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Scientia Horticulturae 149 (2013) 9–12

Contents lists available at SciVerse ScienceDirect

Scientia Horticulturae

journa l h o me page: www.elsev ier .com/ locate /sc ihor t i

nterspecific hybridization and hybrid seed yield of winter squashCucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.)ines for rootstock breeding

nur Karaagac a,1, Ahmet Balkayab,∗

Black Sea Agricultural Research Institute, Samsun, TurkeyOndokuz Mayıs University, Faculty of Agriculture, Department of Horticulture, Samsun, Turkey

r t i c l e i n f o

rticle history:eceived 21 October 2011eceived in revised form 23 October 2012ccepted 24 October 2012

eywords:nterspecific hybridizationumpkinsootstock

a b s t r a c t

The use of grafted seedlings in Cucurbits has increased in recent years as interspecific hybrids betweenCucurbita maxima Duch. and Cucurbita moschata Duch. have become the preferred rootstock for water-melon, melon and cucumber. The interspecific hybrid seed production of C. maxima × C. moschata mainlydepends on genotype compatibility. In this study, different interspecific hybridization combinations wereevaluated in order to obtain C. maxima × C. moschata rootstocks. The field experiment of this study wascarried out in the C ars amba district of Samsun Province in 2009. The initial genetic materials were inbredand purified up to the S5 generation. These genotypes were selected based on plant vigor, hypocotyls char-acteristics and seed yields. A total of 234 pollinations of different combinations between twelve C. maxima

reedingucurbita

lines and eleven C. moschata lines were performed. 79 interspecific hybrid fruits were obtained from thesehybridizations. Crossing incompatibility was found to be highest in the MO8 lines (C. moschata) in all com-binations. The MA4, MA9 and MA12 winter squash (C. maxima) lines were determined to be promisingones for obtaining hybrid seed yield. In conclusion, the MA9 × MO8, MA12 × MO2 and MA4 × MO8 hybridcombinations were the most promising candidates for rootstock breeding. As a result of this study, theseselected combinations will be used in the development of promising new rootstock cultivars.

. Introduction

The production of grafted plants first began in Japan and Korean the late 1920s with watermelon (Citrullus lanatus (Thunb.) Mat-um. & Nakai var. lanatus) grafted onto gourd rootstock (Davist al., 2008). The use of grafted seedlings in Cucurbits has increasedreatly in recent years in many of the major vegetable producingegions of the world. More than 700 million grafted seedlings werestimated to have been produced in 2008 in Korea and Japan aloneLee et al., 2010). The use of grafted seedlings is expected to increaseapidly throughout the world during the next few decades (Davist al., 2008; Lee et al., 2010). Cucurbit plants are grafted onto variousootstock species and varieties using a range of grafting methods.ucurbit crops that are commonly grafted include watermelon,

elon and cucumber. The most common rootstocks for water-elon are bottle gourd, interspecific hybrids between Cucurbitaaxima and Cucurbita moschata and wild watermelon (C. lanatus

∗ Corresponding author. Tel.: +90 362 312 19 19/1383; fax: +90 362 457 60 34.E-mail addresses: onurkaraagac@hotmail.com (O. Karaagac ),

balkaya@omu.edu.tr (A. Balkaya).1 Tel.: +90 362 256 05 14; fax: +90 362 256 05 16.

304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2012.10.021

© 2012 Elsevier B.V. All rights reserved.

var. citroides) (Davis et al., 2008). The compatibility of watermelonwith any of these rootstocks is generally high, although there is vari-ability within the species (Yamamuro and Marukawa, 1974). Themost commonly used Cucurbita spp. rootstock is an interspecific C.maxima × C. moschata hybrid (Colla et al., 2010).

The use of rootstocks has been shown to enhance the vigor of thescion through the resistance to soil pathogens and tolerance to lowsoil temperatures and or salinity (Ruiz et al., 1997). The practice ofbreeding to combine traits from different germplasms into desir-able rootstock genotypes is increasing in the private sector (Kinget al., 2010). Breeders have long been interested in interspecificcrosses between major Cucurbita species (Baggett, 1979). Interspe-cific crosses are an effective way to create new germplasms. In thegenus Cucurbita, several attempts have been made to produce inter-specific hybrids between five cultivated species: Cucurbita pepo, C.maxima, C. moschata, Cucurbita argyrosperma and Cucurbita ficifolia(Korakot et al., 2010). Whitaker and Davis (1962) reported that C.moschata was difficult to crossbreed with C. pepo, C. maxima and C.mixta. The results of previous studies showed that there are some

crossing barriers between C. maxima and C. moschata (Depei, 2000;Yongan et al., 2002a). Bemis and Nelson (1963) studied interspe-cific hybridization in ten species within the genus Cucurbita. Theyreported that 43 interspecific crosses were successful at setting

1 ntia Horticulturae 149 (2013) 9–12

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Table 1Results of interspecies hybridization in genotypes with C. maxima as the femaleparent in different combinations (mean ± standard deviation).

