Comparison of ploidy level screening methods in
watermelon:
Citrullus lanatus
(Thunb.)
Matsum. and Nakai
N. Sari
a,*, K. Abak
a, M. Pitrat
ba
Department of Horticulture, Faculty of Agriculture, C,ukurova University, 01330 Adana, Turkey
b
Unite 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±18mm; diameter, 10±12mm and number of chloroplasts of
the guard cells, 6±7 and in the diploids they were 23±24mm, 18mm 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 breeding tools, among which production of doubled haploids is of great interest for rapidly
* Corresponding author. Tel.: +90-3223386497; fax: +90-3223386388
E-mail address:nesari@pamuk.cc.cu.edu.tr (N. Sari)
producing pure lines conferring time saving (Dickson and Wallace, 1986) and increased efficiency (Griffing, 1975; Gallais, 1978; Snape, 1982; Demarly and Sibi, 1989), in the improvement of cultivars. Doubled haploids can also be used in mutation 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 breeding cycle lasts a long time. Consequently, to produce pure lines in the shortest possible time span a collobarative study was set up between the Department of Horticulture, Faculty of Agriculture, C,ukurova University, Adana, Turkey and Vegetable Breeding Station of INRA-Montfavet, France to obtain irradiated-pollen-induced parthenogenetic haploid embryos which were raised into complete haploid plants (GuÈrsoÈz et al., 1991; Sari et al., 1994).
It has long been known that in chromosome number or ploidy level determination, the classic method of counting chromosomes is the most accurate, in addition to which, other methods of indirect approach can be used satisfactorily.
De Laat et al. (1987) proposed that chromosome counting technique requires a well equipped laboratory and a qualified work team and some species pose difficulties that argue against the use of this technique. They considered that the chloroplast number could be an alternative approach to chromosome scoring. However, in cases where ploidy differences were low, the technique was not effectively useful, and they suggested using the flow cytometry technique. Brown et al. (1991) compared various techniques and proposed that flow cytometry was the most effective one. Flow cytometry is a powerful technique for estimating plant nuclear DNA content because it permits sensitive measurements of florescence intensity of large numbers of stained nuclei within seconds (Arumuganathan and Earle, 1991). This technique was used successfully in melon (Cuny et al., 1992; Abak et al., 1996). Dore (1986) studied Brussels sprout to determine if stomata length measurements could be an alternative method to classical chromosome scoring using haploid, diploid and triploid plants and reported that the stomata length of Brussels sprout was 14mm in haploid plants, 20mm in diploid plants and 24mm in triploid plants. It was concluded that stomata length could be an alternative method to chromosome scoring. Rode and Dumas de Vaulx (1987) measured the stomata length of carrot to determine ploidy level. They reported that the stomata length was 15.2 and 19.7mm in haploid and diploid plants, respectively. Brown et al. (1991) counted the chloroplast numbers in haploid sugarbeet obtained via in vitro gynogenesis and compared them to diploid sugarbeet plants. They noted that there were 16 chloroplasts in diploid plants (2n2x18) while in haploids (nx9) it was
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, direct methods, 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 Karasi obtained by haploid parthenogenesis after pollination with gamma irradiated pollen from a60Co source (Sari, 1994) were used together with diploid plants of
the same cultivars.
Sugar Baby and Halep Karasi are two old and open pollinated cultivars used for a long time in Turkey. Sugar Baby has medium sized, round fruit. Seeds are small and light brown. Biomass is weak, leaf colour is dark green. It is an early cultivar and time for ripening is between 33 and 36 days after flowering. Flower type is monocious. Halep Karasi is an old cultivar introduced from Allepo (Syrian) to Turkey. It has medium sized, round fruits. Seeds are big, black and black speckled. 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 morphological observations were used to determine the ploidy levels. While plants grown in vitro conditions were used for chromosome counting and flow cytometry, plants grown in situ were used for determinations of stomata size and chloroplast number in guard cells and for morphological observations.
2.1. Chromosome counting in root tips
Chromosome counting was done using five plants of each genotype either multiplied by microcuttings or raised from seeds sown in vitro. The process given below 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. in the morning and rinsing with tap water to separate the remnants of agar, Keeping the roots in alfa-monobromonaftalen solution for three hours at room temperature,
Draining off the alfa-monobromonaftalen solution and rinsing the roots three times with distilled water,
Keeping the roots in glacial acetic acid for 1/2 h,
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 and staining 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 light microscope magnified by 2010 and 4010 and the chromosome counting
and photographing were done with a 10010 objective and eyepiece
combination, respectively.
2.2. Flow cytometry
This part of the work was carried out at the INRA-Plant Breeding Station in Clermond-Ferrand of France. In this study, a Partec CII Chemunex flow cytometer was used. A piece of leaf approximately 1 cm2 in size from each genotype was cut into pieces using 1 ml of buffer prepared by the Chemunex SA company. Five plants from in vitro material of each genotype were used. Then the suspension was filtered into small tubes through a Sartorius Minisart NML microfilter with a permeability of about 0.2mm. For the coloration of these samples they were put into the flow cytometer after addition of one drop of DAPI and the DNA histograms were created. The cytometer was arranged usingLolium perenne L., then calibrated with a suspension of diploid control watermelon (2n2x22).
2.3. Stomata size and chloroplast number of guard cells
For this aim, 10 haploid plants per genotype were transferred from tubes to pots 25 cm in width and 40 cm depth. Simultaneously 10 seeds of diploid plants per genotype were sown into pots as controls. From the top of both diploid and haploid plants, seventh or eighth leaves were cut and taken into the laboratory. A certain amount of epidermal cells were obtained from the underside of each leaf by tearing with a nail and were then rubbed on to a microscope slide by a razor blade. In order to measure stomata diameter and length, the under surface of a leaf was placed onto a microscope slide after the addition of one drop of tap water, the cover glass was then closed (DoreÂ, 1986). In the chloroplast scoring method 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 were measured magnifying by 4010 objective and oculars, respectively. Obtained
Measurements and scoring were performed for 10 leaves of 10 plants per genotype.
