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The aim of this research was carried out to rapid multiplication of Curcuma aromatica salisb with large quantities, uniform and good quality, stored and transported easily which are advantageous for in vitro multiplication. Experiment comprised: the effect of tree concentration Benzyl adenin (3, 5, 7 mg/L) and six concentration sucrose (20, 40, 60, 80, 100, 120 mg/L) on the in vitro microrhizome production were investigated.
AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 EFFECT OF BENZYL ADENINE AND SUCROSE FOR IN VITRO MICRORHIZOME PRODUCTION IN CURCUMA AROMATICA SALISB Nguyen Thi Thuy Diem1 An Giang University, VNU - HCM Information: Received: 04/10/2018 Accepted: 13/08/2019 Published: 11/2019 Keywords: Sucrose, microrhizomes, in vitro, Curcuma aromatica, Benzyl adenin ABSTRACT The aim of this research was carried out to rapid multiplication of Curcuma aromatica salisb with large quantities, uniform and good quality, stored and transported easily which are advantageous for in vitro multiplication Experiment comprised: the effect of tree concentration Benzyl adenin (3, 5, mg/L) and six concentration sucrose (20, 40, 60, 80, 100, 120 mg/L) on the in vitro microrhizome production were investigated The experiment was arranged in a completely randomized form, two-factor, 18 treatments and replications The results indicated that shoots of Curcuma aromatica salisb cultured on MS medium supplemented with 80 g/L sucrose and BA - mg/L for to be the most suitable medium for in vitro microrhizomes induction of Curcuma aromatica with time sooner, the rate of in vitro tuber formation, the number of tubers, tuber weight and tuber diameter reached the highest after 16 weeks of culture INTRODUCTION types On the other hand, white turmeric was demonstrated diffent from essenttial oil into white turmeric plant such as anti- inflammatory, anti platelet aggregation, cough suppression and kidney protection (Sikha et al., 2015) In nature, white turmeric occurs from rhizome, after about 210 day to establish the rhizome breed (Ravindran et al., 2007) The rhizome has a long domance and germinates the rainy season Beside, the work to maintain source species of white turmeric annually is both costly and effort Often, the disease on the plant such as rhizome is a negative ordor (by Pythium sp.) and spot leaf by Taphrina species and Collectrichum are very damaging to the preservation process breed in field causing the lack of the breed resource in the culture (Nayak & Naik, 2006) Beside, white turmeric is building plan for planting area in An Giang province to make material area This object White turmeric (Curcuma aromatic Salisb) is a rare medicinal herb plant species, in Zinger family – Zingiberaceae, this is projecting to develop herbs plant source in An Giang province from now onwards to the year 2020 Studies show the biological active substances extracted from rhizome in white turmeric contain group acticve antioxidant such as curcuminoid, flavonoid, terpenoid In the adopted antioxidant mechanism, the manufatured product from white turmeric have biological effects such as anti- inflammatory stomach medication from wine (Jeon et al., 2010) The situation artery antherogenesis is reduced (Lee et al., 2010), prevent to injury carcinogenesis on skin by ultraviolet ray (Panich et al., 2010) The power antioxidant mechanism, white turmeric plant has potential to become effect medicine treatment assist for many cancer 17 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 is new so the white turmeric breed source is seen early So, the cultural technology is set up to produce breed and quality guaranteed to support seedlings free from disease, uniform to request breed produce In making microrhizomes in vitro by method micropropagation to processing and good achieved on object such as Solanum tuberosum L., Dioscorea composita, Gladiolus spp., Zingiber officinale Rosc, Curcuma longa,…(Gopal et al., 1998; Alizade et al., 1998; Dương Tấn Nhựt et al., 2007; Sharma & Singh, 1995; Sunitibala et al., 2001) Currently, the studies on microrhizome in vitro have not been processed in Viet Nam through the seedling source for production to support the farmer disease medicine for 60 minutes in clean rice husk ash After 15 – 20 days of incubation, the shoots of white turmeric began to appear MATERIALS AND METHODS The shoots were divided from rhizome washed, roots cut and leaves cleaned, 1/2 leaf cut and clean washed Next, the shoots were soaked in light soap after immature buds were cleaned