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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 97 The effects of explant rotation medium types JA and GA 3 additions on in vitro microtuber production from potato Solanum tuberosum L. Seray KENAR 1 Gökçen BAYSAL FURTANA 2 ġeküre ġebnem ELLĠALTIOĞLU 3 Rukiye TIPIRDAMAZ 4 14 HacettepeUniversity Faculty of Science Department of Biology Beytepe Ankara 2 Gazi University Faculty of Science Department of Biology BeĢevler Ankara 3 Ankara University Faculty of Agriculture Department of Horticulture DıĢkapı Ankara Corresponding author: tuzhacettepe.edu.tr Abstract — This study investigates the effects on the in vitro microtuber formations of Solanum tuberosum L. cv. Marfona species effected by the direction of planting the explants horizontal or vertical the type of medium solid or two-phase adding Jasmonic acid JA 0.0 10 ng/L 1 µg/L and 0.2 mg/L and Gibberellic acid GA 3 0.0 and 0.2 mg/L. The cultures were incubated in a climate chamber at 22-25 o C and were subjected to a light intensity of 145 µmol m -2 s -1 for 8 hours in light and 16 hours in dark photoperiods short day for 4-6 weeks. Microtuber production was inhibited when GA 3 was added. The maximum number of microtubers was observed when the explants were planted vertically and were grown in two-phase medium which did not contain GA 3 and had 10 ng/L JA present. It was determined that two-phase medium with 0.2 mg/L JA but without GA 3 was the most favourable medium for tuber growth for both height and width. The best microtuber formation on single node explants were observed to occur in the short day photoperiod 8 hours light/16 hours dark in a two-phase medium that contained 0.2 mg/L JA without the addition of GA 3 . The results shows that the effect caused by JA works antagonistically with that of GA 3 thus causing the resulting microtuber formation observed. Keywords — Potato In vitro Microtuberization Two-Phase Medium Jasmonic Acid GA 3 . I. INTRODUCTION The potato Solanum tuberosum L. is the fourth most important vegetable after rice wheat and corn in food and industry amongst the food sources for the world’s population 1 2. When potato tubers are grown by vegetative propagation using conventional methods the viruses on the main plant are easily transferred on to the new vegetative organs and tubers and causes serious damage to potato production. Due to seed tuber growth being significantly affected by environmental conditions and that the possibility of disease becomes high in addition to high costs microtubers are emphasized as an alternative seed source 3. The microtubers that are produced from seedlings with no viral infections that were obtained from meristem cultures and propagated in vitro are considered to have significant advantages in potato seed production. The success of in vitro microtuber production in the studies conducted in this field is based on a series of factors. Amongst these nutrient media solutions the chemicals used like activated charcoal sucrose concentration growth regulators such as Gibberellic acid Jasmonic acid and temperature environmental factors like light intensity and genotype factors can be included 4-12. In literature it is emphasized that due to the plant growth regulators Gibberellic acid GA 3 Abscisic acid ABA Ethylene 2-chloroethyl trimethylamonium chloride CCC and Jasmonic acid JA acting on each other and on environmental factors they are effective on the formation and development of vegetative storage organs like tubers 13-16. The physiological mechanisms and their related hormones that determine potato tuber growth and development are still not fully known. The gibberellins that are effective in many physiological processes including plant stem development seed germination breaking of bud dormancy and fruit growth also effect the development of potato tubers. Environmental factors like photoperiod and temperature regulate gibberellin biosynthesis 15. In recent years the effect of Jasmonic acid and methyl esters on tuber stimulation has been the focus of studies 17-19. In addition to acting as a signal molecule for activating an immune response in case of pathogen invasions JA also acts as a regulator for many physiological and developmental such as root development tuberization ageing and pollen

