Callus Induction and Somatic Embryogenesis in Muscadine and Seedless Bunch Grapes (vitis) from Immature Ovule Culture

Pre-embryogenic calli were obtained from the culture of fertilized immature ovules of muscadine ( Vitis rotundifolia Michx.) and seedless bunch grapes ( Vitis vinifera L.) on: 1) Murashige & Turcker (MT, 1969) basal medium supplemented with 4.4 μM 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 1 μM BA (6-benzyladenine) with 6.0% (w/v) sucrose; 2) Chee and Pool (CP, 1978) basal medium supplemented with 5.0 μM NAA ( α -naphthaleneacetic acid) and 1.0 μM BA with 3.0% sucrose; and 3) Nitch and Nitch (NN, 1969) medium supplemented with 9.0 μM 2, 4-D and 4.0 μM BA with 3.0% sucrose, respectively. Somatic embryogenesis, with a success rate of 1.1 to 2.2%, was achieved in seedless bunch grape cultivars ‘Autumn Royal Seedless’, ‘Crimson Seedless’, and with muscadine grape cultivars ‘Alachua’, ‘Summit’, and ‘Tara’ when the callus was subcultured on those NN media with or without plant growth regulators (PGR). The frequency of somatic embryogenesis was genotype dependent and greater in callus derived from white crystal callus. Embryogenic calli maintained their regeneration capacity for 24 months. Somatic embryos proliferated rapidly and germinated and developed into normal plantlets after transfer to Lloyd and McCown Woody Plant (WP) medium supplemented with 1 μM BA and 1.5% (w/v) sucrose. Somatic embryogenesis is an efficient tool for rapid propagation, genetic transformation, somatic hybridization, and somaclonal variation (Gray and Purohit, 1991; Kuksova et al., 1997; Martinelli and Gribaudo, 2001). It provides cytological and genetic stability of regenerated plants (Bennici et al., 2004). Although somatic embryogenesis may occur spontaneously in some plant species, in grape, it is usually induced in tissue culture medium potentially from almost any part of the plant body (Kantharajah and Golegaonkar, 2003). Successful somatic embryogenesis and subsequent maintenance as well as plant regeneration has been described in several species including Vitis vinifera , V. longii , and V. rupestris (Gray and Meredith, 1992; Reisch and Pratt, 1996) and V. rotundifolia (Robacker, 1993). More recently, other cultivars have been shown to be competent for somatic embryo production. It was first reported in V. vinifera L. cv ‘Cabernet Sauvignon’ by using nucellar tissue (Mullins and Srinivasan, 1976) and thereafter with a variety of grape explants such as leaf segments (Robacker, 1993), zygotic embryos (Emershad and Ramming, 1994), shoot tips (Barlass and Skene, 1978), tendrils (Salunkhe et al., 1999), ovules (Srinivasan and Mullins, 1980), anthers (Salunkhe et al., 1999; Stamp and Meredith, 1988), and ovaries (Gray and Mortensen, 1987). The power of somatic embryogenesis as a propagation tool becomes especially apparent when the goal is to generate large numbers of propagules of a plant species that is either rare or has some unusual feature (Barlass and Skene, 1978). Embryogenic cultures are increasingly finding application as a source of material amenable to production of genetically transformed plants (Kirkkert et al., 2001). A vigorous and efficient embryogenic system, besides providing a framework for genetic manipulation, could also facilitate development of compact storage, packaging and distribution methods for elite varieties through the formation of synthetic seeds (Gray and Mortensen, 1987). Successful application of tissue culture technique in breeding programs, transformation experiments or other investigations presupposes the development of a reproducible in vitro plant regeneration system with appropriate culture conditions (Coutos-Thevenot et al., 1992). Although somatic embryogenesis and plant regeneration have been reported from a variety of explants of bunch grapes, somatic embryogenesis in V. rotundifolia has only been achieved from leaf and petioles (Robacker, 1993; Xu et al., 1995). In the present study, a reproducible regeneration protocol for efficient plant recovery via somatic embryogenesis in V. rotundifolia on MT media will be described which is characterized by well-defined stages of embryo development, maturation, germination and plant regeneration. Material and Methods Plant materials and preparation of ovules . Seedless berries of V. vinifera grapes ‘Autumn Royal Seedless’, ‘Crimson Seedless’, ‘Fantasy Seedless’, ‘Flame Seedless’, ‘Princess Seedless’, and ‘Thompson Seedless’ were harvested about 6 weeks after anthesis. Immature berries of muscadine grape cultivars ‘Alachua’, ‘Cowart’, ‘Summit’, and ‘Tara’ were harvested from the experimental vineyard at the Center for Viticulture and Small Fruit Research, Florida Agriculture and Mechanical University. Berries were surface sterilized by immersion in 70% alcohol for 1 min, then transferred to 20% (v/v) sodium hypochloride with a few drops of Tween 20 for 15 min, and washed three times with sterilized distilled water for 15 min. After sterilization, ovules were extracted from berries and scratched slightly on the surface under sterile conditions then placed onto three different media in 15 × 100 mm Petri dishes. Twenty ovules were placed on each petri dish, and 10 Petri dishes were used per treatment. In vitro culture conditions. The media used were 1) Murashige and Turcker (MT, 1969) supplemented with 4.4 μM 2, 4-D and 1 μM BA, 2) Chee and Pool (CP, 1983) supplemented with 5.0 μM NAA and 1.0 μM BA, 3) Nitch and Nitch (NN, 1969) supplemented with 9.0 μM 2, 4-D and 4.0 μM BA. pH was adjusted to 5.7 prior to addition of agar and sterilized at 121°C and 104 kPa pressure for 20 min. Cultures were maintained at 25 ± 2°C under cool-white fluorescent lamps providing with 16 h photoperiod. Cultures were evaluated after four weeks of culture, and observed on a weekly basis. *Corresponding author; e-mail: [email protected]