116 Inflorescence and bunch architecture Australian Journal of Grape and Wine Research 10, 116–124, 2003 Introduction Differences in inflorescence or bunch architecture bet- ween grapevine cultivars can have major implications for disease control in the vineyard. For example, the sus- ceptibility of different grape cultivars to Botrytis bunch rot, caused by Botrytis cinerea, is closely correlated with bunch architecture. Cultivars with tight (compact) bunches develop severe rot whereas those with loose (open) bunches are less susceptible (Weaver et al. 1962, Marois et al. 1986, Ferreira and Marais 1987, Vail and Marois 1991, Percival et al. 1993, Smithyman et al. 1998, Vail et al. 1998, Zabadal and Dittmer 1998). This heightened susceptibility in tight bunches is most likely due to the combined effects of increased water retention and pro- longed drying after rain events (Vail and Marois 1991). Development of epicuticular wax is also reduced where berries touch and these areas are more susceptible to B. cinerea infection than non-contact zones (Marois et al. 1986). Investigation of bunch architecture will assist in determining which physical features have the greatest influence on bunch compactness. Grapevine inflorescence primordia growth begins within latent buds that form in the previous vegetation period, and this initiation process has been extensively studied (Alexander and Woodham 1962, Srinivasan and Mullins 1976, 1981, Morrison 1991). A recent review by May (2000) emphasised the importance of traits such as the number of branches per inflorescence, flower and berry number on inflorescence, and bunch morphology. Environmental factors, especially day-length and tem- perature, also influence inflorescence initiation and devel- opment (Buttrose and Hale 1973, Sugiura et al. 1975, Srinivasan and Mullins 1980a, Kubota et al. 2001). Tendrils are the most closely related organs to inflores- cences with a similar meristematic origin, but have a divergent developmental pathway (Srinivasan and Mullins 1979, 1980b, Boss and Thomas 2000). Studies have shown that tendrils may be converted into inflorescences, a process that is genetically controlled (Boss and Thomas 2000, 2002, 2003). However, it is unclear as to what genetic and developmental processes determine differ- ences in inflorescence architecture between cultivars. Inflorescence architecture has been studied in a range of plants including pea (Nomura et al. 1997), potato (Garcia-Maroto et al. 2000), rice (Nakagawa et al. 2002) and Arabidopsis (Okada and Shimura 1994, Bradley et al. 1997, Ratcliffe et al. 1999, Altamura et al. 2001, Goosey and Sharrock 2001). These studies indicated that muta- tions resulting in compact or elongated inflorescences might result from different rates of cell division, cell Inflorescence and bunch architecture development in Vitis vinifera L. YURI N. SHAVRUKOV, IAN B. DRY and MARK R.THOMAS 1 CSIRO Plant Industry, PO Box 350, Glen Osmond, SA 5064,Australia, and Cooperative Research Centre for Viticulture, PO Box 145, Glen Osmond, SA 5064, Australia 1 Corresponding author: Dr M.R. Thomas, facsimile +61 8 8303 8601, email [email protected]Abstract Inflorescence development from budburst to harvest was analysed in four cultivars of grapevine. Two cultivars with tight or compact bunches (Riesling, Chardonnay) and two with loose or open bunches (Exotic and Sultana) were selected to define differences in bunch development for future genetic analysis. A range of phenotypic characters for both inflorescence and shoot architecture were measured. Differences in the rate of rachis elongation rates were observed between tight and loose bunch cultivars commencing at the earliest stages of inflorescence development after budburst. At anthesis, five phenotypic characters showed significant differences between tight and loose cultivars: (1) total inflorescence length, (2) node number per inflorescence rachis, (3) combined length of two consecutive internodes of the rachis and (4) shoot node position at which the inflorescence was present and (5) mature tendril length. A quantitative estimate of bunch compactness was calculated at bunch maturity. Exotic and Sultana had significantly more open space than did compact bunch cultivars Riesling and Chardonnay. Comparison of flower number at anthesis and berry number at maturity indicated that the proportion of berries set was similar in all cultivars studied and, therefore, did not contribute to variability in bunch openness between cultivars. Internode length of the inflorescence rachis was the major trait responsible for inflorescence openness. Cellular studies using SEM, fluorescence microscopy and DNA content demonstrated that differences in rachis internode lengths were mostly associated with cell expansion. Keywords: grapevine, rachis, internode, elongation, anthesis, epidermis, parenchyma, cell expansion.
