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<p><i>Bulbophyllum griffithii</i> is a rare and threatened medicinal orchid that requires conservation. However, in situ approaches alone may not be sufficient to preserve germplasm and species continuation. Therefore, effective ex situ conservation measures are required to protect orchid resources. The present study is aimed at devising an efficient protocol for in vitro mass multiplication of <i>B. griffithii</i> through asymbiotic seed germination. The methodologies include nutrient media screening, growth parameter comparisons, genetic fidelity analysis and acclimatization. The use of Mitra medium resulted in the highest seed germination percentage (73.51 ± 0.81%) and protocorm development of stage III (enlarged protocorm with a leaf: 0.19 ± 0.09%). Chitosan supplementation at 7&#xa0;mg/L significantly improved the germination percentage (94.21 ± 1.18%) and protocorm development (stage III: 4.91 ± 1.27%). Further, chitosan and activated charcoal (0.2%) in the medium led to the best plantlet development, viz., number of leaves (5.40 ± 0.08), leaf length (1.24 ± 0.06&#xa0;cm), number of roots (4.80 ± 0.06), root length (1.92 ± 0.14&#xa0;cm) and multiple shoot numbers (2.40 ± 0.09). However, incorporation of thidiazuron (4 µM) in Mitra yielded the highest number of initiation of protocorm-like-bodies (PLBs) (5.00 ± 0.09). Genetic fidelity analysis of the in vitro – raised plantlets using start codon targeted (SCoT) and inter simple sequence repeats (ISSR) markers indicated 8.44% polymorphism and 91.56% monomorphism among PLB-derived in vitro plantlets. Maximum acclimatization survivability of plantlets (70.15%) was recorded in a compost mixture containing charcoal pieces, coconut husks, brick chips, and pine bark pieces in a ratio of 1:1:1:1. The hardened plants were transplanted to natural wild habitats. This study is significant, as it adds to the limited number of protocols available for micropropagation of <i>Bulbophyllum</i> spp. It may also aid in future research in metabolomics, proteomics and genomics of the genus.</p>

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In vitro regeneration and acclimatization strategies for germplasm conservation of Bulbophyllum griffithii (Lindl.) Rchb.f, a rare and threatened medicinal orchid

  • 期刊论文

  • 发表日期：

    2025年11月20日

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  • Mihin Targu,
  • Suman Kumaria

摘要

Bulbophyllum griffithii is a rare and threatened medicinal orchid that requires conservation. However, in situ approaches alone may not be sufficient to preserve germplasm and species continuation. Therefore, effective ex situ conservation measures are required to protect orchid resources. The present study is aimed at devising an efficient protocol for in vitro mass multiplication of B. griffithii through asymbiotic seed germination. The methodologies include nutrient media screening, growth parameter comparisons, genetic fidelity analysis and acclimatization. The use of Mitra medium resulted in the highest seed germination percentage (73.51 ± 0.81%) and protocorm development of stage III (enlarged protocorm with a leaf: 0.19 ± 0.09%). Chitosan supplementation at 7 mg/L significantly improved the germination percentage (94.21 ± 1.18%) and protocorm development (stage III: 4.91 ± 1.27%). Further, chitosan and activated charcoal (0.2%) in the medium led to the best plantlet development, viz., number of leaves (5.40 ± 0.08), leaf length (1.24 ± 0.06 cm), number of roots (4.80 ± 0.06), root length (1.92 ± 0.14 cm) and multiple shoot numbers (2.40 ± 0.09). However, incorporation of thidiazuron (4 µM) in Mitra yielded the highest number of initiation of protocorm-like-bodies (PLBs) (5.00 ± 0.09). Genetic fidelity analysis of the in vitro – raised plantlets using start codon targeted (SCoT) and inter simple sequence repeats (ISSR) markers indicated 8.44% polymorphism and 91.56% monomorphism among PLB-derived in vitro plantlets. Maximum acclimatization survivability of plantlets (70.15%) was recorded in a compost mixture containing charcoal pieces, coconut husks, brick chips, and pine bark pieces in a ratio of 1:1:1:1. The hardened plants were transplanted to natural wild habitats. This study is significant, as it adds to the limited number of protocols available for micropropagation of Bulbophyllum spp. It may also aid in future research in metabolomics, proteomics and genomics of the genus.