RESEARCH ARTICLE ISSN In vitro growth of potato plant (in vitro tuberization) *Corresponding author:

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1 INT J CURR SCI 2015, 17: E RESEARCH ARTICLE ISSN In vitro growth of potato plant (in vitro tuberization) Ndacyayisenga Joseph a, M. Anbazhagan b * and S. Srinivasan C a,c Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar , Tamil Nadu, India b Department of Botany, Tissue Culture Laboratory, Annamalai University, Annamalainagar , Tamil Nadu, India Abstract *Corresponding author: anbungm@yahoo.co.in Potato plant is one of the important crops contributing in the staple food of population worldwide. The techniques have adopted for improving the yield of potato crops in return for preventing the shortage of food. The work on potato plant has been conducted as in vitro growth of potato plant (in vitro tuberization) with the main purpose of initiation of potato plant culture in vitro, micropropagation of potato plant, production of microtubers or/and hardening and transfer of plant to the out of laboratory environment (soil). Keywords: potato plant, in vitro, micropropagation Received: 14 th April 2015; Revised: 18 th May; Accepted: 04 th June; IJCS New Liberty Group 2015 Introduction Potato (Solanum tuberosum) is a staple food belonging to the three most important crops worldwide. Potato tubers are radiailly expanded stem axes formed by shortened internodes and nodes (eyes). They originate from underground stolons in a series of developmental processes comprising the cessation of growth at the apex, the swelling of the stolon by subapical radial growth and longitudinal cell divisions of the body (Xu et al., 1998). The latter process is accompanied by accumulation of starch and storage proteins and requires coordinated transcriptional and metabolic changes (Visser et al., 1994; Appeldoorn et al., 1999; Kloosterman et al., 2005; Kloosterman et al., 2008). After formation, potato tubers undergo a period of dormancy which is characterized by the absence of visible bud growth. It is generally accepted that the onset of dormancy coincides with the cessation of meristematic activity during tuber initiation (Burton, 1989). The length of the dormancy period depends on the genetic background and is affected by pre-harvest and post-harvest conditions (Sonnewald, 2001; Suttle, 2004a). With the onset of sprouting the tuber turns into a source organ supporting growth of the developing sprout. This is associated with structural and metabolic changes (Viola et al., 2007) as well as with major changes in gene expression pattern (Ronning et al., 2003; Campbell et al., 2008). Additionally, endogenous plant hormones play a critical role in regulation of dormancy and bud break (Hemberg, 1985; Suttle, 2004a). Potatoes are generally grown from seed potatoes-these are tubers specifically grown to be disease free and provide consistent and healthy plants. To be disease free, the areas where seed potatoes are grown are selected with care. Tuberization in potato is a highly complex developmental process, which may be modified in various ways. Tuberization can be induced under in vitro condition (Hossain and Sultana, 1998). In agricultural practice, the potato production cycle is mainly vegetative, tuber products constituting both an organ of asexual reproduction (seed tubers) and an edible part of the plant. The

