Abstract
In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. The Nepenthes genus, comprising nearly 160 species, presents a remarkable pitcher-shaped trap, leading to great interest among biologists, but the species of this genus are listed as threatened. In this work, we developed a protocol for reproducing Nepenthes mirabilis through shoot regeneration from calli. The cultivation of stem segments of N. mirabilis on MS medium containing thidiazuron induced organogenic calli after 10 weeks. Subcultured calli exposed to 6-benzylaminopurine showed shoot regeneration in 3 weeks with considerable yields (143 shoots/g of calli). Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate. Based on this method, we also developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. Twelve weeks post infection, regenerated shoots were observed at the surface of calli. Their transgenic status was confirmed by PCR and RT-PCR. In conclusion, this study provides an efficient method for regenerating Nepenthes and the first protocol for its stable genetic transformation, a new tool for studying carnivory.
Highlights
In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps
We present in this work an easy-to-follow method for the plant regeneration and preliminary transformation efforts of Nepenthes mirabilis using Agrobacterium tumefaciens
We developed a method for regenerating and genetically transforming Nepenthes mirabilis under in vitro conditions
Summary
In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate Based on this method, we developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. 160 Nepenthes species are widespread in southwestern Asia, with the greatest diversity in Borneo and Sumatra[1,2,3] These carnivorous plants have developed trap-shaped leaves that attract, capture, retain, and allow the digestion of prey such as arthropods. We present in this work an easy-to-follow method for the plant regeneration and preliminary transformation efforts of Nepenthes mirabilis using Agrobacterium tumefaciens
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