Factors influencing induction and maintenance of Vitis rotundifolia Michx. embryogenic cultures

Abstract

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of 'Supreme' and unopened leaves and petioles of 'Delicious'. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of 'Alachua' and 'Carlos' cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in 'Carlos' (379.3) followed by 'Alachua' (350.0) and 'Delicious' (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 lM Benzyladenine (BA). Although high embryo germination rates (80-100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6-47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.

Producing Transgenic Thompson Seedless Grape (Vitis vinifera) Plants using Agrobacterium tumefaciens

“…NN basal medium supplemented with 2.0 mg L -1 2, 4-D and 0.5 mg L -1 TDZ was the most effective for the induction of callus (92%) and shoot formation (+++). Several studies on Vitis rupestris (Olah et al, 2003), Vitis rotundifolia (Dhekney et al, 2011;Li et al, 2014) and Vitis vinifera (Vidal et al, 2009) has been similarly succeeded to obtain regeneration using leaves as explants. In agreement with our results, Mullaw et al (2010) reported that abundant embryogenic callus in wine grape was obtained from leaf and floral explants supplemented with 9.0 μM 2, 4-D +17.0 μM IASP in combination with either 1.0 μM BA or 1.0 μM TDZ in darkness.…”

Genetic Transformation Strategies in Fruit Crops

“…Commonly, SE systems in grapes are initiated from stamens and pistils and responses are variety-dependent. The best developmental stages to initiate embryogenic cultures have been deduced for some genotypes using the basis of phenological stages of inflorescences (Dhekney et al, 2009); whereas stamens and pistils from some cultivars such as ´Pinot Gris´ must be collected at early developmental stages; other genotypes such as ´Merlot´, ´Sauvignon Blanc´ and ´Freedom´ must be induced using explants at more advanced maturity stages. In vitro leaves have been also proposed as source explants for SE induction in grapevines.…”

“…Although lower efficiencies than stamens and pistils have been obtained, the use of unopened leaves (i.e. between 1.5 to 5.0 mm long) placed abaxial side down on Petri dishes supplemented with solid NB2 medium leads to the generation of PE masses that later will regenerate into whole plants in ´Superior Seedless´, ´Thompson Seedless´ and ´Freedom´ genotypes (Dhekney et al, 2009). Alternatively, convenient procedures to introduce material from the field have been reached by proper cultivation of grapevine sterile buds in solid C2D4B medium (Araya et al, 2008;Gray, 1995) by one to four months and then culturing the processed tissues into NB2 solid medium.…”

“…An optimized SE procedure for ´Thompson Seedless´ ( Figure 3a) is not as efficient as applied on ´Red Globe´ (Figure 3b). Analyses of factors affecting competence have been recently reported (Dhekney et al, 2009) by exploring changes in the induction phase. The use of MS and Nitsch macro-and micro-elements supplemented with 6-bencyl aminopurine (BAP) at 4.5 and 8.9 µM plus 2,4-dichlorophenoxy acetic acid (2,4-D) at 8.9 and 4.5 µM, respectively, leaded to most of the 18 evaluated cultivars and eight Vitis hybrids, to produce SE to some degree in that work.…”

“…These masses are the pivot for a new branch of procedures in which solid and liquid cultures are used. In ´Thompson Seedless´ or ´Princess´, bud production by culturing of leaves in C2D4B media prepare cells that will produce as many somatic embryos as required during the phase of cultivation in NB2 medium; these cultivars describe high yield enough to accomplish considerable number of transformation experiments (Dhekney et al, 2009) (Figure 3a). However, such yield is not observed when the strategy is applied on ´Harmony´, ´Freedom´ or ´Red Globe´ genotypes ( Figure 4a).…”