English (pdf)
Article in xml format
Article references
Automatic translation
Send this article by e-mail
Cited by SciELO 
Cited by Google 
Similars in
SciELO 
Similars in Google 
Permalink
Rubus is a widely distributed genus. Its species are found on all types of cultivable soils ranging from tropical to subarctic regions. However, the exact number of species remains unknown because its classification
was last done by Focke (1910-1914), and since then, many species have been described. It is estimated that the number of species of this genus ranges from 600 to 800. Difficulties in its taxonomic classification, particularly of Rubus subgenus in Europe and North America, are due to the prevalence of interspecific hybridizations, polyploidy, and various forms of apomixis (Thompson, 1995). The Andean blackberry (Rubus glaucus Benth.) is distributed in the three mountain ranges of Colombia and has combined characteristics of Idaeobatus and Rubus subgenera. It is a fertile amphiploid or allotetraploid that has resulted from the genome fusion of two species (Jennings, 1988).
In Colombia, blackberry production and cultivated area, but not yield, have increased, and it is considered a fruit with cultivation opportunities in Colombia, both for supply in the domestic market and export. The Colombian National Fruit Plan (PFN, Spanish acronym; 2006-2026) expects a 94.1% increase in cultivated area in Colombia by 2026 (MADR, 2006), i.e., from 10,743 ha in 2008 to 20,631 ha in 2026, which would generate approximately 6,917 direct jobs. This species exhibits great variability in terms of size, color, and fruit quality, which was possibly produced through selection breeding from wild plants in ancient times (Rativa et al., 2016).
The associated producers of blackberry in Colombia have highlighted the need to standardize the supply of planting material, starting with plant breeding schemes that lead to more productive varieties with morphological characteristics that ease agricultural activities; the desired characteristics include absence of spines, tolerance to diseases, and increase in productivity. Therefore, it is necessary to collect, multiply, and characterize possible parent plants and conduct crossing. The latter step warrants studies on the morphology and floral biology of this species for improvement.
In plant breeding activities, the desired characteristics are obtained using existing variability. The understanding of pollination mechanisms in plants is critical for improvement processes. Sexual hybridization, essential in plant breeding, constitutes controlled pollination and implementation of such pollination mechanisms (Frankel and Galun, 2012).
In this study, for advancements in breeding processes for R. glaucus, morphological characteristics and studies on floral biology of this species were conducted as well as pollen viability and stigma receptivity were studied.
MATERIALS AND METHODS
Vegetal material
Experimental plant material (R. glaucus flowers) was obtained in August 2017 from the Botanical Garden of Universidad Tecnológica de Pereira, located at 1,467 masl (4°47'28.2" N 75°41'24.5" W).
Floral morphology
Flowers collected at anthesis (fully opened flowers) were inspected using a Leica EZ4 stereoscope. The morphological characteristics of the flowers, as suggested by Strasburger (1994), were evaluated and classified for external (chalice and corolla) and internal verticils (androecium and gynoecium). Finally, the floral formula of R. glaucus was constructed.
Pollen viability
A total of 25 flowers were collected at anthesis from different plants, and their anthers were detached under a stereoscope to collect pollen grains. To obtain a random sample, 100 pollen grains per sheet were counted for evaluation at each collection time. Pollen collection started at 8:00 am and continued until 1:00 pm with periodic collection conducted every hour (6-hour collection, treatments 1 to 6).
Pollen viability analysis
Pollen viability analysis was conducted using coloration with 2% acetocarmine glycerol as described by Alexander (1969). This procedure can detect non-aborted pollens and indirectly evaluate their viability. Pollens were visualized under a Leica DM750 optical microscope with a photographic head. Pollens were considered viable when they presented reddish coloration and a hyaline halo (Figure 1A). Viable pollens were quantified in percentage by determining viability percentage in 100 pollen grains from 5 flowers; this procedure was performed in triplicates. Statistical analysis was performed using ANOVA after determining normal distribution of data using Shapiro-Wilk test with at 5% significance. Differences among treatments were determined using Tukey test. All statistical analyses were performed using the R3.4.4 software.
Scanning electron microscopy (SEM)
To visualize pollen grains using SEM, flowers were collected at anthesis, and their anthers were detached under a stereoscope. The detached anthers were fixed in a 3% glutaraldehyde solution overnight at 4 °C. Subsequently, the samples were washed three times for 10 min in a 0.1 M PIPES solution (pH 7.2), followed by serial washes in ethanol solutions (50%, 60%, 70%, 80%, 90%, 95%, and 100%; v/v). Subsequently, the samples were transferred into a drying equipment. The specimens, previously visualized by optical microscopy, were observed under a FEI scanning electron microscope (version Quanta 250; Thermo Fisher Scientific).
Stigma receptivity
Stigma receptivity was chemically evaluated with 3% hydrogen peroxide (H2O2). This evaluation involved depositing H2O2 in the stigma of flowers by considering receptive stigmas that rapidly formed bubbles (Zambon et al., 2018). Evaluation of stigma receptivity was performed at pre-anthesis and anthesis stages using a periodicity schedule, which started at 7:00 am and continued until 2:00 pm.
RESULTS AND DISCUSSION
Floral morphology
External verticils. R. glaucus flowers were complete and perfect and had a regular and perianth, actinomorphic structure, with 5 petals and 5 differentiated sepals (heterochlamydeous). The corolla had free petals and sepals (dialipetal and dialisepal, respectively; Figure 2).
