Micro rhizome formation of temulawak (Curcuma xanthorrhiza Roxb.) and genetic stability analysis to repeated sub-cultured plantlets

Abstract

Temulawak (Curcuma xanthorrhiza Roxb.) was used to be propagated from rhizome. High propagul weight will produce high yield. The use of big sized rhizome for seedling will reduce its use for consumption and processing purposes. Therefore, in vitro micro rhizome could be used as propagul alternative for the propagation of temulawak because of its simplicity and high multiplication in shorter time. Plantlet from repeated sub-cultures induced to form micro rhizome. High rate shoot multiplication caused somaclonal variation. In plants propagation, somaclonal variation was not desired because seed uniformity was very important. Therefore, genetic stability of plantlets for get true to type seed from the use of repeated sub-culture in vitro shoot for micro rhizome formation must be studied. This research used RFLP and sequencing method to find the genetic stability of plantlet among sub-cultures generations. RFLP method was used to mother plant’s DNA and plantlet’s DNA from 3rd, 4th, 5th and 12th sub-cultures. Sequencing analysis was used to mother plant’s and 12th sub-cultures plantlet DNA. In vitro micro rhizome formation research consisted of three studies : 1) The effect of physical of media (liquid and solid medium) and sucrose (30, 60, 90, 120 gL-1); 2) The effect of the strength of MS medium (half and full strength) and sucrose (30, 60, 90, 120 gL-1) ; 3) The effect of level of BAP (0, 1, 2, 3 mgL-1) and sucrose (30, 60, 90, 120 gL-1) to plantlet growth and micro rhizome formation. Cultures were incubated in the dark for 16 hours day-1. Electrophoresis band of DNA temulawak in RFLP analysis with restriction enzymes MBo1, Taq1 and Alu1 showed the same pattern between 5 samples DNA. There is possibility of genetic stability of temulawak plantlet between generation 3rd, 4th, 5th, and 12th sub-cultures and mother’s plants DNA. Homology result of sequencing DNA also showed genetic stability for 300 bp DNA. There is genetic sequence equality between DNA mother plants sequence at base number 61-361 and DNA plantlet 12th sub-culture at base sequence number 61- 361. Micro rhizomes of temulawak was formed in liquid half and full strength MS. Interaction BAP and sucrose have significant effect on rhizomes formation at 18 weeks after planting. The higher frequency of rhizomes formation was enhanced in medium with BAP 1 and 2 mgL-1 on sucrose concentration 60, 90, 120 gL-1. The increased of sucrose concentration also had positive effect to higher number, diameter and weight of micro rhizomes 8 weeks after acclimatization.