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 98 development processes. Jasmonates are also potential stimulators for the expression of vegetative storage protein genes 20. It is also reported that JA’s are effective during in vitro microtuberization in addition to playing an important role in the development of vegetative storage organs 16 19 21-24. However JA’s influence on microtuber development and its relationship with other growth regulators are still not fully elucidated. In the studies conducted the time taken for microtuber development the medium conditions for growing microtubers and which hormones chemicals etc. need to be applied in what doses could not be fully determined 18 19 22 25-27. To include JA when commercially producing microtubers more information is needed on the effect of this substance on tuberization and its relationship with other hormones. In this study the relationship between the planting of explants either horizontally or vertically the type of medium either solid or two-phase the addition of Jasmonic acid 0.0 10 ng/L 1 µg/L and 0.2 mg/L and Gibberellic acid 0.0 and 0.2 mg/L into the media and how they affect the development of microtubers growing in vitro on the economically valuable Solanum tuberosum L. cv. Marfona is investigated. II. MATERIALS AND METHODS The study was conducted in four stages: The S. tuberosum tuber shoots in vivo in vitro shoot tip cultures micropropagation and microtuberization. For the purpose of shoot development the potato tubers belonging to the Marfona variety are first washed under tap water then they are placed in a 15 commercial sodium hypochloride NaOCl solution and left there for 20 minutes. They are then washed with distilled water 3 times 28 so that surface sterilization can occur. The tubers that had there surfaces disinfected are then planted into a pot filled with perlite and left to grow in a climate chamber that has a temperature of 30-35 o C 60-65 humidity a light intensity of 145 µmol m -2 s -1 and left for photoperiods of 16 hours in light and 8 hours in dark 29. After 4 weeks successfully grown shoots were obtained Figure 1. a b FIGURE 1. SHOOT DEVELOPMENT ON TUBERS a AFTER 1-2 WEEKS b AFTER 3-4 WEEKS To produce a shoot tip culture the shoot tips obtained from potato tubers were placed in a 70 alcohol solution and shaken for 1 minute. Two drops of Tween-20 were then added and left to wait in a 10 NaOCl solution for 7-8 minutes after which the shoot tips were washed with distilled water 3 times for sterilization 18-28. Shoot tip explants obtained from sterilized shoots were then planted in a modified Murashige and Skoog MS medium 30. Different references 31 32 were used to produce the nutrient media and the final solution consisted of 30 g/L sucrose 7g/L agar 0.2 mg/L GA 3 0.2 mg/L Kinetin 0.2 mg/L Indole Acetic Acid IAA and 100 mg/L Myo-inositol. The solution was adjusted to have a 5.8 pH. Shoot tips were placed in tubes with dimensions of 15x2.5 cm and cultured in 10 mL of Modified MS MMS in each tube. The cultures were then incubated for 2-2.5 months in a climate chamber that had a temperature of 20-22 o C and were subjected to a light intensity of 145 µmol m -2 s -1 with 16 hours of light and 8 hours of dark photoperiods 33. After this period in vitro potato plantlets were obtained Figure 2. Single node explants taken from plantlets obtained from shoot tip cultures were used for micropropagation. The single nodes were planted into tubes of dimensions 15x2.5 cm that contained 1.0 mg/L IAA and 1.0 mg/L in addition to the MMS nutrient media. The MMS nutrient media was adjusted to have a 5.8 pH and contained 7 g/L agar 30 g/L sucrose 60 mg/L myo- inositol 04 mg/L thiamine 1 g/L pyridoxine 34. The cultures were incubated for 4-7 weeks in a controlled climate chamber of 22 o C with 16 hours of light and 8 hours of dark photoperiods and a light intensity of 145 µmol -2 s -1 31-33.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 99 a b FIGURE 2. PLANTS OBTAINED FROM SHOOT TIP CULTURES a 3-4 WEEKS AFTER b 6 WEEKS AFTER To determine the effects of the medium solution the planting position either horizontally or vertically and type of medium whether in a solid or two-phase medium during microtuberization on the development of microtubers single node explants obtained from plants that were grown in vitro were planted either horizontally or vertically into glass jars of dimensions 6.5x7.5 that had 50 mL each of either the solid phasic or two-phase MMS medium Figure 3. The MMS medium used was made up of JA 0 10 ng/L 1 µg/L and 0.2 mg/L GA 3 0 and 0.2 mg/L and the combination of these in addition to 80 g/L sucrose and 7 g/L agar. The MMS medium was also adjusted to have a 5.7 pH 9 26 34. Ten explants were planted into each of the 5 jars used and each experiment was repeated 5 times. The cultures were then incubated in a climate chamber