Adventitious root of T. Paniculatum Gaertn. : a‐d fresh biomass in batch,

32 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 7 14 - 14 + 21 28 - 28 + 35 Total su gar Days 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 7 14 - 14 + 21 28 - 28 + 35 pH Days 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 7 14 - 14 + 21 28 - 28 + 35 Electrica l co nd uctivity m Scm Days Continuous + Buffer Batch + Buffer Continuous + Non Buffer Batch + Non Buffer and cells is commonly used for the production of growth associated products, typically primary metabolites and biomass. The continuous culture technique has also been adopted for the cultivation of several plant cells such as, Coptis japonica Matsubara et al. 8 , Catharanthus roseus Park et al. , and Nicotiana tabacum ashimoto et al. 8 . Total sugar, conductivity and hydrogen ion concentration in the medium Determination of hydrogen ion concentration p , total sugar and electrical conductivity EC of the médium was conducted every seven days. Basically, total sugar, p and EC had decreased, but increased by using buffer of NaCO every two weeks figure . The total sugar value in batch system are different with continous system which was provided new medium in days and 8 days. Meanwhile, p in batch and continuous without buffer decreased until days, except on batch and continuous with buffer periodically in two weeks increased in days and 8 days. For electrical conductivity in the all treatments decreased and increased when was added buffer each two weeks. Figure 3. Adventitious root culture condition for days. Culture condition change in A hydrogen ion p ; B Total sugar; C Electrical conductivity EC . ‐ before added buffer and + after added buffer. Several studies described that liquid medium was examined by electrical conductivity, p and total sugar. The high conductivity of medium showed that the adventitious root unavailable to absorb inorganic compounds. n study of Mariateresa et 32 al . reported that sucrose availability in liquid medium bioreactor . Adelberg et al. also reported that findings of sucrose availability in liquid medium higher than in gelled medium. t has known that sucrose as energy source and important carbon for plant cells growth. Gertlowski and Petersen explained that it can also affect metabolism of the cells and production of metabolites. Lee et al. reported that sucrose is hydrolyzed into two monosaccharides, glucose and fructose, by invertase which bounds extracellular or cell wall during the initial culture period. Meanwhile, electrical conductivity EC values of the residual media progressively increased with increasing salt strength of the culture medium, whereas p values progressively decreased. The EC values of the residual media reflect the uptake of ions by roots and represent an indirect method of biomass estimation Baque et al., ; Cui et al., . n plant tissue culture, p is an important factor affecting biomass and secondary metabolite accumulation. For instance, in root suspension cultures of ginseng, root dry biomass and ginsenoside accumulation were strongly inhibited when p was maintained below . or above . ahn et al., . Wu et al., also reported that the growth of Echinacea roots decreased when p was maintained below or above . Lulu et al. explained that in their study after weeks of culture, the p of the medium declined as the N + level increased. The EC measurements have been used as an indirect method for biomass estimation during the continuous online monitoring of nutrient uptake in plant cell culture bioprocess engineering because of their accuracy and efficiency. Conclusions n this study, we have successfully established adventitious root cultures of T. paniculatum L capacity bioreactors containing mL medium and worked out various treatments continuous + buffer, continuous, batch + buffer, batch . Treatment of continuous system with buffer of NaCO has shown fresh biomass .8 g and dry biomass . g higher than the others. That treatment has produced biomass four fold of initial inoculum. The continuous system provide biomass production better than the others and supplemented of buffer NaCO influence biomass productivity. Acknowledgements Thanks to ndonesia Endowment Fund for Education LPDP , Ministry of Finance, Republic of ndonesia and Plant Physiology Laboratory at Departement of Biology, Faculty of Science and Technology, Airlangga University. References Adelberg, J. W., Delgado, M. P. and Tomkins, J. T. . Spent medium