Transcript
116 Inflorescence and bunch architecture Australian Journal of Grape and Wine Research 10, 116–124, 2003
IntroductionDifferences in inflorescence or bunch architecture bet-ween grapevine cultivars can have major implications fordisease control in the vineyard. For example, the sus-ceptibility of different grape cultivars to Botrytis bunch rot,caused by Botrytis cinerea, is closely correlated with buncharchitecture. Cultivars with tight (compact) bunchesdevelop severe rot whereas those with loose (open)bunches are less susceptible (Weaver et al. 1962, Marois etal. 1986, Ferreira and Marais 1987, Vail and Marois 1991,Percival et al. 1993, Smithyman et al. 1998, Vail et al.1998, Zabadal and Dittmer 1998). This heightened susceptibility in tight bunches is most likely due to thecombined effects of increased water retention and pro-longed drying after rain events (Vail and Marois 1991).Development of epicuticular wax is also reduced whereberries touch and these areas are more susceptible to B.cinerea infection than non-contact zones (Marois et al.1986). Investigation of bunch architecture will assist indetermining which physical features have the greatestinfluence on bunch compactness.
Grapevine inflorescence primordia growth beginswithin latent buds that form in the previous vegetationperiod, and this initiation process has been extensivelystudied (Alexander and Woodham 1962, Srinivasan and
Mullins 1976, 1981, Morrison 1991). A recent review byMay (2000) emphasised the importance of traits such asthe number of branches per inflorescence, flower andberry number on inflorescence, and bunch morphology.Environmental factors, especially day-length and tem-perature, also influence inflorescence initiation and devel-opment (Buttrose and Hale 1973, Sugiura et al. 1975,Srinivasan and Mullins 1980a, Kubota et al. 2001).Tendrils are the most closely related organs to inflores-cences with a similar meristematic origin, but have adivergent developmental pathway (Srinivasan and Mullins1979, 1980b, Boss and Thomas 2000). Studies haveshown that tendrils may be converted into inflorescences,a process that is genetically controlled (Boss and Thomas2000, 2002, 2003). However, it is unclear as to whatgenetic and developmental processes determine differ-ences in inflorescence architecture between cultivars.
Inflorescence architecture has been studied in a rangeof plants including pea (Nomura et al. 1997), potato(Garcia-Maroto et al. 2000), rice (Nakagawa et al. 2002)and Arabidopsis (Okada and Shimura 1994, Bradley et al.1997, Ratcliffe et al. 1999, Altamura et al. 2001, Gooseyand Sharrock 2001). These studies indicated that muta-tions resulting in compact or elongated inflorescencesmight result from different rates of cell division, cell
Inflorescence and bunch architecture development in Vitis vinifera L.
YURI N. SHAVRUKOV, IAN B. DRY and MARK R.THOMAS1
CSIRO Plant Industry, PO Box 350, Glen Osmond, SA 5064,Australia, and Cooperative Research Centre for Viticulture,PO Box 145, Glen Osmond, SA 5064, Australia
Abstract Inflorescence development from budburst to harvest was analysed in four cultivars of grapevine. Twocultivars with tight or compact bunches (Riesling, Chardonnay) and two with loose or open bunches(Exotic and Sultana) were selected to define differences in bunch development for future genetic analysis.A range of phenotypic characters for both inflorescence and shoot architecture were measured. Differencesin the rate of rachis elongation rates were observed between tight and loose bunch cultivars commencingat the earliest stages of inflorescence development after budburst. At anthesis, five phenotypic charactersshowed significant differences between tight and loose cultivars: (1) total inflorescence length, (2) nodenumber per inflorescence rachis, (3) combined length of two consecutive internodes of the rachis and (4)shoot node position at which the inflorescence was present and (5) mature tendril length. A quantitativeestimate of bunch compactness was calculated at bunch maturity. Exotic and Sultana had significantlymore open space than did compact bunch cultivars Riesling and Chardonnay. Comparison of flowernumber at anthesis and berry number at maturity indicated that the proportion of berries set was similarin all cultivars studied and, therefore, did not contribute to variability in bunch openness betweencultivars. Internode length of the inflorescence rachis was the major trait responsible for inflorescenceopenness. Cellular studies using SEM, fluorescence microscopy and DNA content demonstrated thatdifferences in rachis internode lengths were mostly associated with cell expansion.
