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The Effects of Sucrose on in vitro Tuberization of Potato Cultivars

Miheretu Fufa1* and Mulugeta Diro2
1Adami Tullu Agricultural Research Center; Plant Biotechnology Team, P.O. Box 35, Zeway, Ethiopia
2Cascape Project Coordinator, Addis Ababa, Ethiopia
Corresponding Author : Miheretu Fufa
Adami Tullu Agricultural Research Center
Plant Biotechnology Team
P.O. Box 35, Zeway, Ethiopia
E-mail: miheretufufag@gmail.com
Received September 19, 2013; Accepted September 23, 2013; Published September 26, 2013
Citation: Fufa M, Diro M (2013) The Effects of Sucrose on in vitro Tuberization of Potato Cultivars. Adv Crop Sci Tech 1:114. doi:10.4172/2329-8863.1000114
Copyright: © 2013 2013 Fufa M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Two potato varieties namely, ‘Hunde’ and ‘Ararsa’ were tested for in vitro Tuberization response under five levels of sucrose (40, 60, 80, 100 and 120gram litre-1) in completely randomized design with 2×5 factorial combinations. The objective was to determine optimum concentration of sucrose for in vitro Tuberization. In both varieties, among the five concentrations of sucrose, Murashige and Skoog (MS) medium supplemented with 60 gram litter-1 sucrose exhibited a better response than the other concentrations in mean values of microtuber number, diameter, and weight and was found optimum. Accordingly, this medium gave an average value of (1.97 ± 0.02) microtuber number, (3.60 ± 0.04 mm) microtuber diameter, and (0.08 ± 0.002 gram) weight of microtuberin variety ‘Ararsa’ after 42.57 ± 0.58 days of culture. On the other hand, it gave mean value of (2.90 ± 0.031) microtuber number, (2.95 ± 0.01mm) microtuber diameter, and (0.06 ± 0.001 gram) weight of microtuber in variety ‘Hunde’ after 35.67 ± 0.58 days of culture.

Keywords
In vitro tuberization; Microtuber; Potato; Sucrose
Introduction
Potato ( Solanum tuberosum L.) is an important food and cash crop [1]. It ranks first in the world from none-grain crop to ensure food security [2]. It is a high biological value crop that gives an exceptionally high yield, more protein and calories, vitamins, minerals, carbohydrates and iron per unit area per unit time than any other major crops [3].
The conventional propagation of potato is characterized by low multiplication rate and susceptibility to pathogens. Susceptibility to pathogens often leads to poor quality and yields due to degeneration [3]. In Ethiopia, the production of potato is expanding steadily. However, its productivity has shown a decreasing trend [4,5] due to the unavailability of good quality clean seed tubers [6]. This can be avoided through in vitro tuberization, a better alternative to conventional propagation that can produce uniform and identical materials in large scale within a short time [7].
The most critical stimulus influencing in vitro Tuberization is sucrose at high concentration [8]. Sucrose is a cheap, safe and superior agent for in vitro Tuberization [9]. Hence, the present study was initiated with the objective to determine optimum concentration of sucrose for in vitro Tuberization.
Materials and Methods
Single nodal excision from one week old sprouts of the relatively clean tubers of ‘Ararsa’ and ‘Hunde’ potato varieties was used for in vitro Tuberization experiment at Tissue Culture Laboratory of Jimma University College of Agriculture and Veterinary Medicine. The two varieties were tested for Tuberization response under five levels of sucrose (40, 60, 80, 100 and 120 gram liter-1) in completely randomized design with 2×5 factorial combinations.
The pH of the medium was adjusted at 5.8, agar (8 gram liter-1) was added and then the medium was autoclaved at 121°C for 20 minutes at 15 psi. MS basal medium containing gibberellic acid (0.1 milligram liter-1), naphthalene acetic acid (0.01 mg/l) and sucrose (30 g/l) was used for initiation. In the case of in vitro Tuberization, the Murashige and Skoog (MS) (1962) basal medium was prepared for each treatment combination.
All the surface sterilization procedures were carried out under aseptic condition of laminar flow chamber, following the procedure of Naik and Karihaloo [10]. One week old sprouts along buds were excised and used as initial explants. The excised explants were washed 3 times in running tap water with 0.25 millliliter of Tween-20, and then washed thoroughly three times with sterile distilled water and immersed in 70% ethyl alcohol for 10 seconds. The alcohol was removed by three times washing with sterile distilled water. Finally, the sprouts were sterilized with 10% sodium hypochlorite (NaOCl) for 20 minutes before dissection.
After removing the leaves, the excised explants were dissected into single nodes (2 cm long) on a sterile plate. Six explants were cultured into 40 milliliter of an initiation culture medium in culture jar and incubated under a 16 hour photoperiod at 24°C with a light intensity of 2500 lux. The sprouts were allowed to grow into plantlets having nodal segments for 3 to 4 weeks. The multiplication medium was decanted and the plantlets were kept in a conditioning medium before being used for in vitro Tuberization to avoid the carryover effects of hormones. Forty milliliters of in vitro Tuberization medium was dispensed into each culture jar before transferring the culture to the growth room. Finally, the culture was kept at a temperature of 18°C under dark condition.
