluation parameters
The dormancy period should be counted as number of days from haulm cutting to sprouting of 80% of the tubers (12 tubers in each experimental unit) with at least one sprout longer than 2 mm. Tubers should be checked at 10 day-intervals for monitoring sprouting initiation and growth, and to accurately record the dormancy period
- Initial tuber weight (g): Measure the weight of each tuber before the tubers are put into storage
- Intermediate tuber weight (g): Measure the weight of each tuber when dormancy is released
- Final tuber weight (g): Measure the weight of each sprouted tuber of a test clon 45 days after dormancy release in trials conducted under diffused light storage and 60 days in trials conducted under cold storage. Prior to weighing each tuber, remove sprouts carefully.
*Variables for sprout growth should be recorded in each test-clone 45 days after dormancy release under diffused light storage, and 60 days under cold storage. *
- Number of sprouts per tuber: Count the number of sprouts per tuber
- Length of the longest sprout (mm): Measure the longest sprout of each tuber
- Length of lateral axillary sprouts (mm): Measure the length of each lateral axillary sprout of each tuber (mm). Then take the average of lateral axillary sprout length per tuber, i.e, sum the length of the lateral axillary sprouts and divide them into the total number of lateral axillary sprouts.
- Sprout thickness (mm): Measure the thickness of the apical sprout and of one or two lateral axillary sprouts (if present, measure the thickness of the longest ones) of each tuber. Then take the average of sprout thickness per tuber.
Tuber weight loss
Calculate per tuber, the percentage of weight loss through periderm and lenticels, also known as “weight-loss percentage of unsprouted tubers”
% of weight loss-_unsprouted tuber = initial_tuber-weight - intermediate_ tuber-weight x 100
Estimate mean percentage of weight loss of unsprouted tubers per test clone and replication
Calculate per tuber, the percentage of weight loss due mainly to number of sprouts and sprout growth, also known as “weight-loss percentage of sprouted tubers”
Estimate mean percentage of weight loss of sprouted tubers per test clone and replication
Estimate the mean per clone and replication of each sprout growth variable.
Example for “length of lateral sprouts”
Analysis of Variance
Use the data “number of days from haulm cutting to sprouting (dormancy period)”, and means per test clone and replication of “percentage of weight loss of unsprouted tubers”, “percentage of weight loss of sprouted tubers”, “number of sprouts per tuber”, “length of the longest sprout”, “length of lateral axillary sprouts” and “sprout thickness” to perform the ANOVA for a CRD. Estimate the means and standard errors of the analyzed variables for each test clone. It could be of interest to compare means between test clones for some variables. LSD can be used for this purpose. The analysis can be run in R or in any other statistical software.
Dormancy period: Tubers stored at low temperatures such as cold storage (2-4oC) have longer period of dormancy than those stored at higher temperatures such as diffused light storage at ambient temperature (18-20oC). Accordingly, three categories could be suggested to document test-clones for their dormancy period (Table 1). However, ranges of dormancy period within each category may vary depending on tuber growing conditions, and temperature conditions under other storage systems i.e. traditional storage.
Table 1. Categories and dormancy periods suggested for documenting advanced clones
Cultivars with longer dormancy period are believed to perform better under non-refrigerated storage conditions*.* Consequently, cultivars with a long dormancy period and significantly lower percentages of weight loss of unsprouted tubers could be suitable for fresh consumption or processing after long periods of storage (up to 3 months or somewhat longer). Comparison of test clones should be performed for this variable.
Weight loss of sprouted tubers is of great concern as cultivars with a high percentage of weight loss age faster and consequently show disadvantages over young seed during their growing season, such as less vigor, shorter tuber bulking and smaller tubers (Pavlista, 2004). Documenting this variable along with dormancy period provide a good reference for deciding the best storage conditions and time for planting of new varieties. Comparison of test clones should be performed for this variable. Clones with less percentage of weight loss of sprouted tubers maintain an adequate condition for planting for a longer time.
A single apical sprout or an average less than 2 sprouts per tuber observed at the end of the test indicate the prevalence of an apical dominance, even if the additional sprout shows a growth rate almost similar to that of the apical sprout. An average of less than 3 developed sprouts, of which one is the apical, may still be considered a partial dominance, while an average number of 3 or more sprouts per tuber indicates the absence of apical dominance or a pattern of multiple sprouts. However, remember that the number of sprouts per tuber is a genetic characteristic. Multiple sprouting cultivars are desired over those with apical dominance as they give rise to plants with several stems and consequently with greater yields. On the other hand, the presence of multiple sprouts is usually accompanied by greater weight loss of sprouted tubers, thus this latter parameter should be taken into account in multiple sprouting cultivars for deciding best storage conditions or storage period.
Length of the longest sprout along with mean length of lateral axillary sprouts are good indicators for determining the sprout pattern of a cultivar, viz, a negative correlation exist between number of sprouts and length of the longest sprout (Singh and Ezekiel, 2003). Pande et al. (2007) observed examples of cultivars with prominent apical dominance that clearly showed a faster rate of growth of their apical sprout and of multiple sprouting cultivars that observed a slower rate of growth of their longest sprout.
Finally, mean sprout thickness though not correlated with sprout number, nor with length of the longest sprout or dormancy period is not less important, as vigorous sprout growth is associated with a greater resistance to infection to certain diseases such as Rhizoctonia and blackleg. Since sprouts under diffused light storage are generally more vigorous, measure of sprout thickness is better recommended under this storage system.
Implementing dormancy and sprouting pattern protocol – Experiences from Uzbekistan
A preliminary trial was conducted in Tashkent (Uzbekistan) for assessing dormancy period and sprouting pattern of 17 clones comprising 12 advanced and elite CIP clones, and 5 varieties from INTA, Argentina. The growing season was from the first week of July to mid-October 2008 (second season at Tashkent, starting with long and ending with short photoperiods, and high to mild temperatures).