Shavrukov, Dry & Thomas Inflorescence and bunch architecture 117
elongation, or both, within the inflorescence rachis. Forexample, the Arabidopsis dwarf mutant compact inflores-cence (cif) has significantly shorter cells within the inflo-rescence than do the wild-type plants (Goosey andSharrock 2001). In contrast, transgenic tobacco plantstransformed with the potato STMADS16 gene displayedmarkedly elongated inflorescence internodes due to sig-nificant increases in cell number per internode, withoutincreases in cell size (Garcia-Maroto et al. 2000).
Accordingly, the main aim of this present study was toidentify the phenotypic characters responsible for differ-ences in bunch architecture between grapevine cultivarswith compact and open bunches as a precursor to theidentification of the gene(s) controlling this character ingrapes. Identification of the genetic basis for an openbunch character could then be used for the genetic manip-ulation of bunch character in elite cultivars, through tra-ditional breeding or genetic technology, to reduce sus-ceptibility to B. cinerea in the field.
Materials and methods
Plant material Four grapevine (Vitis vinifera L.) cultivars – Riesling,Chardonnay, Exotic and Sultana on their own roots –were sampled from the University of Adelaide irrigatedexperimental vineyard at the Waite Campus, Urrbrae,South Australia. Row orientation (north-south) and prun-ing treatments (spur) were similar for all cultivars.
Measurement of phenotypic characters For the study of inflorescence length development overthe growing season, three shoots at a similar stage (1shoot per vine) from each of the four different cultivarswere randomly tagged at budburst. Grapevine growthstages were classified using the modified E-L system(Coombe 1995). Inflorescence length was measured everythree days up until fruit set. Following berry set, the timeinterval between measurements was increased graduallyfrom 3–9 days until berry maturation (60 days after anthe-sis). In the event that several inflorescences were presenton a single shoot, only the basal inflorescence (nearestfrom bottom of the shoot) was used for measurements.
For the comparative study of individual grapevinecharacteristics at anthesis, 30 shoots (one shoot per onevine – designated Group A) with inflorescences at thesame stage of floral development, were chosen at randomfrom each cultivar immediately before capfall, and thefollowing traits recorded: total shoot node number, shootnode number at which the inflorescence was present,shoot length, shoot internode length flanking inflores-cence and tendril length. These inflorescences were thenenclosed in plastic mesh (0.5 mm holes) to collect flowercaps. After capfall was complete, the plastic mesh wasremoved to provide normal conditions for bunch andberry development and the number of flower caps count-ed to estimate flower number per inflorescence. Bunchesremained on the vines until fully developed (60 days afteranthesis). Bunches were then removed, berry numberper bunch counted and rachis length determined usingdigital calipers.
A second group of 20 shoots (one shoot per one vine– designated Group B) with inflorescences at the samestage of floral development were selected from each of thefour cultivars at anthesis (defined as 50% capfall) andthe same measurements of shoot length, shoot internodelength flanking the inflorescence and tendril lengthrecorded as for Group A above. In this case, inflorescenceswere then harvested immediately and measurements ofinflorescence traits at anthesis determined by digitalcalipers. Differences in the timing of measurementsbetween Group A (pre-capfall) and Group B (50% capfall)varied by only 4–5 days and have been pooled as timepoint ‘anthesis’.
Estimation of bunch openness A quantitative measurement of bunch openness at harvestwas developed using a method based on the estimated dif-ference between the morphological and real volume of thebunch. The morphological volume of each bunch wasestimated as the volume of a cone using the standard for-mula:
Vcone = 1/3 πr2 l
where radius r is taken as equivalent to 1/2 of the bunchwidth at the widest point and l = bunch length. Actualvolume of the bunch was estimated by displacement.Bunches were harvested from the vine and immersedcompletely in a glass container filled with water. The vol-ume of water displaced by the bunch was collected and itsweight recorded. Taking into account that the density ofwater equals 1, the weight of water collected was equiv-alent to the volume of water displaced, which representsthe real volume of the bunch. The difference betweenthe morphological bunch volume (Vcone) and the actualbunch volume (Vactual) represents the free space in thebunch between the berries and was converted into relativeper cent of bunch openness using the formula:
Per cent of openness = Vcone – Vactual × 100%Vcone
The per cent of bunch openness has a theoretical rangefrom 0% (no free space within bunch) to a maximumvalue approaching 100% (no berries on rachis – all freespace).