for 4-6 weeks at a temperature of 22-25 o C and a light intensity of 145 µmol m -2 s -1 during an 8 hours light and 16 hours dark photoperiod short day 36. At the end of the incubation period the number of single nodes obtained from explants the number of explants from which microtubers were obtained explant yield number of explants from which microtubers were obtianed/total number of explants and the microtuber yield number of microtubers/total number of explants were determined for each group. The data was shown as percentages in a chart. The statistical analysis of the microtuber numbers and weights were conducted by using the SPSS packet programme. According to variance analysis and statistical test results each variable was calculated to have a least significant difference LSD value between the significant 1 and 5 percentage value range. To compare of the groups Oneway-ANOVA were used variance analysis and were followed by the Duncan’s Multiple Range Test for comparing data in individual groups as well as between groups 37. III. RESULTS AND DISCUSSION The results of the effect of the direction of explant planting either horizontally or vertically Figure 3 type of medium solid and two-phase Figure 3 medium composition JA 0 10 ng/L 1 µg/L and 0.2 mg/L and GA 3 0 and 0.2 mg/L on microtuber development from single-node potato explants are summarized in Table 1. It was observed that the effect and relationships of the type of medium the direction in which the explants were planted and medium composition JA and GA 3 were significant p0.05. Table 1 shows that in the control group and those that had JA in their medium microtuber development had occurred while no development was observed when GA 3 was present in the medium. Therefore it was concluded that GA 3 should not be present in media prepared for microtuber development. It was observed that the relationship between explant yield number of explants from which microtubers were obtained/total number of explants and microtuber yield number of microtubers/total number of explants generally increased in a proportional way. Accordingly it was shown that the two- phase groups two-phase–0.2 mg/L JA 36.53 two-phase–1 µg/L JA 45.28 and two-phase–10 ng/L JA 44.4 which gave a high microtuber percentage yield also gave a high percentage yield of the explants 30.76 28.3 and 38.09 respectively which the microtubers were obtained from. It was also observed that two-phase–control 9.09 Horizontal–10 ng/L JA + 0.2 mg/L GA 3 6.84 and Vertical–10 ng/L JA + 0.2 mg/L GA 3 10.76 medium groups which resulted in low microtuber yields additionally generated low explant percentage yields 7.27 5.47 6.15 respectively.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 100 FIGURE 3. THE PLANTING DIRECTION OF SINGLE NODE EXPLANTS Highest tuber yields were observed in two-phase–1 µg/L JA two-phase–10 ng/L JA and two-phase–0.2 mg/L JA media groups with percentage yield results being 45.28 44.4 and 36.53 respectively. Therefore it was concluded that two- phase media were more suitable for microtuber development. When the microtuber weights in Table 1 are analyzed it was observed that microtuber weight medium composition and direction of planting of explants had a significant relationship p0.05. The highest values obtained with respect to microtuber weight were 176 mg 118 mg and 97 mg for two-phase–control two-phase–0.2 mg/L JA and vertical–0.2 mg/L JA media groups and planting directions respectively. The effects of horizontal and vertical planting directions on microtuber weight and microtuber yield was not significant. TABLE 1 DATA SHOWING THE EFFECTS OF MEDIA COMPOSITION AND PLANTING DIRECTION OF SEEDLINGS ON MICROTUBER DEVELOPMENT Planting Direction JA GA 3 0.2 mg/L Total explant number The number of explants from which microtubers were obtained and explant yield The number of microtubers and microtuber yield Average Microtuber weight mg Horizontal - - - - 66 0 0 0 Vertical - 70 0 0 0 Two-phase - 55 4 7.27 5 909 176±57 c Horizontal 02 mg/L - 64 12 1875 132031 37±9 a + 80 0 0 0 Vertical - 68 16 2352 182647 97±26 ab + 93 0 0 0 Two-phase - 52 16 3076 193653 118±26 bc + 0 0 0 0 Horizontal 1 µg/L - 48 12 25 153125 35±8 a + 72 0 0 0 Vertical - 40 6 1304 820 45±7 a + 64 0 0 0 Two-phase - 53 15 283 244528 55±14 ab + 30 0 0 0 Horizontal 10 ng/L - 69 0 0 0 + 73 4 547 5684 24±5 a Vertical - 69 0 0 0 + 65 4 615 71076 32±9 a Two-phase - 63 24 3809 28444 38±8 a + 41 0 0 0 Horizontal - + 66 0 0 0 Vertical + 70 0 0 0 Two-phase + 41 0 0 0 TOTAL 1412 114 807 142 1005 49±5 Means followed by the different letter in the same column are significantly different by Duncan’s test P ≤ 0.05.