Brassinosteroids and gibberellic acid: effects on in vitro pollen germination in grapevine

摘要

Many physiological processes related to plant growth and development are under the influence of growth regulators, which also have an impact on pollen germination. In this study, the effects of two brassinosteroid compounds, epibrassinolide and 22S,23S-homobrassinolide, and gibberellic acid (GA3) on in vitro pollen germination of two table grape cultivars, ‘Italia’ and ‘Cardinal’ (Vitis vinifera L.), were determined. A total of 28 treatments, alone and in combination, were applied to freshly collected pollens which were sown on a basic medium with 1% agar and 20% sucrose. Petri dishes were kept at 26±1°C for 24 hours. Counting of the germinated pollens revealed that the effects of these plant hormones were cultivar- and substance-specific. The cultivar ‘Italia’ was not influenced by the treatments (the highest germination ratio being 44.4% from 0.001 mg L-1 epibrassinolide) as opposed to the cultivar ‘Cardinal’. The highest germination ratio in ‘Cardinal’ was about 50% in pollens treated with 25 mg L-1 GA3 + 0.01 mg L-1 epibrassinolide. The control group resulted in 32.38% germination. Combining GA3 with epibrassinolide provided slightly higher germination ratios compared to combining GA3 with 22S,23S-homobrassinolide.? Introduction Pollen germination and pollen tube growth are two of the most important aspects pertaining to pollen quality (Sharafi and Bahmani, 2011). In vitro pollen germination has been investigated at the genetic, physiological and biochemical levels in different plant species. Pollen germination evaluated in vitro might validate germination in vivo (Hormaza and Herrero, 1999).Literature on in vitro pollen germination of horticultural crops is abundant with studies determining basic medium constituents including boric acid, mineral salts or plant growth regulators. However, there are contradicting results on the effects of growth regulators added to the growth medium. Following pollination, pollen germination and pollen tube growth have been shown to be dependent on the gibberellins on site (Plackett et al., 2011). Pharis and King (1985) stated that reproductive development is one of the physiological events regulated by gibberellins. Chauhan and Katiyar (1998) reported that gibberellic acid (GA3) at low concentrations, as opposed to the higher concentrations, promoted pollen germination in Pinus kesiya. B?ll et al. (2009) stated that gibberellins had an influence on pollen germination in seeded vine species. Sotomayor et al. (2012) found important effects of gibberellin on increasing germination ratio of almond pollens. On the other hand, Radovi? et al. (2016) stated that GA3 had less impact on pollen germination of almond compared to auxin. Brassinosteroids, first found in the bee-collected pollens of rape plants (Grove et al., 1979), are the least investigated growth regulators in terms of their effects on pollen germination and growth. Brassinosteroids were shown to improve pollen tube growth in Camelia japonica (Hewitt et al., 1985) and tomato (Singh and Shono, 2003). On the other hand, Ylstra et al. (1995) reported contradicting results in tobacco using sterols. Extensive studies in Arabidopsis have shown that brassinosteroids are important in the regulation of reproductive growth (Kang and Guo, 2011). Thussagunpanit et al. (2013) showed increasing and decreasing effect of 0.01 μM 24-epibrassinolide on pollen viability and germination. Application of 24-epibrassinolide to Arabidopsis pollens resulted in stimulated germination and pollen tube growth (Vogler et al., 2014). So, there is both a lack of information and contradicting results about the effects of brassinosteroids on pollen germination. This study was conducted to determine the effects of two brassinosteroid compounds, epibrassinolide and 22S,23S-homobrassinolide, along with the other growth regulator group, gibberellins, on in vitro pollen germination and growth of grapevine cultivars. Materials and methods 1. Plant materials Pollens of the table grape cultivars ‘Italia’ and ‘Cardinal’ (Vitis vinifera L.) were used as plant materials. Cultivars are white colored and late maturing. The vines were located at the experimental vineyard in the Dardanos Campus of ?anakkale Onsekiz Mart University, ?anakkale, Turkey. 2. Pollen collection and growth regulator treatments Inflorescences were collected in the morning hours at the 23rd stage (full bloom) of Eichhorn and Lorenz (1977) classification. Inflorescences were brought into the lab and all unopened flowers were emasculated. Anthers were later collected in Petri dishes and allowed to burst for 24 hours at room temperature. Pollen germination capability was determined using epibrassinolide (EBR, Sigma E-1641), 22S,23S-homobrassinolide (HBR, Sigma H-1267) and gibberellic acid (GA3, commercial grade 1 mg a.i.) at three concentrations (w/v) with basic 1% agar and 20% sucrose medium. Concentrations applied were GA3 25, 50 and 100 mg L-1; EBR 0.001, 0.01 and 0.1 mg L-1; and HBR 0.00