For those three parameters, statistical analysis was carried out; the split plot experimental design was applied, and the average values were then compared by Tukey test.
2.4. Morphologic development
For the observations of morphological development, 10 haploid plantlets from each cultivar were transferred from tubes to pots in greenhouses. Simultaneously, 10 seeds of diploid plants per genotype were sown into pots and then the following observations were performed. Because only 10 haploid and diploid plants of each genotype were available, replicates were not included in the experiment hence statistical analysis was not carried out only standard deviations were calculated.
The branching period (days): It is the period between the day that the diploid plant seeds were sown and the haploid plantlets were transferred into pots and the day that the plants had branches 5±10 cm in length.
The first male flowering period (days): For diploid plants, it is the duration from sowing to the date that the first male flower opened on the main branch and for in vitro plants, the time from transplanting to the blooming of the first male flower.
The node on which the first male flower bloomed: The rank of the node on which 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 from sowing to the date that the first female flower opened on the main branch and for in vitro plants the time from transplanting to the day the first female flower opened.
The node on which the first female flower is bloomed: The rank of node on which 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 on the first day that a female flower bloomed.
Diameter of the main stem (mm): Stem diameter was measured at the cotyledon level by a digital calipers which was accurate to 0.1 mm.
3. Results
3.1. Results of chromosome counting in root tips
The results of the chromosome scoring of plantlets that were raised in agar medium showed that the plantlets originated from embryos obtained through pollination by irradiated pollen were haploid (n11) (Fig. 1). In roots of haploid
plants, doubling of chromosome number may occur spontaneously. While from the same root tip, a few of the cells were diploid (2n2x22), most were
haploid (n11) (Fig. 2).
3.2. Results of flow cytometry
The results of the DNA survey by flow cytometry are shown in Table 1. It was found that 69±74% of haploid plant cells were haploids. However, rapid chromosome number doubling was observed, as in the case of root tip cells. In the haploid 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 was used to calibrate the machine, haploid cells were not observed; but, 75.7% and 24.3% of the cells were diploid and tetraploid, respectively. The results of flow cytometry related to haploid and diploid watermelon plants are presented in Fig. 3.
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 and chloroplast number of epidermis cells could be reasonable criteria to determine the ploidy level, are presented in Table 2. In haploid and diploid watermelon plants, the stomata length and chloroplast number were found to be significant at 1% level. The differences within the genotypes were not significant, however ploidy level difference was significant. In diploid plants the stomata length reached 24.0mm while in haploids it was about 17.5mm. As for stomata diameter, 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
significant. In diploids the average stomata diameter was about 18.2mm while in haploids it was about 11.3mm. In stomata of haploid plants, a total of 6±7 chloroplasts were found but in diploids it was about 11±12. In Figs. 4 and 5 photos 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 Karasi and 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
*
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 leaf area.
Diploid plants branched in 64 days while haploids branched in 75 days. In diploid plants the first male flower bloomed on the 7th or 9th node, the female flower bloomed on the 10th or 20th node depending on the genotypes, but in
Fig. 4. The stomata and chloroplasts of haploid plants.
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) 77.00.0 55.00.0 21.00.0 52.00.0 18.00.0 37.00.0 1.150.0 4.50.1 ÿ
Halep Karasi (haploid) 73.84.7 68.07.6 26.24.0 66.810.2 29.32.6 67.39.3 1.730.4 10.10.0 ÿ
Sugar Baby (diploid) 66.36.2 64.310.2 6.81.4 69.43.9 10.32.9 51.610.5 3.920.9 39.10.2
Halep Karasi (diploid) 62.24.7 79.411.0 8.71.7 82.111.6 19.93.8 100.018.5 5.830.5 49.40.3
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 23rd or 24th node. In addition, the male and female flowers were smaller and male flowers did not produce pollen in haploid plants. Also in haploid plants, plant length and stem diameter were measured and found to be lower than these of diploids. In haploid plants of the cultivar Sugar Baby, the area of individual leaves was 5 cm2while it was 10 cm2in the cv. Halep Karasi, but in the diploids it was 5±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, indirect techniques were considered as an alternative to the classical method of chromosome counting. When the plantlets were in tubes i.e. young and small, DNA surveys using 1 cm2leaf samples could easily be employed to determine the ploidy 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 methods could be used in watermelon as in Brussel sprout (DoreÂ, 1986), carrot (Rode and Dumas de Vaulx, 1987), sugarbeet (Brown et al., 1991) and pepper (Abak et al., 1998). In haploid watermelon plants the stomata length, stomata diameter and chloroplast number were found to be 17±18, 10±12 and 6±7mm, respectively, while in diploids they were about 23±24, 18 and 11±12mm, respectively.
Distinguishing the plants according to the morphological development was also a useful approach, but waiting until flowering was time consuming.
The results of this study indicated that the ploidy level would be detected succesfully by the four methods mentioned. Among them, chromosome scoring gave perfect results but, it was time consuming and season dependent. Flow cytometry was useful, but needs specific equipment. However, the published plant nuclear isolation protocols worked well for watermelon nuclei isolated from a young in vitro true leaf. To distinguish morphologically the haploids from diploid plants, the plants should be grown under greenhouse conditions and they should be observed at a certain period, thus, it is time consuming. Ploidy level can be determined in a short time by examining epidermal tissue from the under surface of leaves without the requirements of specific equipment and high expenditure.
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 cytometer in this work.
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