Scanning electron microscopy (SEM)Rachises (with berries removed) were fixed in FAA:ethanol 65% (v/v), glacial acetic acid 5% (v/v) and com-mercial formalin 5% (v/v) for three days and then trans-ferred to 80% ethanol. The first and second internodes ofthe central rachis, and the largest internode of the firstbranch were sectioned and dehydrated through a shortethanol series (80%, 95%, 100%) with two changes ofsolution for one hour each. Rachis fragments were thenkept overnight in absolute ethanol under vacuum. Criticalpoint drying was carried out in a Balxers Union oven,placed on an aluminium mount with a sticky surface,and carbon-coated with an SEM Coating Unit E5100(Polaron Equipment, Walford, UK). Epidermal cells wereobserved with an XL30 Philips SEM (Eindhoven, Holland)
118 Inflorescence and bunch architecture Australian Journal of Grape and Wine Research 10, 116–124, 2003
with optional field emission gun scanning. A total of 12images (3 rachises from separate vines per cultivar and 4images per rachis) were analysed and used for calculatingaverage epidermal cell area for each cultivar.
Fluorescence microscopyRachises were fixed in FAA, transferred to 80% ethanoland dehydrated as described above for SEM. Dehydratedinternodes were bisected longitudinally by hand using arazor blade and stained with 0.5% safranin O in methylsalicylate overnight. Sections were mounted in freshmethyl salicylate, covered with glass coverslips andobserved under UV light using a fluorescence Axioplanmicroscope (Zeiss, Germany) with a 365–400 nm filter set.A total of 18 images (3 rachises per cultivar and 6 imagesper rachis) were analysed and used for calculating theaverage parenchyma cell area for each cultivar. Both SEMand fluorescent microscope images were printed and thenthe number of cells and cell area were calculated.
DNA extraction and quantitative measurementBunches were collected at harvest for DNA extraction.The first (longest) internode of each rachis (see Figure 3)was collected and immediately frozen at –80ºC. Frozensamples were ground to a fine powder using a cold coffeegrinder in the presence of liquid nitrogen and stored at–80ºC. DNA extraction was carried out on duplicate 1 gsamples (weighed to an accuracy of ± 10 mg) based on themethod described by Thomas et al. (1993). The DNA con-tent was quantified using a multi-level fluorimetrycounter Wallac1420 (Wallac, Finland) in the range485–535 nm. DNA concentrations were determined induplicate by reference to a linear regression between three
Lambda DNA standards (1 ng, 5 ng and 10 ng).
Estimation of cell numberFor estimation of cell number in a volume unit of 1 cubiccentimetre, five first internodes of a rachis from each cul-tivar (Riesling, Chardonnay, Exotic and Sultana) weremeasured (length and diameter) and weighed. Volume ofinternodes was calculated using the standard cylinder vol-ume formula: Vcyl = lπr2, where l is length and r is radius.Total DNA was extracted from 1 g of internodes and theamount of DNA in a volume unit (1 cubic centimetre) wascalculated. To estimate the number of cells in this volume,the total amount of DNA was divided by the DNA in onenucleus (one cell), where one nucleus of CabernetSauvignon has a haploid (1C) DNA value of 0.53 ± 0.028pg (Thomas et al. 1993). Therefore, the amount of DNA inone diploid cell (2C) was estimated as 1.06 ± 0.056 pg.
Statistical analysisData are presented in tables and graphs as the mean ±standard error. For significance determination the datawere analysed using ANOVA with Fisher’s protected LSD(GenStat-6 software) or a t-test (GraphPad). Appropriategraphs and curves were produced using SigmaPlot 7.101software.
Results
Phenotypic characters measured at floweringSignificant differences (P < 0.001) were only observedbetween both tight (Riesling, Chardonnay) and loose(Exotic, Sultana) cultivars for inflorescence length andthe number of nodes (branches) per rachis (Table 1).Interestingly, a significant difference was also observed
Table 1. Comparison of phenotypic characters at anthesis. Traits labelled (*) show significant differences (P < 0.001)between cultivars with tight and loose inflorescences. Significant differences (P < 0.001) between cultivars are labelled(a), (b) and (c). Data shown are the mean ± standard error calculated from measurements of 50 shoots and 20 inflo-rescences per cultivar at anthesis. Significance was determined using an ANOVA test with Fisher’s protected LSD.