Data collection and analysis
The first, second and third date of formation of microtuber was carefully followed and recorded. 50% days to set microtuber was recorded and used for analysis. The number of microtubers produced by each explant was counted before harvest. The diameter (in millimeter) of each microtuber was measured by Digital Caliper. Immediately after harvest, each microtuber was weighed on sensitive balance to get the mean microtuber weight in gram. After 15 days of light exposure, the microtubers were treated with gibberellic acid (GA3) and incubated in the dark before planting in the green house. The number of the microtubers germinated and established was counted to get their percent survival under in vivo. The data were subjected to the analysis of variance (ANOVA) at 5% level of significance using SAS statistical software Version 9.2 [11]. The REGWQ multiple comparison procedure was used for separating significant means.
Results and Discussion
Analysis of variance (ANOVA) revealed that sucrose and variety interaction had very highly significant effect (α=5%) on days to Tuberization and on the average number, diameter (millimeter) and weight (gram) of microtubers (Table 1). This implied that there is interdependence of sucrose and genotype on in vitro Tuberization of potato. Thus, the response of genotypes to a given level of sucrose was not the same.
Effects of sucrose on days to Tuberization, mean microtuber number and diameter
At 40 gram liter-1 sucrose, both varieties did not produce microtubers. However, when 60 gram liter-1 sucrose was added to growth media, ‘Hunde’ variety produced microtubers in 36 days, which is significantly earlier than that of ‘Ararsa’ variety (43 days). Increasing concentration of sucrose from 60 to 80 gram liter-1 delayed microtuber formation in both varieties, but more pronounced on ‘Ararsa’ variety. This might be due to the marked variation in the responses of plant gene to changing sucrose status. Some genes are induced, some are repressed, and others are minimally affected [12].
Moreover, in both varieties, microtuber number and size get reduced as the concentration of sucrose increased from 60 gram liter-1 to 80 gram liter-1. At 120 gram liter-1 of sucrose, both genotypes did not produce microtubers (Table 1). The absence of microtuber formation at high sucrose concentration might be due to the effect of super optimal level of sucrose that can result in an unfavorable osmotic condition for water uptake, and thus affecting microtuber formation of the seedlings.
Effects of sucrose on mean microtuber weight
In both varieties, a decreasing trend in mean weight (gram) of microtuber was observed as the level of sucrose increased (Figure 1). This might be again due to the effect of high sucrose level on osmotic condition of the culture that affect cell turgidity [12], and hence, microtuber weight.
In both varieties, Murashige and Skoog (MS) medium supplemented with 60 gram liter-1 sucrose exhibited a better response than the other concentrations in mean values of microtuber number, diameter and weight, and was found optimum. Accordingly, this medium gave an average value of (1.97 ± 0.02) microtuber number, (3.60 ± 0.04 mm) microtuber diameter, and (0.08 ± 0.002 g) weight of microtuber in the variety ‘Ararsa’ variety after 42.57 ± 0.58 days of culture. On the other hand, it gave mean value of (2.90 ± 0.031) microtuber number, (2.95 ± 0.01 mm) microtuber diameter, and (0.06 ± 0.001 g) weight of microtuber in variety ‘Hunde’, after 35.67 ± 0.58 days of culture (Table 1)
The present result is in agreement with that of Aslam et al. [13], who found that a medium containing 6% sucrose was optimal in terms of minimum time of induction (34), mean tuber number (1.2) and weight (0.03 gram) of microtubers per single nodal explant in cultivar Desiree. Imani et al. [14] also reported that Murashige and Skoog (MS) medium supplemented with 60 gram liter-1 of sucrose as the best in producing the maximum number (4.20) and size (0.44 centimeter) of micro tubers. Iqbal et al. [9] also recorded similar results on mean numbers of tubers (4.8) on Murashige and Skoog (MS) medium treated with 60 gram liter-1 sucrose. Kanwal et al. [15], on the other hand, reported that Murashige and Skoog (MS) medium supplemented with 30 and 40gram liter-1 sucrose did not produce microtubers.
Conclusion
A protocol for in vitro Tuberization of potato varieties ‘Ararsa’ and ‘Hunde’ from single nodal explant has been developed. The result indicated that in vitro Tuberization of potato was highly dependent on sucrose and genotype interaction.
Murashige and Skoog (MS) medium supplemented with 60 gram liter-1 exhibited fewer days to microtuber formation (35.67 ± 0.58/42.67 ± 0.58), better mean number (2.90 ± 0.031/1.97 ± 0.02), diameter (2.81 ± 0.015/3.60 ± 0.04 millimeter) and fresh weight (0.06 ± 0.001/0.08 ± 0.002 gram) of microtubers in ‘Hunde’ and ‘Ararsa’ varieties, respectively. However, microtuber production needs further improvement since the size of microtubers produced was not large enough. Thus, trying different levels of Benzylaminopurine (BAP) in combination with sucrose and extending the time of harvesting may be helpful to improve the size of the microtuber.
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