Comparison of ploidy level screening methods in

watermelon: Citrullus lanatus (Thunb.)

Matsum. and Nakai

N. Saria,*, K. Abaka, M. Pitratb

aDepartment of Horticulture, Faculty of Agriculture, C,ukurova University,

01330 Adana, TurkeybUnite de GeÂneÂtique et d'AmeÂlioration des Fruits et LeÂgumes,

Institut National de la Recherche Agronomique,

BP 94, 84143 Montfavet Cedex, France

Accepted 6 May 1999

Abstract

Direct (chromosome counting) and indirect (flow cytometry, stomatal size, chloroplast number of

the guard cells and morphological observations) methods were tested in order to determine the

ploidy levels of haploid and diploid watermelon plants of the cultivars Sugar Baby and Halep

Karasi. The results revealed that all the techniques tested can be used successfully. It was

determined that while counting chromosomes is cumbersome, producing plants for morphological

observations requires a long time and flow cytometry is expensive and labour intensive. On the

other hand, measurement of stomata and chloroplast counting methods are simple to use are less

labour intensive and hence can be considered a practical alternative to the others. The data for the

stomata in the haploids were length, 17±18 mm; diameter, 10±12 mm and number of chloroplasts of

the guard cells, 6±7 and in the diploids they were 23±24 mm, 18 mm and 11±12, respectively.

# 1999 Elsevier Science B.V. All rights reserved.

Keywords: Citrullus lanatus; Haploid; Diploid; Ploidy determination

1. Introduction

Plant improvement programs always need more and more effective breedingtools, among which production of doubled haploids is of great interest for rapidly

Scientia Horticulturae 82 (1999) 265±277

* Corresponding author. Tel.: +90-3223386497; fax: +90-3223386388

E-mail address: nesari@pamuk.cc.cu.edu.tr (N. Sari)

0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 7 - 1

producing pure lines conferring time saving (Dickson and Wallace, 1986) andincreased efficiency (Griffing, 1975; Gallais, 1978; Snape, 1982; Demarly andSibi, 1989), in the improvement of cultivars. Doubled haploids can also be used inmutation breeding of crop plants (Reinert and Bajaj, 1977).

World production of watermelon is the highest among the cucurbits(Anonymous, 1997). Since the watermelon plant is cross-pollinated, the breedingcycle lasts a long time. Consequently, to produce pure lines in the shortestpossible time span a collobarative study was set up between the Department ofHorticulture, Faculty of Agriculture, C,ukurova University, Adana, Turkey andVegetable Breeding Station of INRA-Montfavet, France to obtain irradiated-pollen-induced parthenogenetic haploid embryos which were raised intocomplete haploid plants (GuÈrsoÈz et al., 1991; Sari et al., 1994).

It has long been known that in chromosome number or ploidy leveldetermination, the classic method of counting chromosomes is the most accurate,in addition to which, other methods of indirect approach can be usedsatisfactorily.

De Laat et al. (1987) proposed that chromosome counting technique requires awell equipped laboratory and a qualified work team and some species posedifficulties that argue against the use of this technique. They considered that thechloroplast number could be an alternative approach to chromosome scoring.However, in cases where ploidy differences were low, the technique was noteffectively useful, and they suggested using the flow cytometry technique. Brownet al. (1991) compared various techniques and proposed that flow cytometry wasthe most effective one. Flow cytometry is a powerful technique for estimatingplant nuclear DNA content because it permits sensitive measurements offlorescence intensity of large numbers of stained nuclei within seconds(Arumuganathan and Earle, 1991). This technique was used successfully inmelon (Cuny et al., 1992; Abak et al., 1996). Dore (1986) studied Brussels sproutto determine if stomata length measurements could be an alternative method toclassical chromosome scoring using haploid, diploid and triploid plants andreported that the stomata length of Brussels sprout was 14 mm in haploid plants,20 mm in diploid plants and 24 mm in triploid plants. It was concluded thatstomata length could be an alternative method to chromosome scoring. Rode andDumas de Vaulx (1987) measured the stomata length of carrot to determineploidy level. They reported that the stomata length was 15.2 and 19.7 mm inhaploid and diploid plants, respectively. Brown et al. (1991) counted thechloroplast numbers in haploid sugarbeet obtained via in vitro gynogenesis andcompared them to diploid sugarbeet plants. They noted that there were 16chloroplasts in diploid plants (2n � 2x � 18) while in haploids (n � x � 9) it wasabout 9. These researchers also reported that chloroplast number was a goodcriterion to determine ploidy level of sugarbeet in its early developmental stages.In pepper also, stomatal density and especially the number of chloroplasts in