Riesling Chardonnay Exotic Sultana
Shoot characters (50 shoots, Groups A and B)
Total length (mm) 993 ± 32 976 ± 21 1055 ± 45 1077 ± 56Total number of nodes 18.2 ± 1.2 17.3 ± 1.1 18.4 ± 1.2 19.7 ± 1.5Node position of basal inflorescence* 3.5 ± 0.5a 4.0 ± 0.4a 6.0 ± 0.9b 6.0 ± 0.8b
1 P-internode is defined as that part of the inflorescence stem between the attachment of the inflorescence to the shoot and 1st inflorescence branch. 2 Floral group describes a group of flowers on a branchlet where the pedicels were separated by less than an inflorescence internode distance of approximately
2–3 mm.
Shavrukov, Dry & Thomas Inflorescence and bunch architecture 119
between tight and loose bunch cultivars in the position ofthe basal inflorescence on the shoot as well as the lengthof tendrils. Riesling and Chardonnay consistently pro-duced the basal inflorescence at shoot node position 3 or4, whereas Exotic and Sultana did not produce an inflo-rescence until node 6. Tendrils were significantly longer inExotic and Sultana in comparison with Riesling andChardonnay. Some other measured traits were differentbetween individual cultivars but not between the looseand tight classes (Table 1).
Inflorescence length was also measured for each cul-tivar from budburst until berry maturation. There was amajor difference in the rate of increase of inflorescencelength between cultivars with tight inflorescences(Riesling, Chardonnay) and loose inflorescences (Exotic,Sultana) during the period of bunch development prior toanthesis (Figure 1). However, after anthesis, there wereonly minor increases in inflorescence (bunch) length in allcultivars.
One of the major differences between tight and looseinflorescence bunches was the rate of bunch elongationprior to anthesis (Table 1, Figure 1). There were signifi-cantly more nodes per rachis between tight and loosebunch cultivars. However, the difference in total inflores-cence length was not simply due to the presence of morenodes per bunch. More importantly, in terms of buncharchitecture, there is a significant difference in the lengthof each equivalent internode within the bunch betweentight and loose bunch cultivars (Figure 2). Figure 2a showsa comparison of the mean internode length, for each indi-vidual internode, along the rachis of Riesling and Exotic.Both cultivars show a high degree of variability, whichresults from a pattern of alternating internode lengths(long-short-long-short- etc.) as illustrated in Figure 3. Byreplotting the data in Figure 2a, as the sum of the length
of two consecutive internodes, this variability is reducedand the difference in mean internode length becomesobvious (Figure 2b). Thus, one of the major differences inbunch architecture between tight and loose bunch culti-vars is rachis internode length.
Phenotypic characters measured at bunch maturityThe effect of various inflorescence characters on buncharchitecture was most apparent just prior to harvest whenthe berries had reached full size. Figure 4 illustrates theclear differences in bunch architecture between tightbunch cultivars Riesling and Chardonnay and the loosebunch cultivars Exotic and Sultana. However, it is difficultto estimate the relative amount of free space within thesedifferent bunches using visible criteria, and especiallygiven the differences in rachis length and berry sizebetween the different cultivars. In order to obtain a
Figure 1. Inflorescence growth in four cultivars during the2001–2002 season. The accordance between dates of measurementand development (modified E-L stages) are as follows: 19.09. –Stages 8–9 (first leaves are separated); 15.10. – stages 16–17 (10–12leaves are separated); 31.10. – stages 22–23 (near full-bloom); 03.12.– stages 30–31 (berries corn or pea-size); and 02.01. – stages 34-35(berries begin to soften, colour and enlarge). The arrow (↓) indicatesflowering date (50% capfall).
Figure 2. Comparison of rachis internode length in grapevinecultivars Riesling and Exotic at flowering. (a) Length of individualinternodes. (b) Combined length of two consecutive internodes in therachis of the same inflorescence. The mean internode lengths (±standard error) at each internode from the top of the bunch (P-internode) to the bottom node were calculated from 20 bunchesharvested at 50% capfall. For all data present in b, significantdifferences (t-test; P < 0.001) were observed between Riesling andExotic.
