DETERMINATION OF FLORAL DEVELOPMENT STAGES IN ‘CABERNET

SUMMARY Despite relatively intensive work on the development of inflorescence primordia during grapevine growth in season one, some informational gaps are present in the flower and floral organ development in the season two. In addition, concurrents events of phenology and formation of flowers and floral parts has not been dealt with. With the aid of digital imaging, this research had three objectives; a) describe the developmental events that take place during and after bud break in the buds and in the individual flowers in terms of differentiation, b) match these events with phenological stages, and c) determine size-related growth of the floral organs. After careful dissecting and examination of the samples under microscopy, taken ever 5-10 days between March 20 and May 10 in 2016, the results indicated that highly esteemed works regarding the reproductive anatomy of grapevines needed some additional stages to fully describe events in the stamen and pistil primordia


INTRODUCTION
Investigating the sequence and timing of floral development in horticultural species is of importance in terms of determining productivity and deciding on application time of cultural practices.Different from most woody species in horticulture, grape flowers do not attract any special attention at maturity due to their lack of variation in appearance.Therefore, they are not a crucial material of ampelographic identification (Jackes, 1984).On the other hand, the flowering process is unusual and covers two growing seasons in temperate regions.In the first season, an uncommitted primordium (anlage) becomes visible in the apices of latent buds on shoots and, at optimum conditions, inflorescence primordia (IP) might develop from these specialized meristematic structures.In the second season, individual flowers are formed on previously developed inflorescence primordia (Li-Mallet et al., 2016).
The first studies on floral organogenesis in grapevine flowers included micro techniques that involves sectioning of grapevine buds in a serial order (Barnard and Thomas, 1933;Snyder, 1933).With the advent of microscopy, especially epi-illumination microscopy, the development and structures of floral inflorescences and/or individual flowers were examined in different Vitis species (Gerrath and Posluszny, 1988;Timmons et al., 2007).Scanning electron microscopy has enabled researchers to 3-D visualize the sequential developmental events in the flowers both in Vitis species, including the wild grape (Caporali et al., 2003;Spada et al., 2003;Ramos et al., 2014) and Vitis vinifera L. cultivars, such as 'Sultana' (Scholefield and Ward, 1975), 'Shiraz' (Srinivasan and Mullins, 1976), 'Chardonnay' (Watt et al., 2008) and 'Pinot noir' (Jones et al., 2009).However, these studies were only descriptive without providing quantification or size information.
Literature on the dimensions of grape flower in the Vitaceae family at full development stage before anthesis state that its size depends on species, being 2 mm in Vitis berlandieri Planch.and 6-7 mm in Vitis labrusca L. (Keller, 2015).In one study, Caporali et al. (2003), presented through SEM observations some dimension information on pistil and anther length of wild grape, V. vinifera subsp.sylvestris (C.C.Gmel.)Hegi.Noyce et al. (2015Noyce et al. ( , 2016) ) conducted a series of studies in order to provide more detail into development of IP in cv.'Chardonnay' (V.vinifera) using dissecting microscope and quantified the number and the size of IP in the buds.To the best of our knowledge, no specific size information regarding the sizes of primordia of flowers and/or floral organs in V. vinifera are presented in previous studies.
Even though grapevine floral morphogenesis has been well understood and explained in detail, the morphological development and size increment over time in the floral, specifically generative, parts has not been indicated.There are some vital gaps in the development of grape inflorescence primordia in the season two after inflorescence differentiation.The most regarded study of Srinivasan and Mullins (1981) described flower development in season two, to start with stage 8 and end with stage 11 without any specific reference to stamen and pistil organogenesis between stage 10 and 11.
There are some widely known descriptive systems for identifying growth cycle (phenology) of grapevine, such Baggiolini (1952), Eichhorn and Lorenz (1977), the BBCH system by Lorenz et al. (1995) and Coombe (1995).Unfortunately, none of these systems indicate any relationship between progress of flower development and phenology.This study aimed to unveil primordial development of the reproductive parts in a Vitis vinifera L. flower (cv.'Cabernet Sauvignon') from the beginning of bud break to the completion of pistil formation.This would enable determining the fate of the differentiated inflorescence primordia turning into a complete flower cluster and quantifying the size increase in the individual flower parts well before anthesis.Also, an attempt to associate all these events with the phenological stages in the life cycle of grapevine was also done.

Plant material and study site
Plant material was obtained from Vitis vinifera L. cv.'Cabernet Sauvignon' at the Research Station of Department of Horticulture, located at Dardanos campus of Canakkale Onsekiz Mart University in Çanakkale, Turkey.The grapevines were ten years old, grafted on 5 BB rootstock, with goblet training and winter spur pruning (2-3 buds per spur).The planting density was 2x3 m.Management was all identically applied to all vines, with no irrigation (rain-fed), throughout the season.

Studies on flower morphology
Sampling time was from the end of dormant season (March) to the mid-May 2016.Samples (compound latent buds or individual flowers) were taken every 5-10 days at each sampling date and the phenological stages, between 2-15, were recorded according to the modified Eichorn-Lorenz (E-L) system by Coombe (1995) (Fig. 1).The central tendency of each stage was measured by taking the median count at each sampling time.Blooming took place 3 weeks later the last sampling time.Sampling size was at least 15 buds or flowers per sampling time.