266 N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277

guard cells seem to be reliable characteristics for the estimation of ploidy level(Abak et al., 1998).

In the present study, to determine the ploidy level of watermelon, directmethods, i.e. chromosome counting, and indirect methods, i.e. flow cytometry,stomata size, chloroplast number and plant morphology, were compared.

2. Material and methods

In this study, haploid plants of the cultivars Sugar Baby and Halep Karasiobtained by haploid parthenogenesis after pollination with gamma irradiatedpollen from a 60Co source (Sari, 1994) were used together with diploid plants ofthe same cultivars.

Sugar Baby and Halep Karasi are two old and open pollinated cultivars used fora long time in Turkey. Sugar Baby has medium sized, round fruit. Seeds are smalland light brown. Biomass is weak, leaf colour is dark green. It is an early cultivarand time for ripening is between 33 and 36 days after flowering. Flower type ismonocious. Halep Karasi is an old cultivar introduced from Allepo (Syrian) toTurkey. It has medium sized, round fruits. Seeds are big, black and blackspeckled. Biomass is very strong, leaf colour is dark green. It is a late cultivar.Time from flowering to harvest is 42±48 days. Flower type is andromonocious.

Different methods such as chromosome counting, flow cytometry, determina-tion of stomata size, chloroplast number in guard cells and morphologicalobservations were used to determine the ploidy levels. While plants grown invitro conditions were used for chromosome counting and flow cytometry, plantsgrown in situ were used for determinations of stomata size and chloroplastnumber in guard cells and for morphological observations.

2.1. Chromosome counting in root tips

Chromosome counting was done using five plants of each genotype eithermultiplied by microcuttings or raised from seeds sown in vitro. The process givenbelow was used for chromosome counting in root tips:

Cutting off root tips by 1±2 cm of 8±10-day old in vitro plantlets at 10 a.m. inthe morning and rinsing with tap water to separate the remnants of agar,Keeping the roots in alfa-monobromonaftalen solution for three hours at roomtemperature,Draining off the alfa-monobromonaftalen solution and rinsing the roots threetimes with distilled water,Keeping the roots in glacial acetic acid for 1/2 h,Draining off the glacial acetic acid, rinsing the roots three times with distilledwater and then keeping them in tap water for 5 min,

N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277 267

Hydrolysis of the root tips in 1 N HCl at 608C for 8±10 min,Rinsing the roots for three times with distilled water, drying the surface andstaining with Schiff's reagent (Darlington and La Cour, 1963) for 1 h,Draining off the Schiff's reagent and keeping the roots in tap water for 10 min,Crushing the roots in 1% aceto carmine solution.

Prepared samples according to the above procedure were visualised by a lightmicroscope magnified by 20 � 10 and 40 � 10 and the chromosome countingand photographing were done with a 100 � 10 objective and eyepiececombination, respectively.

2.2. Flow cytometry

This part of the work was carried out at the INRA-Plant Breeding Station inClermond-Ferrand of France. In this study, a Partec CII Chemunex flowcytometer was used. A piece of leaf approximately 1 cm2 in size from eachgenotype was cut into pieces using 1 ml of buffer prepared by the Chemunex SAcompany. Five plants from in vitro material of each genotype were used. Then thesuspension was filtered into small tubes through a Sartorius Minisart NMLmicrofilter with a permeability of about 0.2 mm. For the coloration of thesesamples they were put into the flow cytometer after addition of one drop of DAPIand the DNA histograms were created. The cytometer was arranged using Lolium

perenne L., then calibrated with a suspension of diploid control watermelon(2n � 2x � 22).