Female parents(C. maxima)

Number ofcross

Number offruit set

Number of fruit setwith normal seeds

MA1 18 3 ± 0.38 3 ± 0.38MA3 16 0 ± 0.00 0 ± 0.00MA4 9 3 ± 0.50 3 ± 0.50MA5 15 0 ± 0.00 0 ± 0.00MA8 18 0 ± 0.00 0 ± 0.00MA9 12 9 ± 0.45 6 ± 0.52MA11 36 9 ± 0.44 9 ± 0.44MA12 30 24 ± 0.41 6 ± 0.41MA13 21 15 ± 0.46 0 ± 0.00MA14 17 0 ± 0.00 0 ± 0.00MA15 24 1 ± 0.20 1 ± 0.20

lines (Tables 1 and 2). In the 12 inbred lines with C. maxima as thefemale parent, the highest value of this parameter was recorded inMA12 (80%), MA9 (75%) and MA13 (71%), while MA3, MA, MA8 and

Table 2Results of interspecies hybridization in genotypes with C. moschata as the maleparent in different combinations (mean ± standard deviation).

Male parents(C. moschata)

Number ofcross

Number offruit set

Number of fruit setwith normal seeds

MO1 8 0 ± 0.00 0 ± 0.00MO2 3 3 ± 0.00 3 ± 0.00MO4 12 6 ± 0.52 0 ± 0.00MO5 8 0 ± 0.00 0 ± 0.00MO6 67 15 ± 0.42 0 ± 0.00MO7 9 9 ± 0.00 0 ± 0.00MO8 57 24 ± 0.50 24 ± 0.50MO10 3 1 ± 0.58 1 ± 0.58

0 O. Karaagac , A. Balkaya / Scie

ruit, but that seed development was not uniform in said fruit,nd only 23 of the interspecific crosses were successful at pro-ucing first-generation hybrid plants. Yongan et al. (2002b) foundhat C. moschata × C. maxima and C. argyrosperma × C. maxima wereross-compatible, and that C. maxima may be used as a bridgeor interspecific crosses. Similar results were found by Bingdong1996). In other studies, Yongan et al. (2002a) determined thathe number of normal seeds per fruit was what best defined theompatibility between C. maxima and C. moschata crosses. Korakott al. (2010) developed a novel inbred squash line from interspecificrosses between C. maxima and C. moschata. They did not produceiable plants, although several F1 seeds were obtained when C.oschata was the female and C. maxima was the male parent in

ll combinations.Most of the cucurbit rootstock breeding work has been done

n China, Japan and Korea. These countries have a long history ofootstock breeding. While the most current rootstock cultivars areld releases from 15 years ago, these rootstock breeding studies forucurbits are a new topic in Turkey. Unfortunately, there has beeno comprehensive program for Cucurbita spp. rootstock breeding

n Turkey and there are no reported studies. The main objectivef this study is to develop an inbred line from interspecific crossesetween C. maxima and C. moschata using hybridization. The devel-ped inbred lines may be used for rootstock breeding in the future.

. Materials and methods

This study was carried out cooperatively by the Black Seagricultural Research Institute and the Ondokuz Mayis Universitygriculture Faculty. In this experiment, the 12 inbred lines of C.axima (MA1, MA3, MA4, MA5, MA8, MA9, MA11, MA12, MA13,A14, MA15 and MA20) at the S5 generation, and 11 inbred lines

f C. moschata (MO1, MO2, MO4, MO5, MO6, MO7, MO8, MO10,O11 and MO13) at the S5 generation were used. These geneticaterials, consisting of winter squash and pumpkin populations

ollected from different parts of Turkey, were characterized andelfed by Balkaya et al. (2009, 2010). The C. maxima plants weresed as the female parent and the C. moschata plants were used ashe male parent in diallel crossing treatments.

The field experiment of this study was carried out in the exper-ment areas of the Black Sea Agricultural Research Institute inamsun Province in 2009. The experimental site is located at1◦14′N, 36◦29′E. The soil in the experimental area is sandy loamith a pH of 6.5. The seeds were sown in plastic flats (cell vol-me 150 cm3 and 28 cells per flat) containing a mixture of peatoss:perlite (3:1, v/v) on the 27th of April 2009. Seedlings were

aised in an unheated glasshouse, and 40 seedlings from each geno-ype were planted at the 3–4 leaf stage with spacing of 2.5 m × 3.0 mn the 15th of May 2009. Standard fertilization and weed con-rol practices were applied. Plants were regularly protected withungicides and insecticides throughout the growing season.

