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Species-dependent divergent responses to in vitro somatic embryo induction in Passiflora spp.

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Abstract

Passiflora is a large and widespread genus of tropical plants that includes over 500 species. Organogenesis-based in vitro plant regeneration systems have long been available for the commercially important species Passiflora edulis, the passionfruit, and for a few other related wild species. Recently, somatic embryogenesis from mature zygotic embryos was reported for passionfruit and for a related wild species, P. cincinnata, although the recovery of entire plants was obtained only for the latter. Here we assessed the in vitro morphogenic responses of zygotic embryos of five different Passiflora species (P. alata Curtis, P. crenata Feuillet & Cremers, P. edulis Sims, P. foetida L. and P. gibertii N.E. Brown) cultured in basal Murashige and Skoog (MS) medium supplemented with 4.5 μM 6-benzyladenine (BA) and different concentrations (13.6, 18.1, 22.6 or 27.1 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). We characterized these different responses using light and scanning electron microscopy. Somatic embryos were obtained in MS medium supplemented with 4.5 μM BA and either 13.6 or 18.1 μM 2,4-D for all species, except P. foetida for which only indirect shoot organogenesis was observed. Regeneration of entire plants that could be acclimatized was achieved for all species studied. Additionally, our results indicated that the in vitro conditions that promote somatic embryogenesis in some Passiflora species might induce shoot organogenesis in others, suggesting that the conservation of morphogenetic signals among Passiflora species might be limited by their phylogenetic relatedness.

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Acknowledgments

We acknowledge Prof. E.W. Kitajima and Prof. F. Tanaka for maintaining the electron microscope facility at NAP/MEPA-ESALQ/USP, Piracicaba, Brazil. We also acknowledge funding from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).

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Authors and Affiliations

  1. Faculdade de Ciências Agrárias. Rod. Dourados-Itaum, Universidade Federal da Grande Dourados, Km 12, Cx Postal 533, Dourados, MS, CEP 79804-970, Brazil

    Yara Brito Chaim Jardim Rosa

  2. Departamento de Biologia Vegetal Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), R. Monteiro Lobato, 255, Cx Postal 6109, Campinas, SP, CEP 13418-970, Brazil

    Carolina Cassano Monte Bello & Marcelo Carnier Dornelas

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  1. Yara Brito Chaim Jardim Rosa
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  2. Carolina Cassano Monte Bello
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  3. Marcelo Carnier Dornelas
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Correspondence to Marcelo Carnier Dornelas.

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11240_2014_580_MOESM1_ESM.jpg

Morphogenic responses of mature zygotic embryos of P. alata cultured in vitro. b-g, i and j: Scanning electron microscopy images. a P. alata mature seed from which the outer integument was removed. b Dissected P. alata mature zygotic embryo used as an in vitro culture explant. c and d Zygotic embryo-derived callus after 15 days in culture in M1 (c) or M3 (d) medium. e and f Zygotic embryo-derived callus after 21 days in culture in M1 (c) or M2 (d) medium. Arrows indicate early globular embryos. g and h Zygotic embryo-derived callus after 35 days in culture in M1 (g) or M2 (h) medium. Arrows indicate late globular embryos starting to change shape. i and j Zygotic embryo-derived callus after 35 days in culture in M3 (i) or M4 (j) medium. Only proliferation of a friable callus could be observed. Bars: a and b: 0.8 mm; c, d, f, i and j: 230 μm; e, g and h: 130 μm (JPEG 1316 kb)