with running tap water Explants were then immersed in 70% ethanol for and the explants were rinsed three times with distilled water Next, the explants were soak with 20% Ca(OCl)2 for 15 minutes, after the explants were rinsed three times with distilled water The explants were cut, divided to take the shoot top and culture in bottle contain medium MS not supplemented plant growth regulator to create a material source to use for experimentation in making microrhizome 2.1 Plant Materials 2.3.2 Experimental arrangement The white turmeric rhizome (Curcuma aromatica) to take on Tinh Bien Oriental medicine congress in An Giang province and take it from the lab of Agriculture and Natural Resources, An Giang University to nursery Excised buds of Curcuma aromatica were high about 4-5 cm, initially cultured on MS basal medium After 14 days of culture, the shoots became root formations and were transfered to a culture medium MS supplement with BA and sucrose in different concentrations for microrhizome induction The experiment was arranged in Completely Block Design (CBD), two factor, including concentrations of BA (3, and mg/L) and concentrations of sucrose (20, 40, 60, 80, 100 and 120 g/L) The experiment consists of 18 treatments, each treatment was repeated times, each time was the culture bottle (1 explant/ the culture bottle) 2.2 Medium and culture conditions The medium were used MS basal medium (Musrashige & Sokol, 1962) supplemented with agar (8 g/l), Myo - Inositol (0,1 g/l) and mg/L NAA Depending on how the experiment supplemented with benzyl adenin (BA) and sucrose in different concentrations The pH of the media were adjusted to 5.8 using 1M NaOH or 1M HCl The culture bottles were steam sterilized in an autoclaved at 1210C, atm for 30 2.3.3 Observations recorded for experiments Cultural conditions: The explants were maintained in a growth room under white fluorescent light for a daily photoperiod of 16 hours, with temperature 24 ± 0C The time microrhizome induction (weeks after cultured - WAC), rate tuber formation (%), the number average tuber/explants, the weight microrhizome (g), diameter microrhizome (cm) Observations were done every week starting from the first week after culture initiation to 16 weeks after culture initiation 2.3 Methods 2.3.1 Culture to make source sample starting Curcuma aromatica rhizomes were selected from to months of age, uniform and not diseased Samples were treating with Antracol 70WP 2.3.4 Data analysis 18 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 Data were analyzed using the Statistical SPSS software version 20.0 and analysis of variance (ANOVA) was used to investigate if there is any significant difference A mean separation test was conducted using Duncan multiple range The time set up microrhizome in vitro had interact between BA concentration and sucrose The time set up microrhizome induction in vitro earliest on two experiment to supplemented mg/L BA combine 80 mg/L sucrose and mg/L BA combine 60 mg/L sucrose in weeks after being culturation (Fig 1.), it was statistically different in significance at 1% compared to the remaining treatments RESULT AND DISCUSSION 3.1 The time microrhizome induction in vitro of white turmeric shoots Table Effect of concentration of BA and sucrose on the time microrhizome white turmeric induction in vitro Sucrose concentrations (g/L) Concentrations of BA (mg/L) Average (Sucrose) 20 11.00 d 10.00 e 12.00 c 11.00 b 40 9.00 f 8.00 g 9.00 f 8.67 c 60 6.00 i 5.00 k 7.00 h 6.00 e 80 5.00 k 5.67 j 6.00 i 5.56 f 100 7.00 h 7.00 h 9.00 f 7.67 d 120 13.00 b 13.00 b 15.00 a 13.67 a Average (BA) 8.50 b 8.11 c 9.67 a F (Experiment) ** F (BA) ** F (Sucrose) ** F (BA x Sucrose) ** CV (%) 1.54 Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1% Fig In vitro microrhizomes induction of white turmeric after weeks of incubation on culture medium 19 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 The effect of BA concentration to time induction to microrhizome production in vitro In concentrate mg/L BA has time to show microrhizome in earliest (8.11 weeks after cultured), the difference was statistically significant at 1% compared with mg/L BA and mg/L BA concentration in a culture medium It suggest that the time induction microrhizome of BA early or later then depended on BA concentration in supplemented in a culture medium In the BA concentration suitable for induction microrhizome formation in vitro were mg/L, the microrhizome were formation earlier, average about 8.11 weeks after cultured, the shoots start set up microrhizome in