2.3. Stomata size and chloroplast number of guard cells

For this aim, 10 haploid plants per genotype were transferred from tubes to pots25 cm in width and 40 cm depth. Simultaneously 10 seeds of diploid plants pergenotype were sown into pots as controls. From the top of both diploid andhaploid plants, seventh or eighth leaves were cut and taken into the laboratory. Acertain amount of epidermal cells were obtained from the underside of each leafby tearing with a nail and were then rubbed on to a microscope slide by a razorblade. In order to measure stomata diameter and length, the under surface of aleaf was placed onto a microscope slide after the addition of one drop of tapwater, the cover glass was then closed (DoreÂ, 1986). In the chloroplast scoringmethod a 1% AgNO3 solution was used instead of tap water (Rousselle, 1992).

Under a light microscope, the diameter and length of 4 stomata per leaf weremeasured magnifying by 40 � 10 objective and oculars, respectively. Obtainedvalues were multiplied by 2.439, a coefficient obtained by adjusting the ocularmicrometer, so as to obtain the true length of the stomata. The chloroplast numberin each of the two guard cells of stomata was scored under a light microscope.

268 N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277

Measurements and scoring were performed for 10 leaves of 10 plants pergenotype.

For those three parameters, statistical analysis was carried out; the split plotexperimental design was applied, and the average values were then compared byTukey test.

2.4. Morphologic development

For the observations of morphological development, 10 haploid plantlets fromeach cultivar were transferred from tubes to pots in greenhouses. Simultaneously,10 seeds of diploid plants per genotype were sown into pots and then thefollowing observations were performed. Because only 10 haploid and diploidplants of each genotype were available, replicates were not included in theexperiment hence statistical analysis was not carried out only standard deviationswere calculated.

The branching period (days): It is the period between the day that the diploidplant seeds were sown and the haploid plantlets were transferred into pots and theday that the plants had branches 5±10 cm in length.

The first male flowering period (days): For diploid plants, it is the durationfrom sowing to the date that the first male flower opened on the main branch andfor in vitro plants, the time from transplanting to the blooming of the first maleflower.

The node on which the first male flower bloomed: The rank of the node onwhich the first male flower bloomed was recorded except for the cotyledons(cotyledons were considered as beginning).

The first female flowering period (day): For diploid plants, it is the time fromsowing to the date that the first female flower opened on the main branch and forin vitro plants the time from transplanting to the day the first female floweropened.

The node on which the first female flower is bloomed: The rank of node onwhich the first female flower bloomed was recorded except for the cotyledons(cotyledons were considered as beginning).

Stem length (cm): The main stem length above the cotyledons was measured onthe first day that a female flower bloomed.

Diameter of the main stem (mm): Stem diameter was measured at the cotyledonlevel by a digital calipers which was accurate to �0.1 mm.

Leaf area (cm2): From the top, the eighth leaves of all the haploid and diploidplants were cut and leaf areas were measured on a automatically recordingmachine according to Beadle (1985) principles about two months after planting.The average leaf area was calculated by dividing the total leaf area by the leafnumber of each genotype.

N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277 269

3. Results

3.1. Results of chromosome counting in root tips

The results of the chromosome scoring of plantlets that were raised in agarmedium showed that the plantlets originated from embryos obtained throughpollination by irradiated pollen were haploid (n � 11) (Fig. 1). In roots of haploidplants, doubling of chromosome number may occur spontaneously. While fromthe same root tip, a few of the cells were diploid (2n � 2x � 22), most werehaploid (n � 11) (Fig. 2).