11240_2014_580_MOESM2_ESM.jpg

Morphogenic responses of mature zygotic embryos of P. crenata cultured in vitro. b-j: Scanning electron microscopy images. a P. crenata mature seed from which the outer integument was removed. b Dissected P. crenata mature zygotic embryo used as an in vitro culture explant. c-f Zygotic embryo-derived callus after 15 days in culture in M1 (c), M2 (d), M3 (e) or M4 (f) medium. Arrows in (c) point to proliferation of subepidermal cells that ruptured through the epidermis of the explants; Arrows in (d) point to leaf primordia-like structures. g-i Zygotic embryo-derived callus after 21 days in culture in M1 (g), M2 (h) or M4 (i) medium. Arrows indicate early globular embryos. j Zygotic embryo-derived callus after 35 days in culture in M4 medium. Only proliferation of a friable callus could be observed. Bars: a and b: 0.8 mm; c, d and f-j 130 μm; e: 230 μm (JPEG 1872 kb)

11240_2014_580_MOESM3_ESM.jpg

Morphogenic responses of mature zygotic embryos of P. edulis cultured in vitro. b-j: Scanning electron microscopy images. a P. edulis mature seed from which the outer integument was removed. b Dissected P. edulis mature zygotic embryo used as an in vitro culture explant. c-d Zygotic embryo-derived callus after 15 days in culture in M1 (c) or M4 (d) medium. e–g Zygotic embryo-derived callus after 21 days in culture in M1 (e), M2 (f) or M4 (g) medium. h-j Zygotic embryo-derived callus after 35 days in culture in M1 (h), M2 (i) or M3 (j) medium. Arrows indicate embryo-like structures. Bars: a-d: 0.8 mm; e–g, i and j 130 μm; h: 230 μm (JPEG 1257 kb)

11240_2014_580_MOESM4_ESM.jpg

Morphogenic responses of mature zygotic embryos of P. foetida cultured in vitro. b-j: Scanning electron microscopy images. a P. foetida mature seed from which the outer integument was removed. b Dissected P. foetida mature zygotic embryo used as an in vitro culture explant. c and d Zygotic embryo-derived callus after 21 days in culture in M1 (c) or M2 (d) medium. Arrows in (c) point to meristemoid-like structures; Arrows in (d) point to leaf primordia and shoot meristem-like structures. e and f Zygotic embryo-derived callus after 35 days in culture in M1 (e) or M2 (f) medium. Arrows point to leaf primordia. g and h Zygotic embryo-derived callus after 21 days in culture in M3 (g) or M4 (h) medium. Arrows in (g) point to meristemoid-like structures. i and j Zygotic embryo-derived callus after 35 days in culture in M3 (i) or M4 (j) medium. Arrow in (i) points to meristemoid-like structure. j Only proliferation of a compact callus could be observed in M4 medium. Bars: a and b: 0.8 mm; c, d, f, g and i: 130 μm; e, h and j: 230 μm (JPEG 1417 kb)

11240_2014_580_MOESM5_ESM.jpg

Morphogenic responses of mature zygotic embryos of P. gibertii cultured in vitro. b-j: Scanning electron microscopy images. a P. gibertii mature seed from which the outer integument was removed. b Dissected P. gibertii mature zygotic embryo used as an in vitro culture explant. c and d Zygotic embryo-derived callus after 21 days in culture in M1 (c) or M2 (d) medium. Arrows in (d) point to early embryo-like structures. e–g Zygotic embryo-derived callus after 35 days in culture in M1 (e) or M2 (f) medium. Arrows point to embryo-like structures. g Zygotic embryo-derived callus after 15 days in culture in M3 medium. h and i Zygotic embryo-derived callus after 21 days in culture in M3 (i) or M4 (j) medium. Arrow in (h) points to embryo-like structure. j Zygotic embryo-derived callus after 35 days in culture in M4 medium. Only proliferation of callus could be observed. Bars: a and b: 0.8 mm; c, d and h: 130 μm; e–g, i and j: 230 μm (JPEG 2237 kb)

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Rosa, Y.B.C.J., Bello, C.C.M. & Dornelas, M.C. Species-dependent divergent responses to in vitro somatic embryo induction in Passiflora spp.. Plant Cell Tiss Organ Cult 120, 69–77 (2015). https://doi.org/10.1007/s11240-014-0580-7

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