Figure 2 Floral morphology of Rubus glaucus. A. Regular actinomorphic flower; B. Perianth flower; C. Heterochlamydeous; D. Dialisepal-Dialipetal; E. Intrusive anther; F. Hypogynous ovary
Internal verticils. The flowers were hypogynous with pluricarpelar and apocarpous gynoecium and numerous stigmas and intrusive anthers (Figure 2). Based on the previously described characteristics, the following floral formula was constructed:
The morphological characteristics of R. glaucus reported in this study are consistent with those described by Monasterio-Huelin (1992), who reported the taxonomic characteristics of Rubus in the Iberian Peninsula and Balearic Islands. Monasterio-Huelin (1992) described Rubus species as heterochlamydeous and hermaphrodites wherein the chalice is formed by 5 imbricate pieces with whole lanceolate sepals. Rubus from the Iberian Peninsula and Balearic Islands have white, pink, or red petals depending on the species; R. glaucus petal is white. On the other hand, Cancino-Escalante et al. (2011) described Rubus flowers in Colombia as pentamer flowers with welded sepals at the base; deltoid to ovate-acuminate, usually with obovate; and free; and white, pink, or red petal trichomes. These characteristics completely coincide with those found in the present study.
Pollen viability
Pollen viability data showed a normal distribution and coefficient of variation of 6.77%. Analysis of variance showed highly significant differences in terms of pollen viability according to the collection time. Regarding differences in pollen viability according to the collection time, Tukey's test showed that the highest pollen viability was obtained at 10:00 am, followed by at 9:00 am and then 11:00 am. Pollen viability percentages at 9:00 and 10:00 am were significantly different from each other and different from other collection time. However, pollen viability was similar at 8:00 and 11:00 am. Likewise, pollen viability at 12:00 and 1:00 pm were similar, with the latter having the lowest pollen viability percentage (Table 1).
Evaluation of other Rubus species (Nybom, 1985) has shown 8% viability for triploid species and 54% for tetraploid species, compared with 81% for apomictic diploid species. These results coincide with those found in the present study for R. glaucus, which is a tetraploid species. Other studies, which evaluated pollen viability percentages from first-generation progeny individuals of interspecific crosses of Rubus genus, have reported values between 66.7% and 28.8%, with most frequently reported values being 45% and 60% depending on the parents of each progeny (Nybom, 1995). The previously reported results coincide with those found in the present study.
The relationship between pollens and fruits and seed formation has been recognized since the beginning of agriculture. This information has allowed for artificial pollination and double pollination in angiosperms. Darwin's contribution is also known, and his theory of evolution states that cross-fertilization is not only beneficial in evolutionary terms but is also necessary for maintaining a species' vigor and fertility. Understanding the viability of these gametes is vital for plant breeding.
SEM
R. glaucus pollen grains showed a tricolored structure (Sáenz Laín, 2004), with equatorial diameters ranging between 20 and 25 μm (Figure 3). According to Soejarto and Fonnegra (1972), this pollen size is small; they have reported that most species have a diameter between 15 and 50 μm and that almost all plant species have pollen diameters between 8 to 150 μm. In general, there is a correlation between pollen size and pollinating agents. Similar studies conducted on pollen grains from 9 European Rubus species have described pollen grains to be small, isopolar, and tricolporate (Tomlik-Wyremblewska, 1995) such as those found in R. glaucus.
Stigma viability
Qualitative assessments of stigma receptivity led to the conclusion that most stigmas were receptive at anthesis (fully open flower; Figure 4) and the time of highest stigma receptivity was 12:00 m when the highest and most frequent bubble production on stigma exposure to H2O2 (3%) was observed.
Figure 4 Appearance of stigma and production of bubbles during stigma receptivity test. A. Rubus glaucus multiple stigma (30×); B. Production of bubbles in R. glaucus stigma on reaction with hydrogen peroxide.
Studies on the fertility aspects of other Rubus species have been conducted on American "blackberries" in breeding programs (Ruple et al., 2010), which aimed to evaluate the effects of self-pollination and cross-pollination and the subsequent fruiting in different genotypes of Rubus. They have evaluated different fertility components of these genotypes in controlled conditions, particularly before and after being emasculated. This study led to the conclusion that flowers do not change their stigma receptivity as opposed to being emasculated or not.
A previous study (MacPhail and Kevan, 2005) on ecological aspects related to the frequency of insect visits to the wild species of Rosa genus, a species highly related to Rubus, found that the largest period of insect visits to flowers occurs between 9:00 am and 12:00 m, consistent with the findings of the present study. For R. glaucus, the highest insect activity is observed in the morning, with the highest frequency of Apis mellifera (Hymenoptera) in commercial crops; this denoted that stigma is more receptive at 12:00 m.
Understanding R. glaucus pollination mechanisms is vital to improve breeding processes with controlled pollination. Floral morphology indicates pollination mechanisms of this species and helps to infer its most frequent type of pollinators. In addition, determination of the fertility behavior of internal verticils (androecium and gynoecium) allows improved protocol designing for crosses that formally initiate an improvement program for this species. The last aspect has not been previously reported for R. glaucus and would allow establishment of a new variety, leading the productive sector to be more competitive.
CONCLUSIONS
Regarding floral morphology, R. glaucus is an actinomorphic, perfect, and complete pentamer flower, similar to other flowers of Rubus. Evaluation of its pollen grains led to the conclusion that the highest pollen viability occurs at 10:00 am. Meanwhile, the highest stigma receptivity occurs at 12:00 m, i.e., at anthesis. Based on gamete behavior, controlled pollination protocols can be standardized to initiate breeding programs for this species.
