In vitro germination to overcome dormancy in seeds of ‘Red Globe’, ‘Italia’ and ‘Niagara Rosada’ grapes

Abstract Dormancy in grape seeds slows the progress of many breeding programs of the crop, culminating in low uniformity and low germination percentages. Conventional methods used to overcome dormancy are time-consuming. Thus, in vitro seed germination emerges as a promising alternative to ensure the germination of grape seeds. In this study, we examined the in vitro germination and vigor of seedlings originating from seeds of ‘Red Globe’, ‘Italia’ and ‘Niagara Rosada’ grapes in growth media supplemented with five concentrations of gibberellic acid (GA3) (0, 1.41, 2.83, 4.24 and 5.66 µmol L-1) for 47 days. The use of GA3 increased seed germination percentage and seedling vigor, in the three varieties. Therefore, for the in vitro germination of seeds of ‘Red Globe’ can be used between 1.41 to 4.24 µmol L-1 of GA3. For ‘Italia’ grapes is indicated 1.41 µmol L-1 from GA3 and for ‘Niagara Rosada’ grape is as between 1.41 to 5.66 µmol L-1 from GA3 can be used.

Rev. Bras. Frutic., Jaboticabal, 2019, v. 41, n. 5: (e-495) In grape breeding programs, seeds obtained from controlled crosses are very important materials of propagation (GISBERT et al., 2018). However, the low germination percentage of grape seeds constitutes a major obstacle to breeders (WANG et al., 2009;VAL et al., 2010). In grape seeds, dormancy is the main cause of low germination percentages (CELIK, 2001;WANG et al., 2009). In the search for methods to overcome dormancy in grape seeds, several authors have suggested cold stratification (BRASIL, 2009;WANG et al., 2009) along with exogenous application of gibberellic acid (GA 3 ) (ELLIS et al., 1983;ERGENOGLU et al., 1997;CELIK, 2001). However, in addition to not being effective in inducing germination in many seeds, these treatments require more than 120 days to produce normal seedlings.
In this scenario, the use of plant tissue culture with in vitro germination of grape seeds is a viable alternative to break dormancy, ensure seed germination and provide new genotypes for grape breeding programs. The present study proposes to examine in vitro germination and vigor of seedlings originating from seeds of table grapes in growth media supplemented with five concentrations of GA 3 (0, 1.41, 2.83, 4.24 and 5.66 µmol L -1 ).
The plant material consisted of seeds of three commercial genotypes of table grape, namely, 'Red Globe', 'Italia' and 'Niagara Rosada'. Seeds were removed from the pericarp, washed in running water, disinfected with neutral detergent, left to dry at room temperature for 48 h and then stored in a refrigerator (±4°C) for seven days, until the in vitro germination test was assembled. The experiment was set up as a completely randomized design with a 3 × 5 factorial arrangement in which the in vitro germination of three table grape genotypes was tested in growth media with five concentrations of GA 3 (0, 1.41, 2.83, 4.24 and 5.66 µmol L -1 ). Seven replicates were used, each of which was composed of a culture bottle containing 40 mL of growth medium and five seeds.
Seeds were cut in the micropyle region and in the upper region using cuticle pliers, in accordance with the methodology of Val et al. (2010). In a laminar flow hood, the seeds were then disinfected in 70% alcohol for 30 s, then in for 20 min a 1.0% sodium hypochlorite (NaClO) solution with two drops of Tween ® 20 deposited for every 100 mL and lastly washed three times in autoclaved deionized water. The seeds were inoculated in growth medium constituted by half of the concentrations of MS medium mineral salts (MURASHIGE and SKOOG, 1962), White vitamins, 100 mg L -1 myo-inositol, 30 g L -1 sucrose, 200 mg L -1 polyvinylpyrrolidone (PVP) and five concentrations of GA 3 (Vetec ® ) (0, 1.41, 2.83, 4.24 and 5.66 µmol L -1 ), with the pH adjusted to 5.7, and solidified with 7 g L -1 pure bacteriological agar (Vetec ® ). The growth medium was autoclaved for 20 min at 121 °C and 1.1 atm and 40 mL were distributed per culture bottle. The growth regulator GA 3 was added to the growth medium after autoclaving.