a
b
Dates of measurement
Inflo
resc
ence
leng
th (m
m)
Leng
th o
f int
erno
de in
inflo
resc
ence
(mm
)Le
ngth
of i
nter
node
s in
inflo
resc
ence
(mm
)
Number of internode inflorescences
Number of internode inflorescences
RieslingExotic
RieslingExotic
ExoticSultanaChardonnayRiesling
120 Inflorescence and bunch architecture Australian Journal of Grape and Wine Research 10, 116–124, 2003
quantitative measure of the difference in bunch free spacebetween these different cultivars, a measurement definedas ‘per cent of bunch openness’ was developed using acombination of morphological bunch volume and mea-sured real bunch volume, based on Archimedes law ofhydrostatics as described in the ‘Materials and methods’section. Figure 5 shows the range of bunch openness val-ues estimated for twenty bunches of each cultivar sampledprior to harvest. The mean per cent bunch openness val-ues calculated for each cultivar were: Riesling, 2.1 ± 1.2;Chardonnay, 3.3 ± 1.9; Exotic, 17.0 ± 3.2 and Sultana,19.6 ± 4.3. These values confirmed the differences inbunch openness observed visually (Figure 4).
Another character that may significantly influencebunch openness is the number of flowers and/or berriesset per unit of rachis length. Cultivars that produce eithera low number of flowers and/or have reduced fruit setmay also produce a bunch with more free space inde-pendent of total rachis length. Table 2 summarises datacollected for Riesling, Exotic and Sultana, to assess the
influence of these inflorescence characters on buncharchitecture. All three cultivars showed a similar rela-tionship between flower number per inflorescence andberry number per bunch, indicating that bunch opennesswas not connected with berry set. Figure 6 compares totalnumber of berries per bunch at harvest with the inflores-cence length measured at anthesis. For bunches with thesame total number of berries, the length of the inflores-cence is markedly longer in Exotic bunches than inRiesling bunches, suggesting that flower/berry number isnot a major cause of the inflorescence length differences.
Microscopic analysis of internode cell structureConsistent with intuition, the data presented above pro-vide strong evidence to indicate that the major trait deter-mining grape bunch openness is the rachis internodelength. Differences in values for internode length could
Figure 3. Morphology of a typical graperachis at harvest. Elements designated asfollows: P – P-internode, 1 – first internode inrachis, 2 – second internode in rachis, 3 –third internode, etc.
Figure 4. Comparison of bunch architecture in fully developed bunches of Riesling (R),Chardonnay (C), Exotic (E) and Sultana (S).
Figure 5. Bunch openness in grapevine cultivars. Per cent of bunchopenness (airspace within the bunch) was calculated for twentymature bunches from each cultivar (Group A) as described in the‘Materials and methods’.
Table 2. Comparison of mean flower and berry numberper bunch. Flower number was estimated by collection offlower caps as described in the ‘Materials and methods’.Mean values (± standard error) were calculated frommeasurements collected from 30 bunches.
Number of flowers Number of berries Per centper inflorescence per bunch at harvest berry set
Shavrukov, Dry & Thomas Inflorescence and bunch architecture 121
arise from either difference in the rate of cell divisionand/or cell elongation during inflorescence development.
To address this issue, scanning electron microscopy(SEM) was used to determine the size of epidermal cells inthe central rachis of bunches at harvest in the four culti-vars. Figure 7 shows a comparison of the epidermal cellsfrom contrasting bunch types – Riesling and Exotic. Theirregular shape of the grape epidermal cells did not permitan accurate estimation of the length and the width ofcells. Therefore, average epidermal cell area was calculat-ed by counting the number of cells within a defined imagearea. The rachis internodes of compact bunch typesRiesling and Chardonnay were found to have significant-ly (P < 0.001) smaller epidermal cells than the loose bunchcultivars Exotic and Sultana (Table 3).
Similar results were also obtained by estimating aver-age parenchyma cell area using fluorescence microscopy(Table 3). The parenchyma cells of the rachis internodes ofExotic and Sultana were significantly larger than thosefound in the internodes of Riesling and Chardonnay.
Both SEM and fluorescence microscopy were also usedto compare cell structure of different segments of the cen-tral rachis and the internode of the first branch of therachis within each of studied cultivars to determine the
basis for the alternating pattern of internode lengths(Figure 3). The first internode, second internode andrachis branches showed no significant differences in cellsize within each of the four investigated cultivars (data notshown). This indicates that observed differences in thelength of rachis internodes and branches within the samebunch result from differences in cell number.
Estimation of internode cell number by DNA determinationCell number in grape tissues can be indirectly estimated bymeasuring the total DNA content of the tissue (Ojeda et al.1999). Quantitative measurements of DNA content per 1 gof internode tissue of the first rachis internode of the four
Table 3. Comparison of the mean area of epidermal andcentral parenchyma cells of the rachis internode of dif-ferent grapevine cultivars. Epidermal cells were analysedby SEM and central parenchyma cells were analysed usingfluorescence microscopy as described in ‘Materials andmethods’. Significant difference (P < 0.001) within a col-umn was determined using an ANOVA test with Fisher’sprotected LSD.
