ABSTRACT
Genetic fidelity assessment of micropropagated pepper (Piper nigrum L.) plantlets using ISSR markers

Pei Ee Kho1, 2*, Nur Ardiyana Rejab1, 3, Norzulaani Khalid4, Sim Soon Liang5, Zehnder Jaroop Augustine Mercer2 , Siaw San Hwang6, and Acga Cheng1*
 
Pepper (Piper nigrum L.) is a high-value spice crop, but in vitro propagation can induce somaclonal variation, necessitating genetic fidelity assessment. This study evaluated the genetic fidelity of micropropagated plantlets of a major Malaysian pepper cultivar Semongok Aman, generated via somatic embryogenesis using inter-simple sequence repeat (ISSR) markers. Tissue around the micropylar region in seeds, which were surface-sterilized and cultured under standardized conditions, was used to induce somatic embryogenesis. Twelve regenerated plantlets representing primary, secondary, and tertiary embryogenic stages, along with the field-grown mother plant, were analyzed. Genomic DNA was extracted and initially screened with 30 ISSR primers, of which six produced clear and reproducible amplification patterns and were selected for further analysis. These primers generated 64 scorable bands, comprising 54 monomorphic (84.4%) and 10 polymorphic (15.6%) bands. Genetic similarity between regenerated plantlets and the mother plant, calculated using the Jaccard coefficient, ranged from 0.902 to 1.000. Unweighted pair group technique with arithmetic average (UPGMA) cluster analysis grouped all regenerated plantlets with the mother plant at a 91% similarity threshold, while three clusters were resolved at a 95% threshold. Plantlets derived from primary and secondary somatic embryogenesis exhibited high genetic uniformity, whereas those originating from tertiary somatic embryogenesis displayed minor genetic variation. Overall, the findings demonstrate that somatic embryogenesis, particularly at primary and secondary stages, reliably produces true-to-type clones of ‘Semongok Aman’. The optimized ISSR primers offer a robust and cost-effective tool for routine genetic fidelity assessment, supporting large-scale clonal propagation for sustainable pepper production in Malaysia and possibly in other pepper producing countries.
Key words: Genetic fidelity, ISSR markers, micropropagation, Piper nigrum, somatic embryogenesis.
1Universiti Malaya, Faculty of Science, Institute of Biological Sciences, 50603 Kuala Lumpur, Malaysia.
2Malaysian Pepper Board, Department of Research, 93450 Kuching, Sarawak, Malaysia.
3Universiti Malaya, Centre for Research in Biotechnology for Agriculture, 50603 Kuala Lumpur, Malaysia.
4International University of Malaya-Wales, Faculty of Art and Science, 50480 Kuala Lumpur, Malaysia.
5Academy of Sciences Malaysia, Kuala Lumpur 50480, Malaysia.
6Swinburne University of Technology, Faculty of Engineering Computing and Science, Kuching, Sarawak 93350, Malaysia.
*Corresponding authors (khope@mpb.gov.my; acgacheng@um.edu.my)
Received: 26 February 2026; Accepted: 5 June 2026, Available online: 3 August 2026.