mg/ L BA concentration and mg/L BA concentration after 8.50- 9.67 weeks after cultured the new microrhizome was establish When the sucrose concentration were increase from 20 – 80 g/L then the time induction microrhizome production to shorten from 11 weeks after cultured to decrease 5.56 weeks after cultured But, when the sucrose concentration were increased continuous up 100 – 120 g/L then the time induction microrhizome production from 7.67 weeks after cultured to increase 13.67 weeks after cultured show new microrhizome In general, the time induction microrhizome production were MS medium supplement mg/L NAA combine – mg/L BA and 60 -80 g/L sucrose in earliest, the microrhizome was show – weeks after culturation 3.2 The rate explant were established microrhizome of shoot white turmeric The results on table 2, the rate of explant to microrhizome established in 16 weeks after cultured to have interact between BA concentration and sucrose The explant culture rate were highest in experiment MS supplemented mg/L NAA with mg/L BA and 80 g/L sucroser to achieve 91.67%, the difference was statistically significant at 1% The sucrose concentration to effect come on the time induction microrhizome production in vitro The sucrose on 80 g/L concentration were time induction production earliest was 5.56 weeks after cultured, the difference was statistically significant at 1% compared the sucrose concentration in the other in a culture medium Table Effect of BA concentration and sucrose on the explant rate microrhizome formation (%) in white turmeric in vitro in the 16 weeks after cultured Average (Sucrose) Concentrations of BA (mg/L) Sucrose oncentration (g/L) 20 25.00 d 25.00 d 25.00 d 25.00 c 40 25.00 d 25.00 d 41.67 d 30.56 c 60 41.67 d 66.67 bc 25.00 d 44.44 b 80 91.67 a 75.00 b 58.33 c 75.00 a 100 41.67 d 75.00 b 33.33 d 50.00 b 120 25.00 d 25.00 d 25.00 d 25.00 c Average (BA) 41.67 b 48.61 a 34.72 c F (Experiment) ** F (BA) ** F (Sucrose g) ** F (BA x Sucrose) ** CV (%) 21.60 Note: The following letters were the same, the difference was not statistically significant: ** = the difference was statistically significant at 1% 20 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 The sucrose concentration to effect come the time induction microrhizome production in vitro In the sucrose 80 g/L concentration for the explant rate microrhizome formation achieved highest 75%, the difference was statistically significant at 1% compared the sucrose concentration different in the culture medium BA concentration to effect to the number of microrhizome In mg/L BA concentration for the number of microrhizome to achieve highest was 1.94 microrhizome/explants, the difference was statistically significant at 1% as compared to BA concentration mg/L and mg/L in the culture medium The BA concentration to effect come the explants rate microrhizome formation in vitro On the mg/L BA concentration for the explants rate microrhizome formation were highest (48.61%), the difference was statistically significant at 1% compared with MS medium supplemented mg/L BA and mg/L BA in the culture medium The sucrose concentration to effect come number microrhizome in vitro The sucrose concentration was 80g/L achive then the number highest baby microrhizome was microrhizome/explants, the difference was statistically significant at 1% as compared the sucrose concentration other in the culture medium In the result to table 2, when BA concentration to increase from mg/L to mg/L, the explants rate microrhizome formation to increase from 41.67% to 48.61% When BA concentration increase on mg/L, the explants rate microrhizome formation to go down 34.72% In the sucrose concentration, when the sucroser concentration to increase from 20 to 80 g/L then the explants rate microrhizome formation to increase from 25% to 75% But when the sucrose concentrate increased on 100 -120 g/L then the explant rate microrhizome formation go to down form 50% to 25% It suggest that, the BA concentration and high sucrose were inhibition to microrhizome formation in vitro The compared between experiment, the results on table 3, the number of microrhizome to highest increase in mg/L BA concentration with 80 g/L sucrose then achieved 3.67 microrhizome/ explants, the difference was statistically significant at 1% as compared the other experiment In general, the results in table 3, when increased BA concentration from mg/L to mg/ L, the number of microrhizome to increase from 1.67 microrhizome/ explants to 1.94 microrhizome/ explants When increase BA concentration up to mg/L, the