analysis for liquidculture micropropagation of emerocallis on Murashige and Skoog medium, n Vitro Cell. Dev. Biol. Plant Vol. , No. , pp. – . Baque, M. A., Shiragi, M. . K., Moh, S. ., Lee, E. J. and Paek, K. Y. . Production of biomass and bioactive compounds by adventitious root suspension cultures of Morinda citrifolia L. in a liquid‐phase airlift balloon‐type bioreactor. n Vitro Cell. Dev. Biol., Vol. , pp. – . Choi, S. M., Son, S. ., Yun, S. R. and Paek, K. Y. . Pilot‐scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell Tiss. Org. Cult., Vol. pp. 8 – . Cui, X. ., Chakrabarty, D., Lee, E. J. and Paek, K. Y. . Production of adventitious roots and secondary metabolites by Hypericum perforatum L. in a bioreactor. Bioresour. Technol., Vol. , pp. 8– . Cui, . Y., Murthy, . N., Moh, S. ., Cui, Y. Y., Lee, E. J. and Paek, K. Y. Production of biomass and bioactive compounds in protocorm cultures of Dendrobium 327 candidum Wall ex Lindl. Using ballon type bubble bioreactors. nd. Crops and Products, Vol. , pp. 8‐ . Davis, J. M. ed. Basic Cell Culture: A Practical Approach. p. ‐ . New York: Oxford University Press. Freshney, R. . ed. Animal Cell Culture: A Practical Approach. nd ed. p. . New York: Oxford University Press. Gertlowski, C. and Petersen, M. . nfluence of the carbon source on growth androsmarinic acid production in suspension cultures of Coleus blumei. Plant Cell Tissue Org. Cult., Vol. , No. , pp. 8 – . ahn, E. J. and Paek, K.Y. . Multiplication of Chrysanthemum shoots in bioreactors as affected by culture method and inoculation density of single node stems. Plant Cell Tissue Org. Cult., Vol.8 , pp. – . ahn, E. J., Kim, Y. S., Yu, K. W., Jeong, C. S. and Paek, K. Y. . Adventitious root cultures of Panax ginseng C. V. Meyer and ginsenoside production through large‐ scale bioreactor system. J. Plant. Biotechnol., Vol. , pp. – . ashimoto, T., Azechi, S., Sugita, S. and Suzuki, K. 8 . Large scale production of tobacco cells by continuous cultivation. n: A. Fujiwara ed . Plant Tissue Culture, p. ‐ . Tokyo: Japan Association of Plant Tissue Culture. Jo, E. A., Murthy, . N., ahn, E. J. and Paek, K. Y. 8 . Micropropagation of Alocasia amazonica using semisolid and liquid cultures. n Vitro Cell. Dev. Biol. Plant., Vol. No. , pp. – . Kieran, P. M., O’Donnell, . J., Malone, D. M. and MacLoughlin P. F. . Fluid shear effects on suspension cultures of Morinda citrifolia. Biotechnol. Bioeng., Vol. , pp. ‐ . Kim, Y. S., ahn, E. J., Yeung, E. C. And Paek, K. Y. . Lateral root development and saponin accumulation as affected by BA or NAA in adventitious root cultures of Panax ginseng C.A. Meyer. n Vitro Cel. Develop. Biol. – Plant., Vol. , pp. – . Lee, E. J., Moh, S. . and Paek K. Y. . nfluence of inoculum density and aeration volume on biomass and bioactive compound production in bulb‐type bubble bioreactor cultures of Eleutherococcus koreanum Nakai. Bioresour. Technol., Vol. , pp. – . Lee, E. J., Mobin, M., ahn, E. J. and Paek, K. Y. . Effect of sucrose, inoculums density, auxins, and aeration volume on cell growth of Gymnema sylvestre. J. Plant Biol. Vol. , pp. ‐ . Lulu, T., Park, S. Y., brahim, R. and Paek, K. Y. . Production of biomass and bioactive compounds from adventitious roots by optimization of culturing conditions of Eurycoma longifolia in ballon‐type bubble bioreactor sistem. J Biosci and Bioeng., Vol. , No. , pp. ‐ . Manuhara, Y. S. W., Kristanti, A. N. and Utami, E. S. W. . Optimization of Culture Conditions of Talinum paniculatum Gaertn. Adventitious Roots in Balloon Type Bubble Bioreactor Using Aeration Rate and nitial noculum Density. Asian Journal of Biological Sciences Vol. 8, No. , pp.8 ‐ . Mariateresa, C., Maria, C. S. C. and Giuseppe, C. . nfluence of ozone treatments on in vitro propagation of Aloe barbadensis in continuous immersion bioreactor. Industrial Crops and Products, Vol. , pp. ‐ . Matsubara, K., Kitani, S., Yoshioka, T., Morimoto, T., Fujita, Y. and Yamada, Y. 8 . igh density culture of Coptis japonica cells increases berberine production. J. Chem. Technol. Biotechnol., Vol. , pp. ‐ . Meijer, J. J., ten oopen, . J. G., Luyben, K. C. A. M. and Libbenga K. R. . Effects of hydrodynamic stress on cultured plant cells: a literature survey. Enzyme Microb. Technol., Vol. , pp. ‐ 8. Murashige, T. and Skoog, F. . A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, Vol. , pp. ‐ . 