Genetic diversity analysis and development of in Vitro regeneration system in selected clones of tea (Camellia sinensis l.) plant

Abstract

The present study was carried out with an aim to analyze the genetic diversity of eighteen tea (Camellia sinensis L.) clones as well as to develop an in vitro regeneration system for selected tea clones of Bangladesh. Genetic diversity among the 18 tea clones was analyzed using Random Amplification of Polymorphic DNA (RAPD) markers. Twenty arbitrary oligonucleotide primers were used for this purpose. All the investigated primers exhibited significant amplification following PCR analysis. The primers amplified reproducible and scorable bands all across the investigated tea clones. A total of 755 bands were produced in all the 18 tea clones with an average of 37.75 RAPD bands per primer. Among all the bands 97.41% were polymorphic in nature. The molecular size of the amplified DNA fragments ranged from 250 to 5000 bp. The highest number of amplified products was recorded in case of the primer OPA-7 (95 bands). Whereas primer CO2, OPA-1 and OPA-2 produced 71, 58 and 52 bands respectively. The lowest number of bands was found in case of primer B06 (14 bands). Ten unique clone specific bands were obtained from the genome of the 18 tea clones. The values of pairwise genetic distance among the clones ranged from 24.0 to 59.0, indicating the presence of wide range of genetic diversity. The highest genetic distance (59.0) was found between the clone BT13 and BT1, whereas that of the lowest (24.0) was obtained between the clone BT18 and BT5. Dendrogram based on Nei’s genetic distance was constructed using Unweighted Pair Group of Arithmetic Mean (UPGMA) where the 18 tea clones were grouped into two major clusters: BT9 and BT13 in cluster 1 and the remainder of 16 clones in cluster 2. The cluster 2 is further sub-divided into many sub-clusters. Cluster analysis revealed that the clone BT5 is closely related to BT18. An in vitro regeneration system for the clones of tea plant was also developed through direct and indirect organogenesis using shoot tip, nodal segment and cotyledon explants. MS medium supplemented with 2.0 mg/l BAP and 0.1 mg/l IBA was found to be effective for the initiation and multiplication of shoots directly from the shoot tip and nodal segment explants. Elongation of such multiple shoots was effected by the addition of 0.5 mg/l GA3 in the shoot multiplication medium. For indirect organogenesis, the highest amount of callus was obtained using MS medium containing 4.0 mg/l 2, 4-D from de-embryonated mature and immature cotyledon explants. Moreover, morphological differentiation of these calli was achieved when these explants were cultured on the MS medium supplemented with 2.5 mg/l BAP and 0.1 mg/l Kn. The highest root induction was achieved when micro shoots were treated with 500 mg/l of IBA for 30 min before transferring them to the soil. After proper hardening rooted plantlets were established in the soil. Genetic variability was assessed among the micropropagated plants derived from the in vitro culture of BT2 clone using eleven RAPD primers. A total of 601 bands ranging from 300 bp to 2900 bp were produced within the examined 17 micropropagated and 1 field grown plants. Among the 11 primers, 7 produced 504 monomorphic bands for all the plants. However, remaining 97 bands were polymorphic in nature and maximum were amplified for three (S4, S14 and S15) micro-propagated plants. The dendrogram tree showed 18 plants were closely grouped into 2 main clusters where the mother plant BT2 (S18) had separately made a position with the cluster 1. The micropropagated plant S14 and S4 each separately had created their position under 2 nd main cluster whereas the remaining plants were arranged under sub cluster1. Moreover, a lower level of genetic distance (0-14) was revealed among the micropropagated plants from the genetic distance matrix.