number of microrhizome in vitro become go to down only achieve 1.39 microrhizome/ explants For sucrose, when the sucrose concentration to increase from 20 to 80 g/ L then the number of microrhizome to increase from microrhizome/explants to microrhizome/ explants But when the sucrose content up to 100 120 g/L then the number microrhizome formation go to down from microrhizome/ explants to microrhizome/explants So, the white turmeric shoots to culture on MS medium contain mg/L NAA to supplemented – mg/L BA concentration combine with 80 g/L sucrose are microrhizome to achieve highest (Fig.2) Finally, white turmeric shoots to culture on the MS medium to have mg/L NAA supplemented mg/L – mg/L BA combine with 80 g/L sucrose for the explants rate microrhizome formation was highest to achieve from 75% to 91.67% 3.3 The number of shoot white turmeric in vitro to increase In the 16 weeks after cultured, the results on table 3, the number of microrhizome to effected of BA concentration and sucroser content 21 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 Fig The development of microrhizome in vitro on the white turmeric shoot on medium supplemented -5 mg/ L BA and 80 g/ L sucrose at 16 weeks after cultured Table Effect of BA concentration and sucrose on the number of to increase on microrhizome in vitro after 16 weeks after cultured Sucrose concentration (g/L) BA concentration (mg/L) 20 1.00 d 1.00 d 1.00 d 1.00 c 40 1.00 d 1.00 d 1.67 d 1.22 c 60 1.67 d 2.67 bc 1.00 d 1.78 b 80 3.67 a 3.00 b 2.33 c 3.00 a 100 1.67 d 3.00 b 1.33 d 2.00 b 120 1.00 d 1.00 d 1.00 d 1.00 c 1.67 b 1.94 a 1.39 c Average (BA) F (Experiment) ** F (BA) ** F (Sucrose) ** F (BA x Sucrose) ** CV (%) Average (Sucrose) 21.63 Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1% 22 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 3.4 The weight of microrhizome white turmeric in vitro statistically significant at 1% as compared the other experiment Between on the experiment to contain 80g/ L sucrose combine with BA concentration and mg/L then microrhizome in vitro weight to achieve 0.93 gram/ microrhizome, the different was not statistically significant The results on table represent the 16 weeks after cultured, the sucrose content to have effect on weight increase of microrhizome in vitro The sucrose content on 80 g/L to achieve highest weight is 0.98 gram/microrhizome, the difference was statistically significant at 1% as compared the sucrose other in the cultural medium The weight microrhizome in vitro on sucrose content to 40 mg/L and 100 mg/L not the different was statistically significant The microrhizome in vitro weight on BA concentration (3, and 7) the different was statistically significant, the microrhizome in vitro average weight archive 0.55 gram/microrhizome after 16 weeks cultured In general, the BA concentration not effect to the weight increase of microrhizome in vitro The results were statistically significant about rhizome in vitro weight to increase all experiments The rhizome in vitro weight to highest achieve on MS medium supplemented mg/L BA and 80 g/L sucrose (1,08 gram/microrhizome), the different was So, the experiment to supplemented 80 g/ L sucrose on MS medium with mg/L NAA combine BA concentration -7 mg/ L for microrhizome in vitro weight was highest Table Effect of BA concentration and sucrose on up to weight (gram/microrhizome) white turmeric in vitro after 16 weeks cultured Sucrose concentration (g/L) BA concentration (mg/L) Average (Sucrose) 20 0.50 fg 0.39 gh 0.29 hi 0.39 d 40 0.49 fg 0.64 e 0.59 ef 0.57 c 60 0.79 c 0.77 cd 0.59 ef 0.72 b 80 1.08 a 0.93 b 0.93 b 0.98 a 100 0.41 g 0.43 g 0.66 de 0.50 c 120 0.13 j 0.19 ij 0.08 j 0.13 e 0.57 0.56 0.52 Average (BA) F (Experiment) ** F (BA) ns F (Sucrose) ** F (BA x sucrose) ** CV (%) 12.88 Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1%; ns= the difference was not statistically significant 23 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 3.5 The diameter to increase of white turmeric microrhizome The sucrose content to effect on increase diameter microrhizome in vitro The sucrose content in 80g/L to have diameter highest gain was 10.05 mm, the difference was statistically significant at 1% as compared the sucrose content other in the culture medium In the results on table 5, the BA concentration and sucrose content to interact on the increase of diameter of white turmeric in vitro microrhizome The BA concentration to effect on the diameter increase of rhizome in vitro In the BA concentration mg/L, the diameter of microrhizome in