2 agronomic performances of seed tubers are highly dependent on dormancy but also on physiological age referred to the physiological state of the tuber at a given point in its growth. However, the use of seed tubers often exposes plants to infections, especially viral. The use of micro tubers offers many advantages (Abraham Dieme et al., 2013). The production of micro tubers is called in vitro tuberization. Many researchers used different growth regulators for in vitro induction of microtuber in potato (Hossain and Sultana, 1998). The number and size of microtubers produced in vitro depend on many factors, such as optimum concentration of sugar, growth regulators and anti-gibberellin compounds in the culture medium (Tovar et al., 1985; Dodds et al., 1988). Among the substances used to induce microtubers, cournarin, CCC and cytokinins have received adequate attention (Wang and Hu, 1985; Chandra et al., 1988). Cytokinins are believed to have strong promotive effects on tuberization, and to constitute major part of the tuberization stimulus, either alone or in combination with other substances (Palmer and Smith, 1969; Pelacho and Mingo-Castel, 1991). Growth retardant stimulates tuberization of plants under unfavourable environmental conditions (Menzel, 1980) and CCC (Chioro choline chloride) is widely used in tissue culture media to promote microtuber formation (Tovar et al., 1985; Rosell et al., 1987). It induces microtuber formation by a wide range of Solanum genotypes of diverse genetic backgrounds (Estrada et al., 1986). In vitro tuberization has become an important mode of rapid multiplication for pre-basic stock in seed tuber multiplication as well as germplasm exchange. Therefore this experiment will be designed to Materials and Methods Plant material The plant materials (potato tubes) were collected from the shop in Chidambaram, and then they were brought in tissue culture laboratory for sprouting. In this research we did not consider the potato variety because our main objective was to do the in vitro tuberization of potato without considering the variety of potato means by using any variety the reason we did not use the potato from the research institution. This research was conducted in the laboratory of plant tissue culture of Botany Department in Annamalai University. Media In this research we used the MS media and tuberization media (MS media supplemented with 10 mg/l BAP, 8% sucrose and 2 g/l activated charcoal. The composition of MS medium is shown in the table. Table 1. Composition of plant tissue culture m edia Constituents Murashige and Skoog MgSO4.7H2O (1962) 370 MS CaCl2.2H2O 440 KNO NH4NO KH2PO4 170 FeSO4.7H2O* 27.8 MnSO4.4H2O 22.3 Kl 0.83 CoCl2.6H2O ZnSO4.7H2O 8.6 CuSO4.5H2O H3BO3 6.2 Na2MoO4.2H2O 0.25 EDTA disodium salt 37.3 Myo- inositol 100 Thiamine HCl 0.1 Pyridoxine HCl 0.5 Nicotinic acid 0.5 Glycine 2 Sucrose 30 grow the potato plant in vitro (in vitro tuberization).

3 Plant Growth regulator: Hormones now referred to as growth regulators are organic compounds that have been naturally synthesized in higher plants, which influence growth and development. These are usually active at different sites from where they are produced and are only present and active in very small quantities. Two main classes of growth regulators of special importance in plant tissue culture are the auxins and cytokinins, while others are of minor importance viz., gibberellin s, abscisic acid, ethylene etc. During our research, the three types of plant growth regulators have been used namely: Auxin (used in the form of NAA: Naphthalene Acetic Acid); cytokinin (used in the form of BAP: 6-Benzyl Amino Purine) and Gibberellins (used as GA 3 : gibberellic acid). Methods: Preparation of sprouts: To get the potato sprouts used as explants, the potato tubers were grown in the basin containing the sand. After 2 weeks the tubers gave enough sprouts used as explants. That basin was kept in the laboratory condition at the temperature of 25±2 o C under 16 hrs photoperiod. Preparation of culture media: The culture media were prepared prior to the surface sterilization and to the initiation of culture. Preparation of stock solutions: A time consuming and tedious process to weigh the necessary products each time a medium is prepared, concentrated solutions of the desired composition of a medium are used which regulators were prepared in distilled water. Macronutrient stock solution: The stock solution of macronutrients was prepared as 10X. All the macronutrients were dissolved one by one except (CaCl2 for macronutrient stock solution. The stock solution of CaCl2 was prepared separately in order to avoid precipitation. Micronutrient stock solution: A stock solution of all the micronutrients was prepared as 100X. Since copper and cobalt are required in very small quantities, they were prepared firstly to make a separate stock solution of these two salts (100X) and then an appropriate volume was pipetted and put into the main mi c r o n u t r i e n t s t o c k s o l u t i o n. Iron-EDTA: Iron EDTA was added fresh. Vitamins and growth regulators stock solutions: These are simple stock solutions. These were added in the media according to the type of culture (initiation culture or tuberization culture). Surface sterilization: After culture media preparation and before the initiation of culture, the surface sterilization of the explants was done as follows: The sprouts were washed with distilled water and one drop of savlon for 10 mins. They were rinsed twice in double distilled water and dip into 70% alcohol, again they were rinsed thoroughly in distilled water, inside the laminar flow chamber, the sprouts were washed in sterile water once followed by washing in 0.1% mercuric chloride for a minute. The one dilutes adequately. These concentrated solutions material was rinsed in the sterile thrice to remove the are called stock solutions. Simple stock solutions comprise only one constituent at a time. Complex stock solutions comprise several chemicals. Stock solutions of macro and micronutrients, vitamins and growth traces of sterilents. Initiation of culture: After the surface sterilization of the explants, the later were inoculated in culture media priory prepared. This process is called initiation of culture and