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 101 In the experiment in which the effects of explant direction when planting the type of medium and the JA–GA 3 effects on microtuber development were investigated it was found that the highest microtuber yield 45.28 resulted from the two- phase–1 µg/L JA group. The highest value for microtuber weight was found to be approximately 176 mg in the KC two- phase–control group. However when all the data converted to a common denominator were analyzed in addition to considering the criteria for using microtubers as potato seeds it was deduced that the AOC two-phase–0.2 mg/L JA application resulted in high values in regard to microtuber yield and microtuber weight Microtuber yield36.53 and Average Microtuber weight118 mg and thus could be more suitable Figure 4. FIGURE 4. MICROTUBERS OBTAINED FROM DIFFERENT MEDIUM. MICROTUBERS OBTAINED FROM THE a TWO-PHASE MEDIUM CONTAINS 0.2 mg/L JA b TWO-PHASE MEDIUM CONTAINS 1 µg/L JA AND c TWO- PHASE CONTROL MEDIUM In this study the economically valuable Solanum tuberosum L. cv. Marfona was used to develop shoots in vivo after which in vitro shoot tip cultures were developed. The effects of explant planting direction horizontal or vertical media type solid phase or two-phase and the addition of Jasmonic acid 0.0 10 ng/L 1 µg/L and 0.2 mg/L Gibberellic acid 0.0 and 0.2 mg/L were added in to the media on microtuber developments from the micropropagated plantlets the shoot tip cultures were then investigated. Similarly Gopal et al. 32’s results the single nodes taken from plantlets obtained from shoot tip cultures were micropropagated in a MMS medium with 1.0 mg/L IAA and 1.0 mg/L BAP with 7g/L agar 30g/L sugar 60 mg/L myo- inositol 0.4 mg/L thiamine 1 g/L pyridoxine with a 5.8 pH. They were incubated in a climate chamber with a temperature of 22 o C and a light intensity of 145 µmol m -2 s -1 for 16 hours of light and 8 hours of dark photoperiods. In our study whether it were shoot development from in vivo tubers or shoot and plant development in vitro all were subjected to 16 hours of light and 8 hours of dark long day photoperiods. On the other hand it was determined that during microtuberization the highest yield of microtuber development occurred when the single node potato explants were subjected to a short day 8 hours of light and 16 hours of dark photoperiod in a two-phase nutrient medium containing 0.2 mg/L JA. Potato tuber development depends on hormonal and environmental factors. Similarly many relationships between in vitro growth conditions during microtuberization effect microtuber development. Some important factors effecting in vitro microtuberization include glucose concentration in the medium the dose of growth regulators added to the medium type of medium culture type and the conditions of the environment in which the potato cultures are incubated in e.g. temperature photoperiod 34 38-40. Momena et al. 41 observed the effect of different combination with sucrose and some growth regulators Kinetin and BAP on four different potato cultivars and as a result reported that treatment T3 8 Sucrose+4 mg/L Kinetin + 1 mg/L BAP was the best combination for the investigated cultivars. In another study 42 researchers purposed that to find out the suitable combination of sucrose growth regulators benzyladenine and paclobutrazol and medium type. At the end of the study they

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 102 reported that the best microtuberization rate was obtained in MS/2 liquid medium which include 80 g/L sucrose and 0.5 mg/L IBA. It was deduced that the carbohydrate source gibberellins and anti-gibberellin-like substances and their interactions with each other all effected and regulated potato microtuberization. Harmey 43 determined that gibberellic acid strongly inhibited tuber development but that indole acetic acid and maleic hydrazide induced tuberization. It was also determined that when stem component carbohydrate sources were limited in media with less glucose concentrations the addition of growth regulators did not induce tuberization. In another study 44 researchers concluded that GA was an important regulator in tuber development that ABA conversely stimulated tuber developments and that sucrose regulated tuber development by controlling GA levels. Vreugdenhil and Sergeeva 45 deduced that internal gibberellin levels were high during condition where tuber development did not occur and were low during conditions where tuber development did occur. When gibberellin