Epidermal cell area Parenchyma cell area(µm2) (µm2 ×× 102)
Riesling 239.8 ± 27.0a 41.1 ± 6.7a
Chardonnay 251.3 ± 26.5a 47.5 ± 6.1a
Exotic 326.8 ± 25.2b 63.9 ± 9.1b
Sultana 316.9 ± 24.4b 59.5 ± 6.2b
Figure 6. Relationship between inflorescence length at anthesis andberry number for the grape cultivars Riesling and Exotic.
Figure 7. Scanning electron microscope images of epidermal cells inthe first rachis internode of Riesling (a) and Exotic (b). Scale barindicates 50 µm for both images.
Table 4. (A) DNA content and estimated cell number ofthe first rachis internode. Total DNA was extracted frominternode tissue and the concentration measured asdescribed in ‘Materials and methods’. Mean values (±standard error) were calculated from duplicate DNAextractions. Significance (P < 0.001) was determined usingan ANOVA test with Fisher’s protected LSD. (B) Number of cells in a 1 cubic centimetre volume of arachis internode was calculated based on nuclei quantifi-cation as described in ‘Materials and Methods.
A BTotal DNA (µg) Estimated number
extracted from 1 g of cells in 1 cm3
of sample of sample
Riesling 2.68 ± 0.31a 4.72 × 106
Chardonnay 2.27 ± 0.18a 3.58 × 106
Exotic 1.33 ± 0.11b 1.86 × 106
Sultana 1.56 ± 0.12b 2.14 × 106
a
b
Inflorescence length (mm), anthesis
Num
ber o
f ber
ries,
har
vest
RieslingExotic
122 Inflorescence and bunch architecture Australian Journal of Grape and Wine Research 10, 116–124, 2003
different cultivars are presented in Table 4(A). Signifi-cantly higher (P < 0.001) levels of DNA were extracted per1 g of rachis internode from the Riesling and Chardonnaythan from Exotic and Sultana. Similar results wereobtained when the estimated number of cells in a unit vol-ume (1 cubic centimetre) was calculated using publishedinformation (Thomas et al. 1993) on the amount of DNA(2C = 1.06 ± 0.056 pg) in the nuclei of cells of CabernetSauvignon (Table 4B). The lower DNA content and cal-culated nuclei number for Exotic and Sultana compared toRiesling and Chardonnay indicates that rachis cell sizemakes a major contribution to inflorescence length.
These data are in agreement with the microscopicanalysis where Riesling and Chardonnay rachis cells weresignificantly smaller in area and size. Thus, small cell sizein a rachis was associated with a higher number of cells(Riesling and Chardonnay) and a larger cell size was asso-ciated with fewer cells per unit rachis (Exotic andSultana).
DiscussionRiesling and Chardonnay, two important cultivars for pre-mium white wine production in cool climate areas, areprone to rain damage during the harvest period. Bothcultivars also have very tight bunches at maturity, somuch so that berry shape may deform from round toangular during ripening due to inter-berry contact. Thecombination of tight bunch structure and moist climaticconditions results in a high incidence of botrytis bunch rotin the field, which has serious implications for wine qual-ity. In many situations, Botrytis infection may be ade-quately controlled by the application of fungicides.However, if infection occurs close to harvest it may not bepossible to apply fungicides because of the minimumresidue limits required for many markets, and spray pen-etration is always a problem with tight bunches.
A number of studies have been carried out whichdemonstrate that a significant reduction in botrytis bunchrot infection can be achieved by physically or chemicallymodifying bunch compactness. Treatments have includedthe application of gibberellic acid at flowering (Weaver etal. 1962, Hopping 1975, Ari et al. 1996), hand thinning(Barbetti 1980) and specific vine management systems(Zabadal and Dittmer 1998, Smithyman et al. 1998).However, in many of these treatments, bunch compact-ness was reduced through decreases in fruit set. While thishas significant benefits in terms of reduced susceptibilityto bunch rot, it imposes a significant yield penalty. Apreferable approach would be to use a genetic strategy toreduce bunch compactness in susceptible cultivars byaltering bunch architecture. The aim of this work there-fore has been to identify phenotypic characters of grapebunch architecture that contribute to differences in bunchcompactness. To avoid any environmental influences onbunch architecture, all four cultivars were analysed atthe same location and under the same conditions.