4 was performed as follows: The sprouts were inoculated in full strength MS medium supplemented with 0.25 mg/l GA 3 (Gibberellic acid), 0.03 mg/l NAA (Naphthalene Acetic Acid) and 2 mg/l calcium panthotherate and 3% sucrose, the culture were kept under 16 hrs photoperiod and a temperature of 26±3 o C, Subculture was done at three weeks. In vitro tuberization After three weeks in laboratory condition (16 hrs photoperiod and a temperature of 26±3 o C), the small plantlets were obtained and used for in vitro tuberization as follows: Excision of the nodal sections from three weeks old in vitro derived plantlets of six nodes each, after removing the apical and basal parts was done, they were transferred to the tuberization medium (MS media with 10 mg/l BAP,8% sucrose and 2 g/l activated charcoal, the cultures were incubated in a growth chamber with a temperature of 26±3 o C and 16 hrs photoperiod. Results and Discussion Growth of sprouts The potato tubers were grown in basin containing sand in the laboratory conditions, after two weeks the sprouts were appeared and ready for being utilized. The following photos present the potato tuber with the sprouts ready for being served as explants. Collection of explants and Surface sterilization Potato tuber formation is a process regulated by several plant growth regulators and influenced by daylength, temperature and other environmental factors (Hussey and Stacey 1984; Machackova et al., 1998; Xu et al., 1998, Xiquan Gao et al., 2005). Tuberization in potato is a highly complex developmental process, which may be modified in various ways. Tuberization can be induced under in vitro condition. Many researchers used different growth regulators for in vitro induction of microtuber in potato (Hossain and Sultana, 1998, Hoque, 2010). Unlike cereal seeds, tubers are not dehydrated and active metabolism occurs. Consequently, sprout growth is initiated following a period of endodormancy. Therefore, controlling the length of the dormancy period is of considerable economic importance. New strategies are needed because current cold storage techniques or treatment with sprout inhibitors are often problematic. Recently, an interesting alternative approach has been developed involving the production of genetically engineered potatoes with altered metabolic pathways to prolong the dormancy period of the harvested tuber (Uwe Sonnewald, 2001). According to Hoque, for rapid potato sprouting, the clean materials were treated with 300 ppm GA3 and after one week old sprouts can be used as initial explants (Hoque, 2010). According to Aafia alams and Aved Iqbal, the tubers were washed several times with detergent followed by several times rinsed with distilled water, dried and placed in dark room for eight week till sprouting started (Aafia alams and Aved Iqbal, 2010). During our research, the potato tubers were grown in basin containing sand in the laboratory conditions, after two weeks the sprouts were appeared and ready for being utilized. For initiation of culture, according to Hoque, One week old sprouts were used as initial explants. The sprouts were surface sterilized by dipping in 0.5 HgCl 2 solutions for 3-5 mins and then washed several times with autoclaved distilled water.