biosynthesis inhibitors CCC paclobutrazol etc. are used the opposite effect was observed while the addition of gibberellins in to the medium inhibited tuber development. They also found that gibberellin played a role in cellular mechanisms like cell division cell widening and in the lodging of microtubers. In another study gibberellin-photoperiod interaction effecting the regulation of microtuber development in potatoes was investigated 46. It was concluded that S. tuberosum ssp. andigena plants needed short day photoperiods for tuber development and that this process was controlled by gibberellins. It is indicated that jasmonic acid is carried as a signal for tuber induction and that it plays a role in tuber growth and development 47. It was observed that when JA is found in the medium the inhibition effect of GA 3 was removed. The reason for this was because it was thought that JA worked antagonistically against the effects of GAs 39 48. Abdala et al. 49 determined in their study that the highest concentrations of JA was found in leaves while during tuber development the highest concentration was found to occur in the roots. During the process of tuber development the JA concentrations in stolons were found to be drastically reduced. In our study it was observed that in regards to medium type and direction of planting the best microtuber yields and microtuber weight was obtained in the two-phase solid+liquid nutrient medium. Similarly it was observed that in various microtuber production methods the highest yield resulted from two-phase solid propagating-liquid induction systems 9. In another study 50 the development of microtubers in liquid and solid media were compared and it was observed that cultures incubated in a liquid medium resulted in microtubers with more weight than those incubated in a solid medium. In a study conducted by Pelacho et al. 51 however it was found that a half-solid medium resulted in higher tuberization rates and higher tuber weight values than those obtained from liquid media. A reason for the good results in regards to tuberization obtained in our study when a two-phase solid+liquid medium was used might be due to the molecules needed for tuberization found in the medium being able to adsorb more readily on to the plant. It was deduced in our study that the reason for tuber development in media when JA was present while no development was seen with GA 3 and JA together was because of the antagonistic effect of GA 3 on JA. In a similar study it was observed that when gibberellins were added externally it had a negative effect on tuber development although internal GA 3 supported tuber development 45. Tuber formation and its subsequent development is a result of cell division and cell growth. Internal GA 3 is thought to play a role in these cell divisions 44. As GA 3 is an inhibitor of the glycoprotein patatin which is associated with microtuberization adding GA 3 to the external environment makes the concentration levels too high for the inhibition to be tolerated and thus microtuber formation is blocked 52. Additionally Jasik and Mantell 26 observed the effect of JA on three types of yam Dioscorea and as a results confirmed that JA supported microtuberization. In another study 46 researchers determined that gibberellins participated in the 8 hours light–16 hours dark photoperiod that encouraged potato tuberization and that their role was as a regulator showing gibberellins to have a relationship to tuber formation in a negative way. In our study the results of the microtuberization experiments showed that when looking at the effects of explant planting direction medium type and JA–GA 3 interactions the highest microtuber yield 45.28 was obtained from the two-phase 1 µg/L JA containing medium. In regards to microtuber weight the highest weight value 176 mg/tuber resulted from the two- phase control medium. In similar studies 16 53 the researchers obtained a higher number of microtubers with greater

