Development and Applications of Somatic Embryogenesis in Grapevine (Vitis spp.)

Abstract

Somatic embryogenesis (SE) provides alternative methodologies for the propagation of grapevine (Vitis spp.) cultivars, conservation of their germplasm resources, and crop improvement. In this review, the current state of knowledge regarding grapevine SE as applied to these technologies is presented, with a focus on the benefits, challenges, and limitations of this method. The paper provides a comprehensive overview of the different steps involved in the grapevine SE process, including callus induction, maintenance of embryogenic cultures, and the production of plantlets. Additionally, the review explores the development of high-health plant material through SE; the molecular and biochemical mechanisms underlying SE, including the regulation of gene expression, hormone signaling pathways, and metabolic pathways; as well as its use in crop improvement programs. The review concludes by highlighting the future directions for grapevine SE research, including the development of new and improved protocols, the integration of SE with other plant tissue culture techniques, and the application of SE for the production of elite grapevine cultivars, for the conservation of endangered grapevine species as well as for cultivars with unique traits that are valuable for breeding programs.

Keywords: chimera; conservation; germplasm; in vitro culture; mutagenesis; propagation; somaclonal variation; tissue culture; transformation.

Figure 1

Somatic embryogenesis and plant regeneration from immature flower tissues in Vitis vinifera. (A) Immature flowers collected a few days before opening and stored at 4 °C. (B) Different floral tissues utilized to induce embryogenic cell lines (anthers, pistils, stigmas/styles, ovaries, and whole flowers). (C) Callus generated from a pistil (arrow) after 3 months of culture on embryogenic medium. (D) Somatic embryos regenerated after 4–6 months of culture initiation at the surface of explant-derived callus. (E) Different developmental stages of somatic embryos regenerated in vitro. The response to somatic embryo induction can also vary based on the organ/tissue types. Anthers, ovaries, leaves, petioles, tendrils, and nodal sections are the explants that are most frequently used for SE induction in grapevine [36]. A remarkable number of genotypes have been regenerated through anther culture [130] with a high success rate. Nevertheless, some authors report that, depending on the stage of growth of reproductive organs, the response can change. For instance, according to Vidal et al. [99], the regeneration from the ovaries was around two times greater than that from the anthers when ovaries were cultured in later stages of development. According to a recent study by San Pedro et al. [103], mature seeds can also be used as explants for SE induction by holding cut seeds for five months in media supplemented with TDZ. However, zygotic seed-derived somatic embryos are not useful for clonal propagation. Embryogenic callus induction has also been achieved using nodal segments, leaf discs [131], petioles, stem nodal explants [115,132], and whole flowers [66], even though they are less commonly used.

Figure 2

Development of somatic embryos and plantlets from cell suspension cultures of grapevine. (A) Proembryogenic masses (200–400 mg) are used for culture initiation. (B) Liquid cultures are maintained in 250 mL Erlenmeyer flasks containing 50 mL of liquid culture medium. (C) Images of cells growing in liquid culture and somatic embryos (globule and heart-shaped stages) differentiated after 40 days of initiation of culture. (D) Somatic embryos, collected by filtration after 2–3 months from the start of culture using a nylon mesh filter (2 mm), are incubated on growth regulator-free liquid medium. (E) Germination of the embryos occurs after approximately 30 days of culture on growth regulator-free solid medium. (F) The plantlets are acclimated in Jiffy pots and reach about 15 cm in height in about 40–60 days.

Figure 3

A scheme for using embryogenic cultures for mutation induction and screening. (a) Embryogenic culture establishment. (b) Optimizing mutagen dose through growth reduction studies [58]. (c) Development of somatic embryos after treatment with optimized mutagen dose. (d) Initial germination. (e) Screening the germinated embryos for the trait of interest in vitro. (f) and (g) Testing mutagenized populations in greenhouse and field conditions, respectively, for traits difficult to screen in vitro, such as bunch architecture, vine growth, fruit quality in table grapes, etc. Bars: (a,c,d) = 1 mm; (e) = 5 mm.

Figure 4

A scheme for transposon activation, tagging mutants and their recovery using embryogenic cultures of grapevine. (a) Cell culture established and subjected to stress for transposon activation. (b) Cells subjected to stress. (c) Somatic embryos (SEs) generated from stressed embryogenic cells. (d) Secondary SEs induced from primary SEs, with the clusters serially numbered. (e) Part of the labelled secondary SEs cryopreserved. (f) Another part of SEs subjected to RNAseq for transposon tagging. (g) Identified mutants of interest recovered from cryopreservation and regenerated. Bars: (b) = 10 µm; (c,d,g) = 1 mm.