328 Paek, K. Y., Murthy, . N., ahn, E. J. and Zhong, J. J. . Large scale culture of ginseng adventitious roots for production of ginsenosides. Adv. Biochem. Eng. Biotechnol., Vol. , pp. – . Paek, K. Y., Chakrabarty, D. and ahn, E. J. . Application of bioreactor systems for large‐scale production of horticultural and medicinal plants. Plant Cell Tissue Organ Cult., Vol. 8 , pp. 8 – . Park, . ., Choi, S. K., Kang, J. K. and Lee, . Y. . Enhancement of producing catharanthine by suspension growth of Catharanthus roseus. Biotechnol. Lett. Vol. , pp. ‐ 8. Praveen, N. and Murthy, . N. . Establishment of cell suspension cultures of Withania sominifera for the production of withanolide A. Bioresour. Technol. Vol. , pp. – . Sajc, L., Grubisic, D. and Novakovic, G. V. . Bioreactors for plant engineering: an outlook for further research. Biochem. Engineer. J., Vol. , pp.8 – . Scragg, A. ., Allan, E. J. and Leckie, F. 88 . Effect of shear on the viability of plant cell suspensions. Enzyme Microb. Technol., Vol. , pp. ‐ . Shaik, S., Dewir, Y. ., Singh, N. and Nicholas, A. . Micropropagation and bioreactorstudies of the medicinally important plant Lessertia Sutherlandia frutescens L. S. Afr. J. Bot., Vol. , pp. 8 – 8 . Shiao, T. and Doran, P.M. . Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures. J. Biotechnol., Vol. 8 , pp. – . Sivakumar, G. . Bioreactor technology: A novel industrial tool for high‐tech production of bioactive molecules and biopharmaceuticals from plant roots, Biotechnol. J., Vol. , pp. – . Sivakumar, G., Yu, K. W. and Paek, K. Y. . Biosafe ginseng: A novel source for human well being. Eng. Life Sci., Vol. , pp. – . Vogelmann, ., Bischof, A., Pape, D. and Wagner, F. 8 . Some aspects of mass cultivation, in: A.W. Alfermann, E. Reinhard Eds. , Production of Natural Compounds by Cell Culture Methods, pp. – . Munich: GSF. Wongasmuth, M. and Doran, P. M. . Foaming and cell floatation in suspended plant cell cultures and the effect of chemical antifoams. Biotechnol. Bioeng., Vol. , pp. 8 ‐ 88. Wu, C. . Murthy, . N., ahn, E. J. and Paek K. Y. . Enhanced production of caftaric acid, chlorogenic acid and cichoric acid in suspension cultures of Echinacea purpurea by the manipulation of incubation temperature and photoperiod. Biochem Eng J., Vol. , pp. – . Wu, C. ., Dewir, Y. ., ahn, E. J. and Paek, K. Y. . Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots of Echinacea angustifolia. J. Plant Biol., Vol. , pp. – . Yu, K. W., Gao, W. Y., ahn, E. J. and Paek, K. Y. . Effects of macro elements and nitrogen source on adventitious root growth and ginsenoside production in ginseng Panax ginseng C.A. Meyer . J. Plant Biol. Vol. , pp. – 8 . Zhong, J. J., Seki, T., Kinoshita, S. and Yoshida, T. . Effects of surfactants on cell growth and pigment production in suspension cultures of Perilla frutescens. World J. Microbiol. Biotechnol., Vol. 8, pp. ‐ . Zhong, J. J., Fujiyama, K., Seki, T. and Yoshida, T. . A quantitative analysis of shear effects on cell suspension and cell cultures of Perilla frutescens in bioreactors. Biotechnol. Bioeng., Vol. , pp. ‐ . Zolman, B. K., Yoder, A. and Bartel, B. . Genetic analysis of indole‐ ‐butyric acid responses in Arabidopsis thaliana reveals four mutant classes. Genetics, Vol. , pp. – . Joint Scientific Symposium IJJSS 2016 Chiba, 20‐24 November 2016 329 Leaf Callus Induction of Moringa oleifera with Addition Various Concentration Napthalene Acetic Acid NAA and N 6 ‐ furfuryladenine Kinetin in MS Medium in vitro Muhammad Evan N a , Erlyta Vivi P b a,b Institut Teknologi Sepuluh Nopember, Kampus ITS Sukolilo, Surabaya 60111, Indonesia b Second affiliation, Address, City and Postcode, Country Abstract Moringa oleifera is the plants which have high nutritional content of minerals and vitamins. n ndonesia, the cultivation of M. oleifera still utilizing vegetative propagation. Therefore, the resulting seedlings are limited and have a low quality of seeds. To solve the problems, rapid M. oleifera propagation and sustainable high quality of seeds through in‐vitro culture are needed. This study aimed to determine the effect of somatic embryogenesis induction of M. oleifera leaves due to the adding variation of plant growth regulators concentration of Napthalene Acetic Acid NAA and Kinetin in MS medium in‐vitro. This study was conducted using a completely randomized design with four treatments, are M MS , M MS + NAA . mgL + Kinetin mgL , M MS + NAA mgL + Kinetin mgL , and M MS + NAA mgL + Kinetin . mgL and there were three replication of each treatments. Parameters observed that were the rate of induction time, color and texture of callus. Data were analyzed using analysis of descriptive. The results showed that the addition of plant growth regulators NAA and Kinetin at various concentration affected callus growth of M. oleifera leaves on MS medium in‐vitro. The medium concentration of M MS + NAA . mgL + Kinetin mgL was the optimal concentration on callus induction on fourteenth day , the callus has white color and compact texture. Keywords callus induction; kinetin; Moringa oleifera; Napthalene Acetic Acid NAA

