Five genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under two in vitro culture conditions (continuos darkness and light). Cultures maintained under continuos darkness (with a temperatue of 170C) had a higher yield with a greater number of microtuber (1.394/plantlet) than those maintained under long days (16 h of light/day) combined with temperature of 200C. In the last case the microtubers were higher, with with the highest average weight of 0.602 g. Three phytohormones it was used: coumarine, benzylaminopurine and salicylic acid. Regarding the average number of microtubers obtained when it was used coumarine and salicylic acid, the highest average number of microtubers was recorded (1,135 microtubers/plantlet).Â  The study had been performed also on two fractions of the of sugar quantity (80 and 140 g/l), but they had less influence compared to the varieties

Chiru, Nicoleta

Cioloca, Mihaela

Nistor, Andreea

Popa, Monica

English

Andreea Nistor

Nicoleta Chiru

Mihaela Cioloca

Monica Popa

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Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 77 -  RESEARCHES CONCERNING IMPROVING METHODS OF IN VITRO MICROTUBERS PRODUCTION  Nistor Andreea, Nicoleta Chiru, Mihaela Cioloca, Monica Popa National Institute of Research and Development for Potato and Sugar Beet Brasov, Street Fundăturii no 2, Postal code: 500470, Brasov, Romania, Phone: +40/0268/474131, Fax: +40/0268/476608; tican_andreea@yahoo.com  Abstract. Five genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under two in vitro culture conditions (continuos darkness and light). Cultures maintained under continuos darkness (with a temperatue of 170C) had a higher yield with a greater number of microtuber (1.394/plantlet) than those maintained under long days (16 h of light/day) combined with temperature of 200C. In the last case the microtubers were higher, with with the highest average weight of 0.602 g. Three phytohormones it was used: coumarine, benzylaminopurine and salicylic acid. Regarding the average number of microtubers obtained when it was used coumarine and salicylic acid, the highest average number of microtubers was recorded (1,135 microtubers/plantlet).  The study had been performed also on two fractions of the of sugar quantity (80 and 140 g/l), but they had less influence compared to the varieties.  Keywords: plantlets, microcuttings, phytohormones, varieties, microtuberization conditions, microtubers  INTRODUCTION  Tissue culture represents the starting point for seed potato production. Seed potato production technology is based on in vitro multiplication, plantlets regeneration or microtubers production. Microtuberisation is a highly complex process, which modified in various ways, increases the capacity of plantlets to produce microtubers. Scheme for seed potato production involves: microtubers production with a high biological value corresponding from phytosanitary point of view and they are the first generation of potato seed, starting from in vitro plantlets; minitubers production by planting of plantlets and microtubers "in the protected spaces" and it is use a substrate consisting of a mixture of organic and inorganic material and under certain culture conditions; obtaining of  first clonal links, by planting minitubers in clonal field. Over time there have been performed extensive physiological researches which show that microtuberization is dependent on a number of factors, such as photoperiod, phytohormones combination, the composition of the nutrient medium, etc.. Influence of light. Wang and Hu (1982), Wattimena et al. (1983), Ortiz-Montiel and Lozoya-Saldana (1987), Forti et al. (1991) have issued the idea that on darkness microtuberization was rapidly. Slimmon et al., 1989, Gopal et al., 1998 observed an increase in the microtubers weight on light conditions compared with induction of microtubers under darkness continuous (Gopal et al., 1997, 1998). Influence of nutrient composition. Ewing and Struik (1992) notified that sugar - is the most effective factor in the formation of microtuber; Khuri and Moorby (1996) have highlighted its role in inducing of microtubers, but at high concentrations; Radouani (1997) specified the effectiveness of coumarin in microtuberization; Gopal et al. (1998), on researches conducted over 22 potato genotypes, have reported that the cytokinin and benzylaminopurine influenced the number and size of microtubers formed. Handro et al Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 78 -  (1997) have shown that salicylic acid promotes tuberization. Clarke et al (2001) have revealed that the salicylic acid in small doses stimulate cell proliferation; General objective of the research is the improving method of microtuberization inducing the specific objectives are: identifying the optimal conditions for induction microtuberization and the best media of microtuberization;  identification of varieties suitable to microtuberizare; promoting Romanian varieties that responded significantly at improved method of microtuberization  The research aim was: establishment of optimum values for the factors involved in potato microtuberization, such as rate of growth and multiplication of the different genotypes are not diminished by the recalcitrant reactions to the culture medium; introduction of microtubers in scheme of potato seed production (figure 1).                                         