Comparison of a range of phenotypic characteristicsbetween tight and open bunch cultivars at anthesis indi-cated that only two inflorescence characters, (1) totalinflorescence length and (2) number of nodes per rachis,
were significantly different (Table 1). Marked differencesin the rate of inflorescence primordium growth wereobserved in the earliest stages of development (modifiedE-L stages 8–9) (Coombe 1995) and continued up untilanthesis (Figure 1). Rachis elongation following anthesiswas minor. This indicates that the genetic program con-trolling inflorescence growth and elongation occurs at theearly stages of bunch development. In contrast, there wasno significant difference in shoot length between the dif-ferent cultivars, indicating that endogenous factors influ-encing the rate of inflorescence growth may be organspecific and not associated with vegetative growth of theshoot and the apical meristem.
At bunch maturity there are clear visual differencesbetween the tight and open bunch cultivars (Figure 4).However, it is important to be able to quantify these dif-ferences. To this end, we developed a rapid and inexpen-sive method for estimating per cent bunch openness(Figure 5), which can be used to assess the segregation ofthis character in grapevine breeding populations. On aver-age, free space between berries within Exotic and Sultanabunches was found to be 5- to 9-fold more than berries ofRiesling and Chardonnay bunches, which would have amajor impact on the degree of airflow between the berries.Analysis of flower number at anthesis (Table 2) and berrynumber at maturity (Figure 6) indicated that the observeddifferences in bunch openness could not be accountedfor by differences in berry number at maturity. Thus itwould appear, for the cultivars examined in this study,that the major factor determining bunch openness is therate of elongation of the rachis internodes during theperiod of bunch development prior to anthesis.
Differences in internode length could result from dif-ferences in (1) cell elongation (expansion); (2) cell divisionor (3) both. Microscopic analysis of both external (epi-dermis) and internal (parenchyma) cells of the rachisinternodes demonstrated the mean cell area to be signif-icantly larger in cultivars with longer internodes (Table 3).Measurements of cell number based on DNA content indi-cated that the shorter rachis internodes of Riesling andChardonnay actually contained more cells per unit vol-ume of rachis tissue than the longer internodes of Exoticand Sultana (Table 4). Therefore, we conclude that cellexpansion is the major factor responsible for the increasedelongation of rachis internodes in the loose bunch culti-vars Exotic and Sultana.
The genetics of inflorescence development have beenstudied in the model plant Arabidopsis. Some Arabidopsismutants, such as, crm (corymbosa), shi (short internodes),fir (fireworks) and cif (compact inflorescence), have short-er inflorescence cell size in comparison with wild-typeplants (Komeda et al. 1998, Fridborg et al. 1999,Nakamura et al. 2000, Goosey and Sharrock 2001). Incontrast, another mutant, sturdy, with a short but thickinflorescence, had increased cell number with cell sizeunaffected (Huang et al. 2001), while the inflorescence ofthe mutant bp (brevipedicellus) was characterised by bothreduced cell size and cell number, leading the authors tothe conclusion that both mechanisms (cell size and cellnumber) were involved in inflorescence internode elon-
Shavrukov, Dry & Thomas Inflorescence and bunch architecture 123
gation (Douglas et al. 2002, Venglat et al. 2002). Thus, allvariants of altered inflorescence elongation are present inArabidopsis mutants and by implication similar variationshould exist in V. vinifera.
In conclusion, the results of this study indicate that amajor character determining bunch compactness ingrapevine is rachis internode length, as determined bythe rate of internode cell expansion prior to anthesis.Further research to determine the genetic basis for intern-ode elongation in grape inflorescences is under way toidentify the gene(s) controlling bunch architecture.
AcknowledgementsThis work was supported in part by the CommonwealthCooperative Research Centre Program, and specificallythe Cooperative Research Centre for Viticulture (CRCV)and the Grape and Wine Research and Development Cor-poration (GWRDC). We wish to thank Stuart McClure,Susan Johnson and Meredith Wallwork for microscopyassistance, Don Mackenzie for technical assistance andChris Davies for helpful discussions. We are also verygrateful to the Editor and anonymous reviewers for com-ments and suggested improvements to the manuscript.
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Manuscript received: 22 September 2003Revised manuscript received: 11 February 2004
