EFFECTS OF DIFFERENT TEMPERATURES AND HORMONE TREATMENTS ON BREAKING DORMANCY IN POTATO TUBERS

Mini propagation is a new method to produce potato. A factorial experiment based on completely randomized block design with three replications was conducted to investigate the effect of two temperature levels (18 and 25°C) and six hormone treatments (H1: 0% of ethanol + 0 mg/l of gibberellic acid, H2: 0% of ethanol + 10 mg/l of gibberellic acid, H3: 0.5% of ethanol + 0 mg/l of gibberellic acid, H4: 0.5% of ethanol + 10 mg/l of gibberellic acid, H5: 0.5% of ethanol + 20 mg/l of gibberellic acid, H6: 0.5% of ethanol + 30 mg/l of gibberellic acid) on mini tubers of two varieties of potato (Agria and Santé). The longest sprouts were about 14.13 mm long and they were produced by Santé variety at 18°C and H1 hormone level. Sprouting percentage was higher in Santé (75.69%) than in Agria (59.72%). The highest average percentage of sprouted tubers (95%) was achieved at the temperature of 18°C and with H4 hormone treatment. Santé had the shortest dormancy duration by using H1 hormone level at 18°C. Applying H6 hormone level at 18°C produced more sprouts per tuber in Santé. Santé was better than Agria with or without hormone treatment in respect of all measured traits. Higher GA concentration accelerates tuber sprouting in both varieties.


Introduction
Potato (Solanum tuberosum L.) is ranked fourth in the list of major world food crops.Potatoes are propagated in Iran traditionally using tubers.Low propagation rate, low efficiency, high risk of disease and pest transmission, demanding intensive care for propagation and high amounts of tubers (because of their larger size) are some of the undesirable aspects of using tubers for growing new plants.Mini propagation refers to the in vitro production of miniature potato tubers (mini tubers) either directly from auxiliary buds or stolons which grow from leaf axils in mini propagated potato plantlets (Espinoza et al., 1986).A great problem in using mini tubers is dormancy.Freshly harvested mini tubers like field-grown tubers, exhibit a period of dormancy; they do not readily sprout even if environmental conditions are favorable, including optimum temperature and humidity (Knowlesand Kumar, 2002).Dormancy duration is different in different potato varieties (Claassens et al., 2005).It could depend on the tuber growth conditions (Mohammadi et al., 2014).Response of different varieties to chemical components is different too (Emilson, 1999).Mini tubers have a 2-to 3-month-long dormancy phase which is related to the variety and light treatments (Sarkar et al., 1997).
Mini tubers have low sprouting rate which results in tuber rot and decreased sprouting rate.In vitro produced mini tubers have advantages compared with conventional field-grown tubers; they have high quality, produce higher yield and are aseptic.Freshly harvested mini tubers exhibit a period of dormancy (Kasrawi and Al-Fayyad, 1989;Lommen, 1995).Habib (1999) stated that mini tubers have a higher ABA (abscisic acid) content than field-grown tubers, which results in longer rest period.Dormancy breaking results in uniform tuber sprouting.It also decreases growing season and increases yield (Otroshy and Struik, 2006;Alexopouloset al., 2007a;Mohammadi et al., 2014).Treating tubers with chemical components breaks dormancy.The research on dormancy in potato tubers has been reviewed by Claassens and Vreugdenhil (2000) and Suttle (2004).It has been shown that gibberellin (GA) can break dormancy in tubers.According to Carlo et al. (1996) the ratio of ABA to GA is a regulating factor in tuber dormancy.If the ratio is in favor of GA, sprouting will happen and dormancy will be terminated.Oktay (2011) reported that GA breaks sprout dormancy in potato.Dogonadze et al. (2000) investigated the effect of GA and auxin on ABA and ethylene synthesis in dormant sprouts of potato.Results showed that internal content of ABA decreased by using GA.Salimi et al. (2009) showed that GA breaks dormancy of two potato varieties by 35-72%.Rehman et al. (2001) investigated the effect of different pre-treatment components on potato tubers.They reported that dormancy duration was shortened in all varieties by applying chemical compounds.Sasani et al. (2009) conducted an experiment to examine the interaction between hormones and temperature on mini tuber dormancy.The longest sprouts were produced by applying 5 mg/l of GA at 10°C.GA is responsible for cell elongation and may play a role in stimulating cell division in meristimic areas (Galun, 2010).Alexopoulos (2007a) reported that interaction between GA and thiourea accelerates sprout emergence and decreases dormancy duration.The potential of ethanol to break dormancy in tubers was demonstrated too (Petel et al., 1993).Ethanol treatment, combined with a low level of sucrose resulted in the growth of sprouts (Claassens et al., 2005).In some regions of Iran, it is possible to grow potato two times a year.Thus, it is necessary to have tubers with short dormancy duration.This experiment was designed to find out the best concentrations of GA and ethanol and favorable temperature for successful breaking of dormancy in potato mini tubers.