The crop was dehaulmed 100 days after planting and harvested 10 days later. Tubers were cured for 14 days.
The trial was performed under two storage systems: Traditional storage (cellar storage) with monthly mean maximum temperatures ranging from 14oC to 11oC, and minimums from 7oC to 4oC, and cold storage (2-4oC).
Ninety tubers of each of 17 clones were allocated to experimental units of 30 tubers placed in trays and randomized in three replications following a random complete block design (RCBD). A RCBD was chosen because the experiment‘s objectives were to test associations between variables in addition to documenting. The trial lasted 3.8 months in cellar storage and 5.7 months in cold storage.
Performance of clones under cellar and cold storage are shown in tables 2 and 3, respectively.
Table 2. Dormancy period and sprouting behavior of 17 clones under cellar storage (Tmax range 11-14oC Tmin range 4-7oC )
Table 3. Dormancy period and sprouting behavior of 17 clones under cold storage (2-4oC )
Dormancy period under cellar storage ranged from 77 to 115 days while that under cold storage ranged from 99 to 174 days. There was a relatively high positive correlation (0.61) for dormancy period between storage systems indicating that clones with longer and shorter dormancy period under one system will also be those with longer and shorter dormancy under the other system. However this was not always the case. Clone 390663.8 that was among those with longest dormancy period under cold storage was not among those with the same tendency under cellar storage (see tables 2 and 3 for comparison).
Dormancy periods of the test clones under cellar storage were well suited to the ranges proposed for dormancy categories under diffused light in Table 1. According to those categories, the test clones could only be grouped into medium and long dormancy. Likewise, under cold storage, the test clones fell into the same categories, which agrees with the positive correlation found between dormancy period of the test clones under the two storage conditions. Clone 390663.8 was the exception as it was categorized as medium dormant under cellar storage and long dormant under cold-storage.
As opposed to the lack of correlation found between dormancy period and bulking maturity in previous studies (Beukema and van der Zaag, 1979), a low but statistically significant negative correlation (-0.40 under cold storage, and -0.20 under cellar storage) was found in this study. This result indicates that there is some tendency toward long dormancy in early bulking clones. However, further studies need to be performed to confirm this observation as very few clones were tested.
Mean sprout number per tuber was only assessed under the cellar storage system. This variable ranged from 1.7 to 6.3 sprouts per tuber. No statistical differences for mean number of sprouts were found among clones with 1.7 to 2.3 sprouts per tuber indicating that apical dominance was prevalent among them (see yellow cells for mean number of sprouts per tuber in table 2). On the other hand, clones with more than a mean number of 2.3 sprouts per tuber showed complete absence of apical dominance. Genetic differences may account for the number of sprouts per tuber among clones with absence of apical dominance.
A relatively high negative correlation (-0.51) was found between dormancy period and length of the longest sprout, indicating that clones with shorter dormancy often show a greater length of their longest sprout. However, this was not the case of the clone with the shortest dormancy period, 392797.22, whose longest sprout reached barely 9mm by the time the trial was over. The short length of its longest sprout can be attributed to the multiple sprouting pattern of the clone (4.7 sprouts/tuber). It has been shown in previous studies that multiple sprouting cultivars observe a slower rate of growth of their longest sprout (Pande et al., 2007). Despite these observations, no correlation was found between number of sprouts and length of the longest sprout in the present work. The negative correlation found between dormancy period and length of the longest sprout may have contributed to the absence of correlation between these two variables. For instance, clone 720150 that had the longest dormancy period under cellar storage was, as expected, among those with shortest length of their longest sprout. On the other hand, based on its apical sprouting pattern (1.7 sprouts/tuber) (Table 3), a faster rate of growth of their apical sprout would also have been expected, but because of its longest dormancy period, the growth of its apical sprout was initiated late and barely reached 8 mm by the time the trial was over (Table 3). A different case was observed for clone 388676.1 that despite its multiple sprouting pattern (6.3 sprouts/tuber) was among those with longest length of its longest sprout (20mm) (Table 3). The relatively short dormancy of this clone (95 days) may account for its long length of its longest sprout by the time the trial was over. However, since length of lateral sprouts were not recorded in this trial, it was not possible to discern if the apical sprout growth was slow or fast based upon the difference between length of the longest sprout and mean length of lateral sprouts. The influence of the number of sprouts on the length of the longest sprout can be better observed by estimating the difference between mean length of lateral sprouts and length of the longest (apical) one. When in addition to the apical sprout there are one or two lateral sprouts in some or several tubers of a clone, this difference allows determination of prevalence of apical dominance. Measurement should be performed on every lateral sprout of a tuber and an average calculated to then estimate mean length of lateral sprouts in an experimental unit.
Finally, percentage of weight loss per tuber showed similar ranges in both storage systems, from 5.0 to 8.0% in cellar storage and from 4.6 to 7.5% s in cold storage. Considering that 80% of the tuber content is water these values can be disregarded. Weight of sprouted tubers in this trial was measured without removing the sprouts, which may have contributed significantly to the final weight of the tuber. Hence, as proposed in the present protocol, weight of sprouted tubers must be measured after removing sprouts carefully.
The trial conducted under traditional and cold storage in Tashkent contributed to the characterization of dormancy period and sprouting pattern of 17 advanced and elite clones and to document them as shown in table 4.
Table 4. Dormancy and sprouting pattern behavior of 17 advanced and elite clones (Tashkent, Uzbekistan)
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Dormancy is considered released when 80% of the tubers have at least one sprout longer than 2 mm (van Ittersum and Scholte, 1992)