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Efficient somatic embryogenesis from meristematic explants in grapevine (Vitis vinifera L.) cv. Chardonnay: an improved protocol

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Abstract

Key message

The use of elevated concentrations of hormones enables effective somatic embryogenesis from nodes of in vitro plants and do not impair embryo viability, in grapevine, a clonally propagated woody crop.

Abstract

Somatic embryogenesis (SE) has great importance for propagation and transformation of woody perennial plants. In Vitis vinifera L., the most cultivated grape species, somatic embryos are generally obtained from floral organs (stamens, ovaries, whole flowers) not easily available. Moreover, the efficiency of SE induction from reproductive organs greatly depends on the physiological state of explants which is hardly controlled. We report here on a culture system for the efficient and reproducible induction of SE and the production of good quality embryos, based on the use of nodal explants excised from in vitro grown plantlets easily obtained all year round. This method involves a first culture step on a callus inducing medium (CIM) containing unusually elevated concentrations of auxin and cytokinin. SE efficiencies from 10.3 to 20.2 % were reproducibly attained with a combination of 18 μM of 2,4-dichlorophenoxyacetic acid (2,4-D) and 9 μM of 6-benzylaminopurine (BAP) in CIM. Callus formation and embryogenesis induction were preferentially observed in the bud region. Moreover, we showed that internodal stem segments, petioles and leaf fragments were not amenable to SE under the same conditions, strongly suggesting that axillary meristems are involved in the embryogenic response. In addition, we verified that high concentrations of 2,4-D and BAP in CIM did not affect the viability of embryos further generated, nor hamper their regeneration into whole plants.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

ANOVA:

Analysis of variance

BAP:

6-Benzylaminopurine

CIM:

Callus inducing medium

IAA:

Indole-3-acetic acid

MS:

Murashige and Skoog

NOA:

2-Naphtoxyacetic acid

PGR:

Plant growth regulator

SE:

Somatic embryogenesis

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Authors and Affiliations

  1. E.A. 3991, Laboratoire Vigne, Biotechnologies et Environnement, Université de Haute Alsace, 33 rue de Herrlisheim, BP 50568, 68 000, Colmar Cedex 008, France

    Pascale Maillot, Laurence Deglène-Benbrahim & Bernard Walter

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  1. Pascale Maillot
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  2. Laurence Deglène-Benbrahim
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  3. Bernard Walter
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Correspondence to Pascale Maillot.

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Communicated by J. Lin.

Electronic supplementary material

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468_2016_1374_MOESM1_ESM.doc

Results of the analysis of variance (ANOVA) conducted to compare the effect of the callus induction media CIM1 (9 µM 2,4-D/4.5 µM BAP) and CIM2 (18 µM 2,4-D/9 µM BAP) on the efficiency of somatic embryogenesis (SE) induction (DOC 31 kb)

468_2016_1374_MOESM2_ESM.doc

Results of the analysis of variance (ANOVA) conducted to compare the effect of the callus induction media CIM2 (18 µM 2,4-D/9 µM BAP), CIM3 (27 µM 2,4-D/13.5 µM BAP) and CIM4 (36 µM 2,4-D/18 µM BAP) on the efficiency of somatic embryogenesis (SE) induction (DOC 31 kb)

468_2016_1374_MOESM3_ESM.doc

Influence of callus induction media (CIM) containing increasing concentrations of plant growth regulators (PGRs) on the efficiency of somatic embryogenesis (SE) induction at the individual level: CIM1 (9 µM 2,4-D/4.5 µM BAP), CIM2 (18 µM 2,4-D/9 µM BAP), CIM3 (27 µM 2,4-D/13.5 µM BAP) and CIM4 (36 µM 2,4-D/18 µM BAP). SE efficiency is defined as the percentage of explants having produced embryogenic structures. Exp. = Experiment. No. = Number. Eff. = Efficiency. * = mean of individual efficiencies in each experiment (DOC 107 kb)

468_2016_1374_MOESM4_ESM.doc

Results of the analysis of variance (ANOVA) conducted to compare the effect of the callus induction media CIM1 (9 µM 2,4-D/4.5 µM BAP), CIM2 (18 µM 2,4-D/9 µM BAP), CIM3 (27 µM 2,4-D/13.5 µM BAP) and CIM4 (36 µM 2,4-D/18 µM BAP) on the regeneration ability of generated embryos (DOC 31 kb)

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Maillot, P., Deglène-Benbrahim, L. & Walter, B. Efficient somatic embryogenesis from meristematic explants in grapevine (Vitis vinifera L.) cv. Chardonnay: an improved protocol. Trees 30, 1377–1387 (2016). https://doi.org/10.1007/s00468-016-1374-9

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