5 After culture in MS solid medium (ph = 5.8) in test tube, the bud sprouted into plantlet having 4-5 nodal segments within one month (Hoque, 2010). According to Aafia alams and Aved Iqbal, One week old sprouts were dipped in 15% NaOCl solution for mins, given three washings with autoclaved distilled water and inoculated on prepared MS medium and after 4 weeks of inoculation the buds were sprouted into full plantlets that contained 7-8 nodes (Aafia alams and Aved Iqbal, 2010). During our research, the sprouts were inoculated in full strength MS medium supplemented with 0.25 mg/l GA 3 (Gibberellic acid), 0.03 mg/l NAA (Naphthalene Acetic Acid) and 2 mg/l calcium panthotherate and 3% sucrose, and after three week in culture media and in laboratory condition, they were grown and used as the explants for in vitro tuberization. For in vitro tuberization, Hoque used Shoot apex, sprout and nodal cutting collected from regenerated plantlet as explants and Kinetine (KIN) was used as a induced hormone for microtuber production. He obtained the microtubers after days of incubation (Hoque, 2010). Aafia Alams and

6 Javed Iqbal used single node (one node) and multi node (three nodes) explants, and the MS media were supplemented with sucrose (4, 6, 8, 10 and 12%) either alone or in combination with BA (6-benzylaminopurine) and Kinetin (6-furfurylaminopurine) at varying concentrations (Aafia Aslam and Javed Iqbal, 2010). Zakaria and his colleagues used eight stem segments (each with 3 nodes) explants and medium was based on MS medium (Murashige and Skoog, 1962) supplemented with 80 g/l sucrose, six levels of Benzyl adenine (BA) and three levels Chloro choline chloride (CCC) having control treatment (Zakaria et al., 2008). Maolin Peng et al. showed that an addition 0.5 of active charcoal, the growth period was shortened from 25 days to 9.33 days on average, and the amount of larger potatoes increased 8.54% (Maolin Peng et al., 2012). During our research, we have cut the nodal sections from three weeks old in vitro derived plantlets of six nodes each, after removing the apical and basal parts, then they were transferred to the tuberization medium (MS media with 10 mg/l BAP, 8% sucrose and 2 g/l activated charcoal, and the cultures were incubated in a growth chamber with a temperature of 26±3 o C and 16 hrs photoperiod. Due to the incident of contamination we did not get the results. That contamination was caused by fungi. The contamination normally is easily in plant tissue culture, but due to the lack of time we were not able to rescue the culture. Conclusion Potato is one of the important crops contributing in the staple food of much population all over the world. The world is facing the problem of increasing of population knowing that soil surface for cultivation instead is decreasing, this means that the food, if there is no other strategies to be taken will be in scarcity. In this point the crop pests also are continuing to ravage the crops. One of the strategies for preventing those problems is the improvement of the scientific research especially in the field of plant improvement. In this angle, one can adopt the in vitro tuberization of potatoes. In vitro tuberization is one of the plant micropropagation techniques where the potato microtubers are produced in laboratory condition. During our research named as in vitro growth of potato plant (in vitro tuberization), our main purpose was Initiation of potato plant culture in vitro, micropropagation of potato plant, production of microtubers or/and hardening and transfer of plant to the out of laboratory environment (soil). References Aafia Aslam, Javed Iqbal (2010). Combined effect of cytokinin and sucrose on in vitro tuberization parameters of two cultivars i.e. diamante and red norland of potato (solanum tuberosum). Pak J Bot 42(2): Abraham Dieme, Mame Ourèye Sy (2013). Environmental, morphological and physiological factors analyzes for optimization of potato (Solanum tuberosum L.) microtuber in vitro germination. Advances in Bioscience and Biotechnology Appeldoorn NJG, De Bruijn SM, Koot-Gronsveld EAM, Visser RGF, Vreugdenhil D, Van der Plas LHW (1999). Developmental changes in enzymes involved in the conversion of hexose phosphate and its subsequent metabolites during early tuberization of potato. Plant Cell and Environment 22: Chandra R, JH Dodds, P Tovar (1988). In vitro tuberization in potato. Newslet. Intl. Assoc. Plant Tissue Cult 55: Dodds JH, Tovar P, Chandra R, Estrella D and Cabello R (1988). Improved methods for in vitro tuber

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