Figure 1. Scheme of potato seed production: a) microtubers; b) planting of microtubers in “protected spaces”, c) plants developed from microtubers; d) minitubers obtained from Romanian varieties; e) clonal field  MATERIALS AND METHOD  Microcuttings resulting from uninodal segmentation were 15 inoculated, in recipients containing Murashige & Skoog (1962) medium enriched with naphthyl acetic acid and 20 g / l sugar; the culture vessels were placed in the growth chamber, ensuring a a a b c c d d e Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 79 -  light and temperature regime required for growth and development of the plantlets. After 4 weeks, in recipients with developed plantlets were applied microtuberization medium with sucrose, as an energy source (it was used in two quantities: 80 and 140 g/l) and phytohormones: coumarin (0.05 g/l), benzylaminopurine (0.006 g/l), salicylic acid (0.024 g/l); microtuberization conditions were continuous darkness (170C) and light (200C). Exposure of culture recipients at microtuberization conditions lasted a period of 3 months. Calculation methods and interpretation of data from experience: the results were transformed by variation analysis; significance of differences was determined by the method of multiple comparisons (Duncan test). Experimental variants: • Polifactorial experience, such as: 2x5x3x2, included the following factors, divided over 3 repetitions: • Experimental factor A- microtuberization conditions with 2 degrees; o a1-continuous darkness; a2-light (photoperiod 16 h light / day). • Experimental factor B - variety, with five degrees:  o b1-Roclas; b2-Rustic; b3-Gared; b4-Kronstad; b5-Nativ. • Experimental factor C - phytohormones, 3 degrees:  o c1-coumarin; c2-benzylaminopurine; c3-salicylic acid. • Experimental factor D - quantity of sugar (g / l) used on medium of microtuberization with 2 degrees: d1-80 g / l; d2-140 g / l. RESULTS AND DISCUSSIONS  Microtuberization under continuous darkness proved to be optimal on microtubers formation in number (1.394) for Roclas variety, which differs significantly from the other variants (table 1) (figure 2, 3). Exposure to light favored varieties Gared (0.602 g) and Kronstad (0.471 g) in microtuberization, average weight of microtubers is significantly different from other variants. Conditions darkness provoked a negative reaction in microtuberization for Nativ variety, reflected by the minimum weight of microtubers (0.209 g) . Table 1  Influence of variety and conditions of microtuberization over the number of microtubers produced / plantlets and their average weight Variety Microtuberization conditions Number of microtubers Duncan test The average weight of microtubers (g) Duncan test Roclas  Continuous darkness 1,394  A  0,297  CD  Light 0,960  BC  0,247  D  Rustic Continuous darkness 1,128  B  0,244  D  Light 1,115  B  0,278  CD  Gared Continuous darkness 1,016  B  0,316  CD  Light 0,993  B  0,602  A  Kronstad Continuous darkness 1,147  B  0,381  BC  Light 0,758  C  0,471  AB  Nativ Continuous darkness 1,155  B  0,209  D  Light 1,155  B  0,459  B                                              Lsd 5% = 0,2192                      Lsd 5% = 0,1335 Means followed by same letters are not significantly different according to Duncan test (p ≤ 0.05) Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 80 -   Fig. 2. Microtuberization on darkness (in left) and light (in right)        Fig. 3. Microtubers produced under                                     continuous darkness                         light Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 81 -  Table 2 Influence of variety on number of microtubers produced / plantlets and their average weight Soiul  Microtubers number Duncan Test The average weight of microtubers (g) Duncan test Roclas  1,177 A 0,272 B Rustic  1,122 AB 0,261 B Gared  1,004 BC 0,459 A Kronstad  0,953 C 0,426 A Nativ  1,155 A 0,334 B                                  Lsd 5% = 0,1302                                  Lsd 5% = 0,07934  Means followed by