The bottles with the seeds were kept in the growth room at a temperature of 27±2 °C, photoperiod of 16:8 (light:dark) and luminosity supplied by Osram ® daylight lamps with a luminous intensity of 54 μmol m -2 s -1 . After 47 days of growth, the seeds were examined for the germination percentage of normal seedlings, abnormal seedlings, seeds with primary root emission and ungerminated seeds (seedlings were considered normal only when showing expanded shoots and roots) and the vigor variables of number of leaves, shoot length, root length, total dry matter and germination speed index (GSI), which was evaluated at a two-day interval, during the 47 days of germination, by observing the number of seeds producing primary roots (MAGUIRE, 1962). The variables were subjected to an initial normality test (Shapiro-Wilk). Subsequently, an analysis of variance was performed and means were separated by the t test (LSD) at P<0.05, using Sisvar statistical software (FERREIRA, 2011).
Dormancy is characterized by a condition in which some seeds do not germinate even when exposed to favorable environmental conditions (BASKIN and BASKIN, 2004). In grape seeds, one of the reported types of dormancy is physical, caused by the thickness and hardness of the seed integument, which preclude the entry of water into the embryo (ELLIS et al., 1983). In in vitro conditions, Val et al. (2010) suggested making cuts on the seed integument to facilitate water imbibing by the embryo. Thus, the cuts made in the micropyle region and in the upper region, in our study, managed to overcome physical dormancy and hydrate the embryo. However, differences were observed for the germination and percentage results across the studied varieties. 'Itlia' grape exhibited the highest percentage of germination into normal seedlings (58.5%), differing statistically from 'Red Globe' (43.9%) and 'Niagara Rosada' (53.6%) ( Figure  1a). At 47 days of germination in vitro, several 'Italia' seeds (16.6%) inoculated in growth medium with 1.41 µmol L -1 GA 3 were still producing primary root (Table 1). Additionally, the highest means observed for the vigorrelated variables were obtained by varieties 'Red Globe' (number of leaves, shoot length and dry matter) and 'Italia' (GSI and number of leaves) ( Table 2). For root length, however, no difference was detected between the three varieties ( Figure 1e).
The use of GA 3 as a plant growth regulator provided a significant increase in seed germination percentage across the grape varieties, which rose from 13% (treatment without GA 3 ) to more than 56%, irrespective of the concentration (Figure 1b). Lack of GA 3 in the growth medium resulted in the highest percentages of ungerminated seeds for all studied varieties (Table 1). The use of GA 3 provided an increase in seedling vigor through GSI for 'Italia' grape and in shoot length for varieties Rev. Bras. Frutic., Jaboticabal, 2019, v. 41, n. 5: (e-495) 'Red Globe' and 'Italia'. Additionally, GA 3 considerably increased germination rate and speed in the three grape varieties (Figure 2). However, addition of 2.83 µmol L -1 of GA 3 to the growth medium led to the emergence of abnormal seedlings (10.4%) (Figure 1d).
A positive effect of GA 3 on the in vitro germination percentage of 'Niagara Rosada' seeds was reported by Val et al. (2010) and in 'Isabella' grape by Celik (2001), in different substrates. In seeds, GA 3 acts on the synthesis and activation of the hydrolytic enzymes present in the endosperm tissue (MOSHKOV et al., 2008).
The conventional method provided by the Rules for Seed Testing (RST) for dormancy breaking in seeds of Vitis vulpina indicates pre-chilling in wet substrate at a temperature of 3-5ºC for a period of three months, to only then set them to germinate (BRASIL, 2009). The entire process may exceed 120 days. By contrast, in vitro germination of grape seeds with GA 3 supplementation is a promising strategy in grape breeding programs, as it allows for breaking seed dormancy, accelerating the germination period and obtaining vigorous seedlings (Figure 3) in 47 days. The significant variability observed among the three varieties was due to the genotypic difference between species. Therefore, for the in vitro germination of seeds of 'Red Globe' can be used between 1.41 to 4.24 μmol L -1 of GA 3 . For 'Italia' grapes is indicated 1.41 μmol L -1 from GA 3 and for 'Niagara Rosada' grape is as between 1.41 to 5.66 μmol L -1 from GA 3 can be used..