vitro achieve highest was 8.10 mm, the difference was statistically significant on 1% as compared the BA concentration mg/L, but the difference was not statistically significant than mg/ L BA to achieve 7.8 mm The result of table suggest that the different was statistically significant about to increase diameter microrhizome in vitro all experiment The diameter of microrhizome in vitro highest gain on MS medium to supplement mg/L BA and 80g/ L sucrose (13.7 mm), the difference was statistically significant at 1% as compared the experiment content other in the culture medium Table The effect of BA concentration and sucrose diameter to increase of microrhizome (mm) on white turmeric in vitro after 16 weeks after cultured Sucrose concentration (g/L) BA concentration (mg/L) Average (Sucrose) 20 8.10 cde 8.00 de 5.60 gh 7.20 c 40 6.40 fg 8.30 bcd 8.40 bcd 7.70 c 60 9.30 b 8.60 bcd 6.90 f 8.20 b 80 13.70 a 9.10 bc 8.80 bcd 10.50 a 100 7.30 ef 8.50 bcd 6.50 fg 7.40 c 120 3.90 i 4.00 i 5.10 h 4.30 d 8.10 a 7.80 a 6.90 b Average (BA) F (Experiment) ** F (BA) ** F (Sucrose) ** F (BA x sucrose) ** CV (%) 7.23 Note: The following letters were the same, the difference was no significant statistically: ** = the difference was statistically significant at 1% In general, the experiments was supplemented – mg/ L BA with 80g/L sucrose in MS medium culture to contain mg/L NAA for the diameter of microrhizome to biggest gain after 16 weeks after cultured (Fig 3.) microrhizome formation of white turmeric in in vitro condition to effect of BA concentration and sucrose content interact The white turmeric shoots were culture on MS medium to supplemented 80g/L BA combine – mg/L BA and mg/L NAA then the time microrhizome induction was earlier, at the same time the In summary, the results of target about microrhizome production in vitro suggest that the 24 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 microrhizome induction, the microrhizome in vitro formation rate, the number of microrhizome production and microrhizome diameter were decreased microrhizome in vitro formation rate, the number of microrhizome production and microrhizome diameter were highest When the BA concentration to increase mg/L combine 100 mg/L and 120 mg/L sucrose then the time Fig The microrhizome diameter of white turmeric shoot were growth on the medium supplemented 3-5 mg/L BA and 80 g/L sucrose in 16 weeks after (cultured) sucrose from – 8% (w/v) was the most important ingredient for formation in vitro tuber on Diascoria composita It suggests that the study topic results concluded that, in the study to show the medium MS supplemented 80 g/L sucrose for the number of microrhizome in vitro was most The number of microrhizome in vitro was shortest on the MS medium contain 20 g/L and 120 g/L sucrose The sucrose was role to promote formation organ store in plant When the sucrose concentrate from 20 g/L to 80 g/L to show the microrhizome in vitro formation to rate, the number microrhizome, the weight microrhizome and diameter microrhizome are increase It suggests that the show of carbon content in sucrose and the white turmeric ingredients contain carbohydrates and sucrose The results of the experiment showed the sucrose concentration in the medium to effect on the formation microrhizome in in vitro condition The result of the experiment suggests that the BA concentration and sucrose content to effect interact on microrhizome in vitro formation On the MS medium contain mg/L NAA to supplemented 80 g/L sucrose with 3-5 mg/L BA for the time microrhizome induction, the microrhizome formation rate, the number of microrhizome productions, the weight and diameter of microrhizome were highest It suggests that this BA concentration to effect on transport carbohydrate content in the white turmeric shoots The result that similar of Nanak (200), the number of microrhizome in vitro to many formation on the medium supplemented 5mg/ L BA on C.aromatica Salisb In Curcuma sp species, the shoots were rhizome formation to view when the medium to supply sucrose concentration was 60 – 90 g/L (Nayak (2000); Sunitabala et al (2001) In addition, the study about the formation zinger in vitro rhizome of Sharma Singh (1995), the sucrose concentration was 75 g/L to effect in rhizome formation Sedigeh et al (1998) to believe that the In summary, the results showed the plant growth regulator was role on cell division, dimensions of shoot germination and the effect on size of 25 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 rhizome The sucrose was effect in the growth process