Material and Methods
The experiment was carried out at Torbat-Heydarieh University from April to June, 2013.The station is at 34°17'N latitude and 59°12'E longitude and 1,333 m altitude.A factorial experiment based on completely randomized block design with three replications was conducted.Seventy-two Petri dishes were selected as growing beds.Each Petri dish contained 4 tubers.Mini tubers of two varieties of potato (Agria, Santé) were considered as the first factor.Tuber characteristics of studied varieties are presented in Table 1.The second factor was temperature (18 and 25°C) and the third factor included different concentrations of GA and ethanol combination (Table 2).Mini tubers of each treatment were separately soaked in desirable solutions at 18°C for two hours.Based on experimental design, four mini tubers of each treatment were placed in Petri dish, and then they were placed in incubators at 18 and 25°C and 85% relative humidity.Sprouts were counted each day.Tubers with a 2-mm sprout were assigned as germinated tubers.Sprouting of more than 80% of total tubers in each treatment was assigned as dormancy break and traits were measured in such tubers.Sprout length was measured by caliper.Sprouts were counted daily and sprouting rate and percentage were calculated (Salimi et al., 2009).Results were analyzed by using SAS version 8.8.Means were compared by using Duncan's multiple range test at p≤0.05 and p≤0.01 significant levels by MSTATC software.

Results and Discussion
The results of analysis of variance of different traits are shown in Table 3.The results showed that the interaction between hormone concentration, temperature and variety was significant on sprout length.The longest sprouts were about 14.13 mm long and were produced by Santé variety at 18°C and H 1 hormone level (Table 4).Our results were in agreement with Otroshy and Struik (2006).Hartmans and van Loon (1987) showed that storage of tubers at higher temperatures increased sprout length.Alexopoulos et al. (2007a) reported that GA is more effective in sprout length than benzyl adenine at the same temperatures.Suttle (2004) found that exogenous GA promoted tuber sprout growth.GA is responsible for cell elongation and may play a role in stimulating cell division in meristimic areas (Galun, 2010).GA promotes cell division and cell elongation in the sub-apical meristem.High GA levels might keep the transversal cortical microtubular cytoskeleton stable so that cells in the sub-apical region divide transversally, and cell elongation will hence result in sprout elongation (Shibaoka, 1993).In each column means with the same letters show no significant differences.
There was a significant difference between varieties in respect of sprouting percentage (p≤0.01).Sprouting percentage was higher in Santé (75.69%) than in Agria (59.72%).Potato species differ in their level of dormancy.Many of these differences among species have been associated with adaptation to the environment of their origin (Mohammadi et al., 2014).The interaction between variety and temperature does not have a significant influence on sprouting rate.This means that both varieties had the same reaction to temperature in respect of producing sprouts.
Interaction between hormone level and temperature has a significant influence on sprouting percentage (p≤0.01)(Table 3).The highest average percentage of sprouted tubers (p≤0.05) was found at the temperature of 18°C and H 4 hormone treatment.H 4 hormone level at 25°C reached 41% which was the lowest percentage (Table 4).Higher temperature produces an increase in low GA concentration in respect of sprout percentage (Table 4).Sasani et al. (2009) reported that high temperature results in higher sprouting percentage.Dormancy is regulated by three growth regulators: abscisic acid, gibberellic acid, and cytokinin.GA stimulates cell division in meristimic tissue and increases the number of sprouts (Galun, 2010).
The interaction between temperature and hormone level shows a significant influence on sprouting rate of different varieties (p≤0.01)(Table 1).This means that different varieties had different responses to various combinations of hormone and temperature levels.The highest rate of sprouting was 0.404 and it was obtained by use of H 1 hormone at 18°C.Alexopoulos et al. (2007b) reported that GA accelerates tuber sprouting at 20°C.Van Ittersum and Scholte (1992) showed that temperature between 30-32°C decreases dormancy duration.They reported that sprouting increased at higher storing temperature.GA stimulates sprouting by activating certain phases of cell division cycle and will accelerate bud growth (Hill, 1980).
The interaction between hormone, temperature and variety significantly influenced tuber dormancy duration (p≤0.05)(Table 3).Santé and Agria tubers have shown the shortest and the longest dormancy duration by using of H 1 hormone at 18°C, respectively (Table 4).The results were in agreement with Van Ittersum and Scholte (1992), who reported that dormancy duration decreased at higher temperatures.Kasrawi and Al-Fayyad (1989) stated that application of GA alone or in combination with thiourea broke dormancy and increased the number and the length of sprout.Sasani et al. (2009) declared that applying high GA concentration breaks tuber dormancy and higher temperature accelerates dormancy break in mini tubers of potato.
When GA is applied on dormant tubers, dormancy can be broken according to Sharad (2008) and Fernie and Willmitzer (2001).GA is responsible for the shortening of the rest period of freshly harvested potatoes, hastening the sprouting and emergence (Aksenova et al., 2013).
In potato, the first signs of the release of bud dormancy are manifested at the cellular level (i.e.mitosis preceding cell division and elongation) and these are evident long before the bud growth is visible.GA may terminate dormancy by activating the synthesis of DNA and RNA (Sharad, 2008).Dormancy breaking results in better tuber sprouting, regulation of sprouting time and decreasing of growing season (Otroshy and Struik, 2006).
The interaction between hormone, temperature and variety has a significant influence on active sprout number (p≤0.01) (Table 3).In Santé variety, H 6 hormone treatment at 18°C produced more sprouts per tuber.The results indicated that a lower concentration of GA at 18°C resulted in lower sprout number (Table 4).The results also showed that 25°C with zero level of GA produced more sprouts in both varieties compared with 18°C (

Table 1 .
Tuber characteristics of studied varieties.

Table 2 .
Different levels of hormone concentrations used as the third factor in the experiment.

Table 3 .
Analysis of variance at different hormone concentrations and temperatures in two potato varieties.

Table 4 .
Comparison of means using Duncan's multiple test.