1. Introduction

Moringa oleifera is one of the most popular plants belonging to the family of Moringaceae Saini et al., . M. oleifera has unique characteristics which attract such toughness tree, easily propagated, and resistant to drought with very low growth’s requirements regard with nutrients, water, and its management which these plants play an important role also in the utilization field of medicine and nutrition Magana, . M. oleifera is one of the promising commodity in the food sector for its rich nutrients and minerals as well as the ability of trees to produce leaves with maximum condition even Corresponding author. Tel.: + ‐8 ‐ 8 ‐ E ‐mail address: muhammadevan gmail.com 330 at the end of the dry season when other food sources are rarely to be found Fuglie, . Every part of the M. oleifera tree has a strong activity as pharmaceuticals. Such as their leaves, roots, seeds, bark, fruit, pods, and flowers act as a stimulant of the heart and blood circulation, antitumor pyretic, anti‐epileptic and anti‐inflammatory Kumar et al., , antiulcer, antipasmodic, diuretics, anti‐hypersensitive, cholesterol lowering, antioxidant, antidiabetic, hepato‐protective, antibateri and anti‐fungal, and some other treatments of different diseases Anwar et al., . n Africa, M. oleifera is used as a food enhancer prenatal nutrition in pregnant women Amin et al., , as well as a program of food sovereignty in Africa and ndia. M. oleifera as a way to combat malnutrition because it contains high vitamins, proteins and minerals. n addition, M. oleifera seeds can be used to purify water Diatta, The study further stated that the purification of water not only reduces solid contaminants, but rather the overall harmful bacteria contained in the water. n ndonesia, the cultivation of M. oleifera has not been done and only based on vegetative propagation grafting or cuttings and generative seeds so the limitations of seeds as planting materials often occur. Planting with an area of approximately , hectares to fulfill the demand requires billion seedlings Jahn, 8 . Limitations of the seedlings should be has an appropriate, economical, and efficient propagation solution. The most appropriate method to apply is utilize tissue culture techniques by taking part of the M. oleifera plant which is not limited to the seed then carried regeneration initiation of embryogenesis, starts induction of somatic embryos for reproduction purposes. Tissue culture itself is the cultivation of a plant tissue explants into complete plants with properties like its parent which explants will be wrapped with a special wrapper that makes explant is not easily damaged and can grow well Marfori, . The combination of Kinetin and NAA as plant growth regulator PGR plays an important role in in‐vitro culture to stimulate growth of certain cells or tissues that have not differentiated. So we need further study to determine the right combination PGR for maximum growth of explants especially M. oleifera leaf explants. This study aimed to describe the effect of somatic embryogenesis induction of Moringa leaves due to the addition of various concentrations of growth regulators Napthalene Acetic Acid NAA and N ‐furfuryladenine Kinetin on MS medium in‐vitro. 2. Materials and Methods 2.1. Sterilization of Explants The study was conducted in April at the Laboratory of Plant Bioscience and Engineering, Department of Biology, Faculty of Mathematics and Natural Science, nstitut Teknologi Sepuluh Nopember. Explants used are Moringa leaf. Sterilization of explants performed in several stages. The first stage is the tools that have been sterilized prepared in LAF. The explants were washed with running water for minutes, soaked in a solution of detergent for minutes, soaked in a solution of clorox for minutes, soaked in a solution of antifungal for minutes, soaked in alcohol for minutes, and then transferred into a glass beaker sterile inside LAF room and soaked in sterile distilled water for minutes.