RESEARCH ARTICLE A Liquid Culture System for Improved Micropropagation of Mature Acacia nilotica (L.) Del. ssp. indica and Ex Vitro Rooting Jitendra Singh Rathore • Manoj K. Rai • Mahendra Phulwaria • N. S. Shekhawat Received: 21 March 2013 / Revised: 9 May 2013 / Accepted: 12 June 2013 Ó The National Academy of Sciences, India 2013 Abstract A micropropagation method using liquid cul- ture medium has been developed for mature Acacia nilot- ica (L.) Del. ssp. indica. Nodal segments obtained from 15 to 20 years old mature trees were used as explants and cultured on 0.8 % agar-gelled Murashige and Skoog medium containing 6-benzylaminopurine for shoot bud induction. Once culture got established, explants were transferred to Murashige and Skoog liquid medium con- taining 6-benzylaminopurine or kinetin for shoot multipli- cation. Shoot multiplication was influenced by plant growth regulators, size of vessels, amount of medium in culture vessels and repeated transfer of mother explants. Murashige and Skoog liquid medium containing 4.4 lM 6-benzylaminopurine was found to be the best for shoot multiplication. The performance of liquid and agar-gelled medium for shoot multiplication was compared. About ten times increase in shoot number in liquid culture medium was achieved. Micropropagated shoots were rooted ex vitro. Shoots treated with 2.46 mM indole-3-butyric acid solution for 1 h followed by 1.41 mM chlorogenic acid for 5 min exhibited highest percent of rooting in the green house. This process of micropropagation of A. nilotica, can be utilized for plant production on a large-scale. Keywords Acacia nilotica Á Acclimatization Á Liquid culture medium Á Plant growth regulators Á Shoot multiplication Introduction Plant tissue culture is employed for propagation at com- mercial level for many plant species. However, use of gelling agent i.e. agar or phytagel, tissue culture grade sucrose, nutrient salts, and plant growth regulators make it more expensive than conventional vegetative propagation like stem cutting [1]. The cost of tissue culture-raised plants can be reduced by amending some of the steps of the production process i.e. culture in liquid medium, use of commercial grade sucrose and ex vitro rooting of in vitro regenerated shoots directly in the green house on soil/ substrate [1–3]. Liquid culture offers many potential advantages over solid cultures like faster growth rates, rapid uptake of nutrients by tissues, dilution of exuded growth inhibitors i.e. phenolics released by explants thus minimizing negative effect on growth [1, 4, 5]. In comparison to liquid cultures, solid medium has many drawbacks, for instance slow rate of bud proliferation and multiplication, non-uniform dispersal of nutrients and growth regulators in medium, sensitivity of the explants to different brand of agar, high costs of manual handling [6]. In vitro propagation using liquid culture has been reported in many plant species [1, 4, 6–11]. Ex vitro rooting of microshoots reduces production cost of micro- propagated plants as the microshoots root in the green house and do not need any additional acclimatization prior to transplanting in the field conditions, and thus require less labor, chemicals and equipments [3, 12]. In many woody plants which are not easy to root and acclimatize, J. S. Rathore Á M. K. Rai Á M. Phulwaria Á N. S. Shekhawat Department of Botany, Biotechnology Centre, Jai Narain Vyas University, Jodhpur 342033, Rajasthan, India J. S. Rathore (&) Department of Botany and Biotechnology, L.M. College of Science and Technology (Autonomous), Jodhpur 342001, Rajasthan, India e-mail: jitendrarathorelmc@gmail.com 123 Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-013-0204-8