of microrhizome in vitro, the development of baby rhizome when the sucrose was highest, the sucrose supplemented nutrient to growth rhizome and rhizome induction, the rhizome was formation and development moisture was provided by water spray twice a day (humid 70 – 80%) The temperature was 28 – 30 C in the nursery step The results showed, after weeks plant on rice husk ash all the white turmeric rhizome in vitro show shoots, leaves and roots ( Fig 5A) After 30 days planted on pot to show the white turmeric from rhizomes in vitro the root system was developed and deep in the ground, the nursery plant was developed with – leaves, the plant was moved out off the pot and planted in black plastic (Fig 5B) 3.6 The Acclimatization of white turmeric in vitro The white turmeric rhizome in vitro were harvested on the medium rhizome production, clean wash agar and nursery on rice husk ash The A B Fig The white turmeric rhizome in vitro to show shoot after weeks cultured (A); the white turmeric from microrhizome in vitro (B) after 30 days planted into pot CONCLUSION Gopal, J., Minocha, J L., & Dhaliwal, H S (1998) Microtuberization in potato (Solanum tuberosum L.) Plant Cell Rep, 17: 794-798 The medium microrhizome production in vitro was MS medium supplemented mg/ L or mg/L BA combine with 80 g/L sucrose Jeon, W Y., Lee, M Y., Shin, I S., Jin, S E., & Ha, H (2015) Curcuma aromatica Water Extract Attenuates Ethanol-Induced Gastritis via Enhancement of Antioxidant Status Eviddence based Complementary and Alternative Medicine, http://dx.doi.org/10.1155/2015/582496 RECOMMENDATION Continue evaluation of the growth and development of rhizome in vitro on nursery stage of the white turmeric REFERENCE Alizadeh, S., Mantell, S H., & Viana, A M (1998) In vitro shoot culture and microtuber induction in steroid yam Dioscorea composita Hemsl Plant Cell Tiss Org Cult 53: 107112 Lee, H S., Lee, M J., Kim, H., & Choi, S K (2010) Curcumin inhibits TNFalpha-induced lectin-like oxidised LDL receptor-1 (LOX-1) expression and suppresses the inflammatory response in human umbilical vein endothelial cells (HUVECs) by an antioxidant mechanism J Enzyme Inhib Med Chem, 25, 720–729 Duong Tan Nhut, Le Thi Diem, Dang Thi Thu Thuy, & Nguyen Duy (2007) Effect of sucrose, IBA, and other culture conditions on in vitro corm formation of Gladiolus spp Journal of biotechnology, vol (1): 67-75 Murashige, T & Skoog, F (1962) A Revised Medium for Rapid Growth and Bio Assays 26 AGU International Journal of Sciences – 2019, Vol (3), 17 – 27 with Tobacco Tissue Cultures Physiologia Plantarum, 15, 473-497 Ravindran, P N., Nirmal, B K., & Sivaraman, K (2007) Turmeric: The genus curcuma Boca Raton, FL: CRC http://dx.doi.org/10.1111/j.13993054.1962.tb08052.x Sharma, T R., & Singh, B M (1995) In vitro microrhizome production in Zingiber officinale Rosc Plant Cell Rep 15: 274-277 Nayak, S (2000) In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb Plant Growth Regulation, 32: 41- 47 Sedigeh, A., Mantell, S H., & Viana, A M (1998) In vitro shoot culture and microtuber induction in the steroid yam dioscorea composite Hemsl Plant Cell Tissue Organ Cult., 53: 107-112 Nayak, S., & Naik, P K (2006) Factors effecting in vitro microrhizome formation and growth in Curcuma longa L and improved field performance of micropropagated Plants ScienceAsia, 32: 31-37 Sikha, A., Harini, A & Hegde Prakash, L (2015) Pharmacological activities of wild turmeric (Curcuma aromatica Salisb): a review Journal of Pharmacognosy and Phytochemistry, 3(5): p 01-04 Panich, U., Kongtaphan, K., Onkoksoong, T., Jaemsak, K., Phadungrakwittaya, R., Thaworn, A., Akarasereenont, P., & Wongkajornsilp, A (2010) Modulation of antioxidant defense by Alpinia galanga and Curcuma aromatica extracts correlates with their inhibition of UVA-induced melanogenesis Cell Biol Toxicol 26, 103– 116 Sunitibala, H., Damayanti, M., & Sharma, G J (2001) In vitro propagation and rhizome formation in Curcuma longa Linn Cytobios 105: 71-82 27 ... 3.1 The time microrhizome induction in vitro of white turmeric shoots Table Effect of concentration of BA and sucrose on the time microrhizome white turmeric induction in vitro Sucrose concentrations... concentration and sucrose content to interact on the increase of diameter of white turmeric in vitro microrhizome The BA concentration to effect on the diameter increase of rhizome in vitro In the BA... (1995) In vitro microrhizome production in Zingiber officinale Rosc Plant Cell Rep 15: 274-277 Nayak, S (2000) In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb Plant