Microscopy and storage of samples
Samples were immediately placed in FAA solution (formalin 10%, ethyl alcohol 50% and glacial acetic acid 5%) for 24 or longer hours.Samples were dissected using an Olympus SZX7 stereo zoom microscope (Olympus Corp., Japan).The overwintering buds were rid of their scales and wooly hairs under the stereo microscope with the help of dissecting needles and mini double lancets.For the microscopic examination of the stamen and pistil primordia structures, sepals and petals were removed with the help of the arrowhead two-edge sharp needles and protruding conical stylet.These operations were done in distilled water to prevent the samples from drying.
Images were photographed using a digital microscope camera (LC20, Olympus DF PLAPO X -4 , Japan) mountable on the microscope.The measurements in µm were enabled by a software program (LC20-Bundle LCmicro, Olympus Corp., Japan).Samples at the same phenological stages were taken for flower and flower organ primordia measurements.Measurements were taken for flower width and length, anther width and length, filament length, pistil length, ovary width and stigma diameter at the sampling times.

Determination of the floral development stages
In order to determine the stages of development of flower and floral primordia, initial guidance was obtained from the work of Srinivasan and Mullins (1981).Since no clear evidence on formation of stamen and pistil primordia was given in this highly respected study, a need to add intermediary stages relating the formation of these primordia existed.Additionally, a link between the phenological stages of the grapevine and morphological stages of floral development in grapevine flowers was tried to be established.

RESULTS AND DISCUSSION
The morphological development of floral primordia in the inflorescence was detected with the aid of microscopy and it was possible to visualize when the floral development occurred in the grapevine cultivar 'Cabernet Sauvignon'.Development of flower and floral organ primordia in this cultivar was depicted in Figure 2.
On April 5 (E-L stages between 7 and 9), the calyx and the petal primordia became visible on inflorescence (denoted as 8.2) (Fig. 2b).At the E-L stage 9, the state that corolla development started but was not yet interdigitated at the top of the flower was caught in our samples on April 10 (Fig. 2c).The progress from the flower initial formation to corolla formation took place in a 10-day period.The next event in the flowers were borders of petal lobes being visible and formation of calyptra, named as the stage 10 according to Srinivasan and Mullins (1981) description.
From this point because there are no explanation or representation as to what happens regarding the stamens and/or pistil, we numbered the following stages as 10.1, 10.2, and so on.Fig. 2d-f shows that on April 20 (E-L stages between 9-12), the calyptra fully formed (stage 10.1) and inside, anthers with two lobes were exposed.They did not have any filament formation yet.The pistil, on the other hand, had already started to develop from gynoecial ring and looked like a pressed balloon filled with water sitting on a flat surface.Style was barely noticeable (Stage 10.1).As the flower continued to grow, on May 5 (E-L stages between 12-15), filaments were formed, and the pistil started to take its shape with a little bit style visibility.A slight asymmetry could be observed on the ovary (stage 10.2, Fig. 2g-i).Within another five days (May 10, E-L stage 15), the flower lost its puffiness and showed a longitudinal growth.At this stage, the stamens and the pistil were fully shaped and elongated (Fig. 2j-l) and the style and the stigma were clearly visible (stage 10.3).
In the earlier studies involving the timing of these developments, flower initiation has been reported to include formation of some part of calyx primordia before bud break (Alleweldt and Ilter 1969;Ağaoğlu 1971), but our data are not able to confirm or deny such results.Due to the possible reasons of difference sampling interval, technological incapacities or simply missed because of their extremely miniature size, we inclined to assume that no floral part develops before bud break.Swanepoel and Archer (1988) stated in their study with V. vinifera cv.'Chenin blanc' that the duration between the appearance of inflorescence and completion of floral development was 20 days.In the current study, this was a bit longer, approximately 30 days, which might have been the result of both the cultivar and the environmental conditions, especially temperature.
Measurements taken on the flower and the flower parts of cv.'Cabernet Sauvignon' were given in Table 1.'Cabernet Sauvignon' flowers increased their width and their length more than 9-and 15-fold, respectively, between stage 8.1 (April 1) and 10.3 (May 10).At first, they were wider than they were longer, but at later stages they grew longitudinally.Signs of generative parts become apparent at the late part of April.Anther width and length were the ones that kept their growth to minimum compared to the other parts of the flower.Filaments, on the other hand, elongated almost 7-fold in a period of 20 days.Gynoecium growth was the most impressive and total pistil length increased from 52.8 to 162 µm, ovary width from 40.4 to 99.8 µm, and stigma diameter from 9.96 to 44.9 µm in twenty days.Literature paid scant attention to the growth rate of the generative parts of the Vitis flower.In an earlier work, Considine and Knox (1979) presented some volume information of the pistil primordia of 'Gordo blanco' (Vitis vinifera L. cv.), reaching 0.1 mm 3 around the time of anthesis.Recently Caporali et al. (2003) provided some size information of the pistil of V. vinifera subsp.sylvestris flowers at stages 7 and 8.However, these studies did not present any size increase over time in the floral parts.

CONCLUSIONS
Here in this study, establishing a link between the phenological stages and the floral primordia development was accomplished for the cv.'Cabernet Sauvignon'.No matter how miniscule and hard to spot in the very compressed shoot at the beginning of the bud break in the spring, appearance of the inflorescence marked the formation of reproductive organs of the flower.Ongoing from this point, the progression of the development in the stamen and the pistil primordia was relatively fast.Full formation of the flower with its complete parts was coincided with the time around 7-8 leaves separated from the shoot and inflorescence was visible.Summation of the intermediate floral stages were as follows: 8.1, differentiation of sepal primordia; 8.2, development of calyx and petal primordia; 10.1, calyptra is almost fully formed, stamens with no filament growth, and the pistil starts to show; 10.2, calyptra fully covers the stamens which have a short filament and pistil with distinguishable style and stigma, and 10.3, extended stamens and pistil with clear style and discoid stigma.
The value of recognition of growth stages for the grapevine is indispensable not only for implementing cultural operations in the vineyard but also providing a unanimous understanding among the human partners in grape growing.In addition, establishing a link between these stages with the flowering process in Vitis vinifera is also of great importance.