BIODIVERSITAS ISSN: 1412-033X Volume 24, Number 7, July 2023 E-ISSN: 2085-4722 Pages: 4168-4174 DOI: 10.13057/biodiv/d240756 Shoot regeneration in Nepenthes mirabilis as affected by flurprimidol and GA3 application MURNI DWIATI ♥ , PUDJI WIDODO, AGUS HERY SUSANTO Faculty of Biology, Universitas Jenderal Soedirman. Jl. Dr. Suparno No. 63, Purwokerto Utara, Banyumas 53122, Central Java, Indonesia. Tel.: +62-281-638794, Fax.: +62-281-631700,  email: murnidwi14@gmail.com Manuscript received: 26 November 2022. Revision accepted: 28 June 2023. Abstract. Dwiati M, Widodo P, Susanto AH. 2023. Shoot regeneration in Nepenthes mirabilis as affected by flurprimidol and GA3 application. Biodiversitas 24: 4168-4174. Nepenthes mirabilis (Lour.) Druce. is a pitcher plant species at a higher risk of extinction mainly due to overexploitation. Previous investigations have shown that in vitro micropropagation can be used as an approach for ex- situ conservation of N. mirabilis. Therefore, this study aimed to assess the new shoot formation of N. mirabilis under in vitro conditions stimulated with the application of flurprimidol and GA3. The three concentrations of flurprimidol and GA3 used as treatments were arranged factorially in a Randomized Complete Block Design (RCBD), with internodes 2, 3, 4, and 5 serving as blocks. Based on the results, F1G0 (flurprimidol of 1 mgL -1 without GA3) had the highest shoot formation, which was not accompanied by sufficiently high contents of chlorophyll, leading to improper shoot development. Despite the smaller number of newly formed shoots in F2G2 (flurprimidol of 2 mgL -1 and GA3 of 2 mgL -1 ), significantly higher chlorophyll contents were observed, enabling better development of shoot. Leaf length and shoot diameter also showed similar results under F2G2, indicating that higher concentrations of exogenous GA3 were necessary for better shoot development. This indicated that appropriate concentrations of both plant growth regulators should be applied for micropropagation of N. mirabilis in support of ex-situ conservation. Keywords: Flurprimidol, GA3, micropropagation, Nepenthes mirabilis INTRODUCTION Nepenthes, also known as pitcher plant, is uniquely carnivorous due to the presence of a sac-like structure at the leaf tip, which is used for trapping insects. This genus consists of 114 species distributed worldwide from East Madagascar to New Caledonia, with over 60 endemic species found in Indonesia (Setiawan et al. 2018). Several species of this plant have been intensively traded and subjected to booming demand from 2007 to 2011, leading to overexploitation in their natural habitats occurred and causing a high risk of extinction. Nepenthes is now categorized as a protected plant based on some regulations such as Indonesian Law Number 5 Year 1990 on Conservation of Biological Resources and Their Ecosystems, Indonesian Government Regulation Number 7 Year 1999 on Preservation of Plant and Animal Species, and Indonesian Government Regulation Number 8 Year 1999 on Use of Wild Plants and Animals. All Nepenthes species are classified as rare according to the International Union for Conservation of Nature (IUCN) and World Conservation Monitoring Centre (WCMC). They are also listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES) (Hernawati et al. 2022). One of Nepenthes species, namely N. mirabilis (Lour.) Druce., which originates from East Kalimantan, is currently at risk of extinction (Handayani and Hadiah 2019). Therefore, to aid the preservation of this species, ex-situ conservation through in vitro propagation outside its natural habitat is required to obtain a large-scale generation of vegetatively propagated plantlets. The propagation of Nepenthes species by using stem cutting without in vitro culture is relatively difficult to carry out. For example, only two shoots resulted from stem cutting in the propagation of N. gymnamphora without in vitro culture (Maulidah 2022). Wahdani (2022) also obtained only two shoots when using stem cutting in the N. adrianii propagation. Therefore, a more efficient procedure using a micro-cutting approach as explant materials in in vitro cultures is needed to obtain a high number of propagated plants. This should be then exposed to a condition where shoot formation is stimulated by increasing synthesis of endogenous cytokinin (Vylíčilová et al. 2020). Flurprimidol is a retardant plant growth regulator that can increase endogenous cytokinin synthesis, making it applicable to stimulate shoot formation in several plant species. It is the most recent plant growth regulator introduced to ornamental plant growers in commercial nurseries in the US. Flurprimidol belongs to the pyrimidine chemical class that has a nitrogen-containing heterocycle compound. This growth regulator inhibits enzymes involved in the gibberellin biosynthetic pathway, which catalyze the oxidation of ent-kaurene to ent-kaurenoic acid, a gibberellin precursor (Neware 2019). According to previous investigations, the application of 200 mgL -1 flurprimidol induced the formation of new shoots from rhizomes or creeping root segments in Solidago altissima and Cayratia japonica. However, the newly formed shoot