3.2. Results of flow cytometry

The results of the DNA survey by flow cytometry are shown in Table 1. It wasfound that 69±74% of haploid plant cells were haploids. However, rapidchromosome number doubling was observed, as in the case of root tip cells. In thehaploid plants, 21±27% and 4±5% of the cells were found diploid and tetraploid,respectively, as determined by flow cytometry. For the diploid plants which wasused to calibrate the machine, haploid cells were not observed; but, 75.7% and24.3% of the cells were diploid and tetraploid, respectively. The results of flowcytometry related to haploid and diploid watermelon plants are presented inFig. 3.

Fig. 1. Chromosome number of haploid plants (n � 11) obtained through pollination by irradiated

pollen.

270 N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277

3.3. Results of stomata size and chloroplast number of guard cells

The results of the study which was performed to determine if stomata size andchloroplast number of epidermis cells could be reasonable criteria to determinethe ploidy level, are presented in Table 2. In haploid and diploid watermelonplants, the stomata length and chloroplast number were found to be significant at1% level. The differences within the genotypes were not significant, howeverploidy level difference was significant. In diploid plants the stomata lengthreached 24.0 mm while in haploids it was about 17.5 mm. As for stomatadiameter, the difference between haploid and diploid plants was determined to be

Fig. 2. Chromosome doubling in root tips of haploid plants obtained through pollination by

irradiated pollen.

Table 1

Frequency of leaf cells in various DNA contents of haploid and diploid plants

Genotypes Number of

cells counted

Ploidy frequency of leaf cells

c 2c 4c

Sugar Baby (haploid) 3606 69.0 26.6 4.4

Halep Karasi (haploid) 2670 74.3 20.8 4.9

Means of haploid 3138 71.7 23.7 4.7

Sugar Baby (diploid) 2898 ± 79.3 20.7

Halep Karasi (diploid) 1943 ± 72.0 28.0

Means of diploid 2421 ± 75.7 24.3

N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277 271

significant. In diploids the average stomata diameter was about 18.2 mm while inhaploids it was about 11.3 mm. In stomata of haploid plants, a total of 6±7chloroplasts were found but in diploids it was about 11±12. In Figs. 4 and 5photos of stomata of epidermis cells related to haploid and diploid plants,respectively, are shown.

3.4. Results of morphologic development

In Table 3, the results related to haploid and diploid plants of Halep Karasiand Sugar Baby cultivars are given; branching, the first male and female

Fig. 3. The results of flow cytometry related to haploid (left) and diploid (right) plants.

Table 2

Stomata length (mm), diameter (mm) and chloroplast number of haploid and diploid watermelon

plants

Genotypes Stomata length

(mm)

Stomata diameter

(mm)

Number of

chloroplast

Sugar Baby (haploid) 17.3 12.1 6.0

Halep Karasi (haploid) 17.6 10.4 7.0

Means of haploids 17.5b 11.3b 6.5b

Sugar Baby (diploid) 23.7 18.2 10.6

Halep Karasi (diploid) 24.2 18.1 12.1

Means of diploids 24.0a 18.2a 11.4a

Tukey (1%)* 2.6 2.7 1.4

* Different letters show the significant differences between the average values according to the

Tukey significance test at the 1% level.

272 N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277

flowering time, nodes on which the first male and female flowers bloomed,length and diameters of the main stem on the first female flowering date and leafarea.

Diploid plants branched in 64 days while haploids branched in 75 days. Indiploid plants the first male flower bloomed on the 7th or 9th node, the femaleflower bloomed on the 10th or 20th node depending on the genotypes, but in

Fig. 4. The stomata and chloroplasts of haploid plants.

Fig. 5. The stomata and chloroplasts of diploid plants.

