In vitro organogenesis and plant regeneration of Passiflora xishuangbannaensis, a species with extremely small populations

Yuan-Yuan Meng,Shi-Jie Song,Sven Landrein

doi:10.1016/j.gecco.2021.e01836

Abstract

Passiflora xishuangbannaensis (Passifloraceae) is endemic to a few sites of Mengyang nature reserve in Yunnan, Xishuangbanna and less than 40 individuals have been recorded. Nine Passiflora species are endemic to Yunnan with most species occurring in South America, making P. xishuangbannaensis highly significant and emblematic to the conservation work in the region. This study is designed to provide the first protocol for in vitro organogenesis and plant regeneration for ex situ conservation and reintroduction for an Asian Passiflora species. Using internodes, petioles and tendrils we optimize calli formation and root elongation using several plant growth regulators, individually or in combination. We also assess the genetic stability of regenerated cells. The maximum callus induction and shoot bud differentiation were both achieved on half Murashige and Skoog basal medium supplemented with 4.44 μM 6-Benzylaminopurine and 1.08 μM 1-Naphthaleneacetic acid. The best rooting was achieved from 30 days old, regenerated shoots on half Murashige and Skoog basal medium supplemented with 1.08 μM 1-Naphthaleneacetic acid. Micropropagated plants were subjected to inter simple sequence repeat markers analyses. Collectively, 86 bands were generated from 6 primers of which 12 bands were polymorphic, showing genetic variation between the regenerated plantlets and the original plant. Response to plant growth regulators was more specific than most other studies using South American species, which could be explained by the morphological and physiological differences between South American and Asian Passiflora species. Because very few individuals remain, sexual reproduction is challenging, aerial shoots are annual (rhizomatous) and because genetic variability can be a consequence of repetitive in-vitro regeneration, we advise ex-situ conservation techniques based on limited regeneration events such as cryopreservation.

Highlights

Passiflora L. is the largest genus in Passifloraceae with more than 600 species (Espinoza et al 2018)
After 45 days of culture, the best results of calli induction are seen with Internodal segments (Ins), Pts and Tds on 1⁄2 MS medium supplemented with 4.44 μM BA and 1.08 μM Naphthaleneacetic Acid (NAA), resulting in a callus induction frequency of 91.67 ± 11.7%, 90.00 ± 14.05% and 88.33 ± 11.25%, respectively (Table 2)
When we did multiple comparisons analysis among treatments and explant types, we found no differences in callus induction among the three explant types or the two treatments (Fig. 2a)

Summary

Introduction

Passiflora L. is the largest genus in Passifloraceae with more than 600 species (Espinoza et al 2018). A total of 38 individuals were counted from 5 sites of which 2 sites did not have plants anymore This species is below the Minimum Viable Population (MVP) size, with less than 5000 individuals in total and less than 500 individuals per population and qualify for the Plant Species with Extremely Small Population PSESP (Sun et al 2019). This species was reported to be self-incompatible (Krosnick 2005), and rarely sets fruit in the wild, causing a decline in natural population size

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Discussion

Conclusion