same letters are not significantly different according to Duncan test (p ≤ 0.05)  Average number of microtubers / plantlets, analyzed through the potential of variety shows the superiority of Roclas and Nativ varieties (1.177; 1.155); the produced microtubers differ significantly from Gared varieties and Kronstad (1.004; 0.953); instead, these varieties have produced microtubers with highest weight (0.459 g, 0.426 g), differing significantly from the other varieties (table 2). Table 3 Influence of phytohormones on the number of  microtubers produced / plantlets and their average weight Phytohormone Microtubers number Duncan Test The average weight of microtubers (g) Duncan test Cumarine  1,135 A 0,316 A Benzylaminopurine  0,977 B 0,388 A Salicylic acid 1,135 A 0,348 A                           Lsd 5% = 0,1376                           Lsd 5% = 0,08387  Means followed by same letters are not significantly different according to Duncan test (p ≤ 0.05)  Analyzing the results through the influence of phytohormones (figure 2), it shows that the effect of coumarin and salicylic acid is superior to benzylaminopurine, proved by the increased number of microtubers; the three phytohormones did not differ significantly in terms of the influence on the average weight of microtubers (table 3).    Fig. 4. Influence of phyohormone in microtuberization (depending by cumarine, benzylaminopurine, salicylic acid – from left to right)  The combined influence of variety and phytohormones in microtuberization indicates achieving of differentiated results:  - salicylic acid prints for Roclas variety high capacity in microtuberization, by obtaining a large number of microtubers (1.330) and benzylaminopurine for Kronstad variety inhibits microtubers production (0.768); Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 82 -  - phytohormones coumarin and salicylic acid stimulates the production of microtubers for Roclas Rustic, Nativ  varieties; for the latter one stimulative effect is given by the BAP, which is not significantly different from the others two phytohormones; - salicylic acid and benzylaminopurine are beneficial to Gared variety by producing microtubers  with increased weight (0.533 g, 0.496 g); in the same manner, benzylaminopurine promotes Kronstad variety (0.467 g). - instead, not the same positive effect on the weight it had coumarin over microtubers of Roclas variety (0.223 g) (table 4).  Table 4 Influence of variety and phytohormones on the number of microtubers produced / plantlets and their average weight Variety  Phytohormone Microtubers number Duncan Test The average weight of microtubers (g) Duncan Test Roclas Cumarine 1,167 ABCD 0,223 E Benzylaminopurine  1,034 BCDE 0,283 DE Salicylic acid 1,330 A 0,310 DE Rustic Cumarine 1,278 AB 0,260 DE Benzylaminopurine  0,916 EF 0,292 DE Salicylic acid 1,171 ABCD 0,232 E Gared Cumarine 0,943 DEF 0,348 BCDE Benzylaminopurine  1,063 BCDE 0,496 AB Salicylic acid 1,007 CDEF 0,533 A Kronstad Cumarine 1,060 BCDE 0,407 ABCD Benzylaminopurine  0,768 F 0,467 ABC Salicylic acid 1,031 CDE 0,402 ABCD Nativ Cumarine 1,227 ABC 0,340 CDE Benzylaminopurine  1,104 ABCDE 0,399 ABCD Salicylic acid 1,134 ABCDE 0,263 DE                                                       Lsd 5% = 0,2468                              Lsd 5% = 0,150  Means followed by same letters are not significantly different according to Duncan test (p ≤ 0.05) Means followed by same letters are not significantly different according to Duncan test (p ≤ 0.05). Influence the sugar quantity used in the nutrient medium is smaller than the influence of variety in microtuberization: - sugar in a quantity of 140 g/l, was established as a good support for the Roclas variety in microtuberization (which induces the formation of 1.262 microtubers / plant); fraction of 80 g/l sugar sustain Rustic, Gared, Kronstad varieties in producing microtubers (1.136, 1.159, 1.017) and for the variety Nativ the two sugar fractions did not differ significantly from viewpoint of obtaining microtubers. - Sugar quantity of 80 g/l favors Gared variety in producing microtubers with highest weight (0.473) which is considered significant compared to Roclas and Rustic and insignificant to Kronstad and Nativ for both graduations of sugar (table 5).  Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 83 -  Table 5 Influence of variety and sugar quantity used in the nutrient medium on the number of   microtubers produced / plantlets and their average weight Variety Sugar quantity (g/l) Microtubers number Duncan Test The average weight of microtubers (g) Duncan Test Roclas  80 1,092 ABC 0,262 CD 140 1,262 A 0,282 BCD Rustic  80 1,136 AB 0,232 D 140 1,108 ABC 0,291 BCD Gared  80 1,159 AB 0,473 A 140 0,849 D 0,445 AB Kronstad  80 1,017 BCD 0,431 ABC 140 0,889 CD 0,421 ABC Nativ  80 1,156 AB 0,334 ABCD 140 1,156 AB 0,334 ABCD                           Lsd 5% = 0,2192                                  Lsd = 0,1804  Means followed by same letters are not significantly different according to Duncan test (p ≤ 0.05). Influence the sugar quantity used in the nutrient medium is smaller than the influence of variety in microtuberization: - sugar in a quantity of 140 g/l, was established as a good support for the Roclas variety in microtuberization (which induces the formation of 1.262 microtubers / plant); fraction of 80 g/l sugar sustain Rustic, Gared, Kronstad varieties in producing microtubers (1.136, 1.159, 1.017) and for the variety Nativ the two sugar fractions did not differ significantly from viewpoint of obtaining microtubers. - Sugar quantity of 80 g/l favors Gared variety in producing microtubers with highest weight (0.473) which is considered significant compared to Roclas and Rustic and insignificant to Kronstad and Nativ for both graduations of sugar (table 5).  CONLUSIONS  Conduct of researches and interpretation of the results indicated that microtuberization depends of the interaction: microtuberization conditions - variety - type of phytohormone - the amount of sugar. Weight of microtubers produced on light conditions, was significantly higher for Gared and Kronstad varieties compared to other varieties (0.602 g and 0.471 g) microtuberization induced in dark variety is beneficial for Roclas when the aim is to produce a large number of microtubers. Addition of salicylic acid and coumarin in microtuberization medium, created the possibility of using these substances as growth regulator, in achieving effective results in microtuberization. Although graduation of sugar factor has less influence compared to variety, beneficial effects presented:  - fraction of 140 g/l sugar in productivity of Roclas variety (1.262 / plant microtubers);  - fraction of 80 g/l sugar stimulated the formation of tubers with higher weight (0.473 g) for  Gared variety. Researches has led to a material with a high biological value:  - Roclas varieties and Nativ-which had the highest number of microtubers / plant (1.177, 1.155);  Agricultura – Ştiinţă şi practică                                         no. 1- 2(89-90)/2014                                         Agriculture - Science and Practice  - 84 -  - Gared and Kronstad varieties, which recorded the highest value of the average weight of microtubers (0.459 g, 0.426 g).  REFERENCES  1. Clarcke JD., Aarts N., Feys B., Dong X. (2001). Constitutive diseas resistance requires EDS I in the Arabidopsis mutans cpr 1 and cpr 6 and is partially in cpr 5, Plant J. 26 (4); p.409-420.  2. Ewing EE and Struik PC. (1992). Tuber formation in potato: Induction, initiation and growth. Hortic Rev 14: 89-98. 3. Forti E, Mandolino G, Ranalli P. (1991). In vitro tuber induction: influence of the variety and of the media. Acta Hortic. 300:127-132. 4. Gopal J, Minocha JL and Dhaliwal HS. (1998). Microtuberization in potato (Solanum tuberosum  L. ) Pl Cell Rep. 17: 794-8. 5. Gopal J, Minocha JL, Sidhu JS. (1997). Comparative performance of potato crops raised from microtubers induced in dark versus microtubers induced in light. Potato Res 40: 407–412. 6. Khuri, S.; Moorby, J. (1996). Nodal segments or microtubers as explants for in vitro microtuber production of potato. Plant Cell, Tissue and Organ Culture vol. 45 issue 3. p. 215 – 222. 7. Handro W., Mello CM., Manzano MA., Floh ES. (1997). Promotive effects of Salicylic 8. acid.R. Bras. Fisiol.Veg. 9 (2), p. 139-142. 9. Radouani, A. (1997). „In vitro” tuberization of potato cultivars, Nicola and Russet Burbank, as influenced by growth regulators, temperature and agitation. M.S. Thesis, University of Minnesota, St. Paul  10. Murashige T, Skoog F. (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473–497. 11. Ortiz-Montiel, G. and H. Lozoya-Saldana. (1987). Potato minitubers: technology validation in Mexico. Am Potato J 64:535–544. 12. Slimmon T, Souza Machado V, Coffin R. (1989). The effect of light  on in vitro microtuberization of potato cultivars. American Potato Journal 66, 843-848.  13. Wang PJ and Hu CY. (1982). In vitro mass tuberization and virus-free seed potato production in Taiwan. Am Po J. 59: 33-37. 14. Wattimena, G., B. McCown and G. Weis. (1983). Comparative field performance of potatoes from microculture. Am Potato J 60:27–33.             

RESEARCHES CONCERNING IMPROVING METHODS OF IN VITRO MICROTUBERS PRODUCTION

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RESEARCHES CONCERNING IMPROVING METHODS OF IN VITRO MICROTUBERS PRODUCTION

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