N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277 273

Table 3

The results of growing and development parameters in haploid and diploid plants

Genotypes BP (day) FMFP (day) FMFN FFFP (day) FFFN SL (cm) MSD (mm) LA (cm2) Pollen

production

Sugar Baby (haploid) 77.0 � 0.0 55.0 � 0.0 21.0 � 0.0 52.0 � 0.0 18.0 � 0.0 37.0 � 0.0 1.15 � 0.0 4.5 � 0.1 ÿHalep Karasi (haploid) 73.8 � 4.7 68.0 � 7.6 26.2 � 4.0 66.8 � 10.2 29.3 � 2.6 67.3 � 9.3 1.73 � 0.4 10.1 � 0.0 ÿSugar Baby (diploid) 66.3 � 6.2 64.3 � 10.2 6.8 � 1.4 69.4 � 3.9 10.3 � 2.9 51.6 � 10.5 3.92 � 0.9 39.1 � 0.2 �Halep Karasi (diploid) 62.2 � 4.7 79.4 � 11.0 8.7 � 1.7 82.1 � 11.6 19.9 � 3.8 100.0 � 18.5 5.83 � 0.5 49.4 � 0.3 �Mean of haploids 75.4 61.5 23.6 59.4 23.6 52.1 1.44 9.0

Mean of diploids 64.3 71.0 7.5 76.8 15.5 74.0 4.81 44.3

Mean of Sugar Baby 71.7 60.0 13.9 60.7 14.2 44.3 3.11 21.8

Mean of Halep Karasi 68.0 73.7 17.5 74.5 24.6 83.7 3.78 29.8

BP: branching period; FMFP: first male flowering period; FMFN: first male flowering node; FFFP: first female flowering period; FFFN: first female

flowering node; SL: stem length; MSD: main stem diameter; LA: leaf area (cm2).

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weakly developed haploid plants, the male or female flowers bloomed on the 23rdor 24th node. In addition, the male and female flowers were smaller and maleflowers did not produce pollen in haploid plants. Also in haploid plants, plantlength and stem diameter were measured and found to be lower than these ofdiploids. In haploid plants of the cultivar Sugar Baby, the area of individual leaveswas 5 cm2 while it was 10 cm2 in the cv. Halep Karasi, but in the diploids it was5±10 times more; i.e. 40 and 50 cm2 for the cultivars mentioned, respectively(Fig. 6).

4. Discussion and conclusion

To distinguish the haploid watermelon plants from the diploids, indirecttechniques were considered as an alternative to the classical method ofchromosome counting. When the plantlets were in tubes i.e. young and small,DNA surveys using 1 cm2 leaf samples could easily be employed to determine theploidy level and flow cytometry was adapted for use on the watermelon crop. But,this technique requires an expensive flow cytometer equipment.

This study showed that stomata measurements and chloroplast scoring methodscould be used in watermelon as in Brussel sprout (DoreÂ, 1986), carrot (Rode andDumas de Vaulx, 1987), sugarbeet (Brown et al., 1991) and pepper (Abak et al.,1998). In haploid watermelon plants the stomata length, stomata diameter andchloroplast number were found to be 17±18, 10±12 and 6±7 mm, respectively,while in diploids they were about 23±24, 18 and 11±12 mm, respectively.

Fig. 6. Leaves of haploid (left) and diploid (right) Sugar Baby plants.

N. Sari et al. / Scientia Horticulturae 82 (1999) 265±277 275

Distinguishing the plants according to the morphological development was alsoa useful approach, but waiting until flowering was time consuming.

The results of this study indicated that the ploidy level would be detectedsuccesfully by the four methods mentioned. Among them, chromosome scoringgave perfect results but, it was time consuming and season dependent. Flowcytometry was useful, but needs specific equipment. However, the publishedplant nuclear isolation protocols worked well for watermelon nuclei isolated froma young in vitro true leaf. To distinguish morphologically the haploids fromdiploid plants, the plants should be grown under greenhouse conditions and theyshould be observed at a certain period, thus, it is time consuming. Ploidy level canbe determined in a short time by examining epidermal tissue from the undersurface of leaves without the requirements of specific equipment and highexpenditure.

Acknowledgements

The authors thank Dr. R. Dumas de Vaulx and Dr. G. Guy from INRA-Clermond Ferrand Plant Breeding Station for allowing use of the flow cytometerin this work.

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