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 103 weight when JA was added to the medium. In potatoes tuberization starts with the widening of the sub-apical meristem of stolons and jasmonates by preventing the longitudinal growth of stolons stimulate the widening of the sub-apical meristem. When the criteria for microtubers that will be used as potato seeds was taken into consideration it was deduced from the results obtained in our study that the medium which gave the highest microtuber yield and weight Microtuber yield36.53 and Avg. Microtuber weight118mg was the 0.2 mg/L JA two-phase medium Figure 4. IV. CONCLUSION To summarize the results:  The best medium type for microtuber formation was a two-phase medium  GA 3 addition to the medium inhibited microtuber formation  JA addition to the medium resulted in positive effects on microtuber formation and this effect resulted from working antagonistically with GA 3  In regards to JA-GA 3 interaction and medium type a two-phase medium with 0.2 mg/L JA but without any GA 3 was found to be the optimal medium. In future studies different substances that encourage tuberization will be beneficial for researching about other and their relationship with photoperiods in developing mechanisms in the production of microtubers. REFERENCES 1 M. S. Zaman A. Quraishi G. Hassan S. Raziuddin Ali A. Khabir and N. Gul “Meristem Culture of potato Solanum tuberosum L. for Production of Virus-Free Plantlets” Online J Biol Sci vol. 110 pp. 898-899 2001. 2 Ü. Güner “Ülkemizde Patateslerde YapılmıĢ Virüs Hastalıkları ile Ġlgili AraĢtırmalar” IV. Ulusal Patates Kongresi Bildiriler Kitabı Özetleri Website:http://www.patates.gov.tr/download.phplngtr 2013. 3 M. E. ÇalıĢkan H. Arıoğlu N. KuĢman and S. ÇalıĢkan “Gerçek Patates Tohumu Teknolojisinin Türkiye’de Verim Potansiyeli ve Uygulanabilirliği” IV. Ulusal Patates Kongresi Bildiriler Kitabı Özetleri. http://www.patates.gov.tr/download. phplngtr 2006. 4 G. Hussey and N.J. Stacey “Factors Affecting the formation of In Vitro Tubers of Potato Solanum tuberosum L.” Ann Bot vo. 53 pp. 565-578 1984. 5 G. Ortiz-Montiel and H. Lozoya-Saldana “Potato Minitubers: Technology Validation in Mexico” Am Potato J vol. 64 pp. 535-544 1987. 6 N. Garner and J. Blake“The Induction and Development of Potato Microtubers In Vitro on Media Free of Growth Regulating Substances” Ann Bot vol. 63: pp. 663-674 1989. 7 E. Esendal “Patates”. Ondokuz Mayıs Üniversitesi Ziraat Fakültesi Yayınları Samsun pp. 49 1990. 8 C. Er and S. Uranbey“NiĢasta ve ġeker Bitkileri” Ankara Üniversitesi Ziraat Fakültesi Yayınları Ankara pp. 458 1998. 9 S. Beyazova “Production of Microtubers in Potato Solanum tuberosum” Middle East Technical Univ. Dept of Biology Ms.C Thesis Ankara 1999. 10 G. Öztürk “Patateste Solanum tuberosum L. In Vitro KoĢullarda Mikro Yumru Üretimine Farklı Besin Ortamlarının Etkisi” Ankara Üniversitesi Fen Bilimleri Enstitüsü Yüksek Lisans Tezi Ankara 2003. 11 FAOSTAT “Food and Agriculture Organization of the United Nation FAO Statistical Data bases” Website: http://www.fao.org 2010. 12 K. G. A.Elaleem R. S. Modawi and M. M. Khalafalla “Microtuber Induction of Two Potato Solanum tuberosum L. Varieties Namely Almera and Diamant” Int Res J Biol Sci vol. 43 pp. 84-89 2015. 13 A. Zaib-Un Nissa and A. Rafiq “Effect of ABA and GA 3 on Tuberization and Some Chemical Constituent of Potato” Plant Cell Physiol vol. 21 pp. 1343-1346 1980. 14 A. K. Bhatia M. L. Pandita and S. C. Khurana “Plant Growth Substances and Sprouting Conditions: II. Effect on Tuber Yield and Multiplication Rate in Seed Potato Production” J Indian Potato Assoc vol. 19 pp. 154-156 1992. 15 S. K. Kim J. T. Kim S. W. Jang S. C. Lee B. H. Lee and I. J. Lee “Exogenous Effect of Gibberellins and Jasmoate on Tuber Enlargement of Dioscorea opposite” Agron Res vol. 3 pp. 39-44 2005. 16 J. Jasik and G. J. Klerk “Effect of Methyl Jasmonate on Morphology and Dormancy Development in Lily Bublets Regenerated In Vitro” J Plant Growth Regul vol. 25 pp. 45-51 2006. 17 K. Pruski P. Duplessis T. Lewis T. Astatkie J. Nowak and P. C. Struik “Jasmonate effect on in vitro tuberization of potato Solanum tuberosum L. cultivars under light and dark conditions” Potato Res vol. 44 pp. 315-325 2002. 18 Z. J. Zhang H. Z. Li W. J. Zhou Y. Takeuchi and K. Yoneyama “Effect of 5-Amino Levulinic Acid on Development and Salt Tolerance of Potato Solanum tuberosum L. Microtubers In Vitro” J Plant Growth Regul vol. 49 pp. 27–34 2006.

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-11 November- 2017 Page | 105 50 G. Rosell F. G. Bertoldi and R.Tizio “In Vitro Mass Tuberisation as A Contribution to Potato Micropropagation” Potato Res vol. 301 pp. 111-116 1987. 51 A. M. Pelacho L. Martin-Closas and J. L. I. Sanfeliu “In Vitro Induction of Potato Tuberization by Organic Acids” Potato Res vol. 42 pp. 585-591 1999. 52 D. J. Hannapel J. C. Miller and W. D. Park “Regulation of Potato Tuber Protein Accumulation by Gibberellic Acid” Plant Physiol vol. 78 pp. 700-703 1985. 53 K. Takahashi K. Fujino Y. Kikuta and Y. Koda “Expansion of Potato Cells in Response to Jasmonic Acid” Plant Sci vol. 100 pp. 3-8 1994.

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