All interspecific pollination combinations were carried outt this experimental site from June 29th until July 27th 2010.emale flowers were isolated with white cloth bags (15 × 10 cm)nd male flowers were collected around noon on the day beforenthesis. Anthers without filaments were excised and mixedqually for each genotype, then placed into small cardboard boxes5 cm × 7 cm × 2 cm). Female flowers were pollinated using pollenn the morning of the following day between 0600 and 0800 h.emale flowers were then isolated with cloth bags again to avoidndesired pollen contamination. The cloth bags were removed on

he 2nd day after pollination. Fruits that developed were harvested5–70 days after hybridization. The fruit set number and fruit setercentage (%) (the number of normal seeds per fruit and the per-entage of abortion seeds/fruit) and seed yields per fruit were

MA20 18 15 ± 0.38 0 ± 0.00

Total 234 79 ± 7.90 28 ± 3.11

measured. In addition, some physical seed traits (seed length (L),width (W) and thickness (T)) were measured on 50 seeds for allcombinations with a high seed yield. Seed weight was also deter-mined by weighing air-dried seeds. A standard germination testwas composed of three replicates of 100 seeds that have been ran-domly selected and are representative of the seed lot being tested.The number of seeds less than 100 was germinated for some hybridgenotypes with less number of seeds. Seed germination rate wereestimated as the peak germination percent/peak count day (ISTA,2004). A statistical evaluation for detailed variables was carriedwith the ANOVA analysis.

3. Results and discussion

A total of 234 pollinations of different combinations betweenthe C. maxima and C. moschata species were performed. The cross-ing affinity between C. maxima and C. moschata varies depending onthe crossing ability of self-lines. Only seventy-nine combinationswere considered successful and resulted in developing embryos.The fruit development of these combinations was stopped at dif-ferent stages (Tables 1 and 2). After hybridization treatments, fruitset was determined for each genotype. However, the fruits of somecombinations were dried 7–10 days later. The main reason of thissituation, somatic cells could not developed at a particular stage asa result parents used in combination with each other were crossingincompatibility. It was found that the fruit-set percentage averagedout at 33.8%. This percentage was different among different self-

MO11 7 0 ± 0.00 0 ± 0.00MO12 15 6 ± 0.51 0 ± 0.00MO13 45 15 ± 0.48 0 ± 0.00

Total 234 79 ± 7.88 28 ± 7.17

O. Karaagac , A. Balkaya / Scientia Horticulturae 149 (2013) 9–12 11

Table 3A summary of the interspecific compatibility of the different interspecific cross-combinations (mean ± standard deviation).

♂ ♀MA1 MA4 MA9 MA11 MA12 MA13 MA15 MA20

MO2 × 25.0y

75.0z× × 361 ± 39.4

6.0 ± 4.1× × ×

MO4 × 50Ab

25.0Ab

× 76.4 ± 10.1Ab

15.5Ab

× 18.0Ab

MO6 0.0Ab

× 15.0 ± 5.0Ab

× 24.0 ± 7.5Ab

8.4 ± 2.2Ab

× 2.5±2.2Ab

MO7 × × × × 28.8 ± 18.9Ab

18.1 ± 10.2Ab

× 7.0 ± 5.4Ab

MO8 116 ± 25.412.0 ± 8.0

365 ± 41.39.6 ± 8.1

207 ± 28.90.0 ± 0.0

122±15.10.0±0.0

148 ± 20.03.0 ± 1.4

54.2 ± 11.4Ab

× 5.4 ± 2.5Ab

MO10 × × × × × × 125.015.2

×

MO12 × × 36.069.1

× 17. ± 4.5Ab

3.0Ab

× 5.9 ± 1.1Ab

MO13 × × 17.0 ± 6.7 × 15.3 ± 5.0 4.5 ± 3.5 × 6.8 ± 0.7

x were

Mmse

(bwiomrottsf

nhOtMasS(

TS

Ab

: Absence of fruit set; y: Seed number/fruit; z: Rate of abortive seed; Ab: All seeds

A14 had the lowest values (Table 1). In the 11 inbred lines with C.oschata as the male parent, MO2 and MO7 had the greatest fruit-

et percentage (100%) (Table 2). It was found that crossing barriersxist between the MO1, MO5 and MO11 pumpkin lines (Table 2).

The hybrid fruits were obtained from 30 different combinationsTable 3). The other remaining seeds of the fruits were found toe abortive or were not well filled out. The number of fruits setith normal seeds was determined for 28 fruits at nine different

nterspecific combinations. In the other fifty-one, the percentagef seeds set was very low, and the embryo did not develop nor-ally. These results showed that C. moschata lines had different

ate affinities to C. maxima. These results are similar to the resultsf the research by Yongan et al. (2002a). Based on these findings,he average seed set was about 12.0% (Tables 1 and 2). The seedraits of 28 interspecific hybrid fruits were evaluated and a higheed yield was obtained in 10 of them. The rate of high seed yieldor interspecific fruit was found to be 4.27% (10 fruits/234 cross).

Hybrid seed yield was found to be high in five different combi-ations with MO8 lines (Table 3). This may be because MO8 linesave a higher interspecific compatibility than the other genotypes.therwise, the hybrid fruits were not obtained at all combinations

hat used male parents of MO1, MO5, MO11 and female parents ofA3, MA5, MA8, MA14. MA4 × MO8 had the highest seed width

t 15.18 mm, followed by MA1 × MO8 at 14.25 mm. The average

eed length for these combinations varied from 17.11 to 23.23 mm.eed thickness measurements ranged between 3.59 and 5.16 mmTable 4). Considerable variability for seed dimensions was found

able 4eed characteristic of the promising interspecific hybrid combinations.

Combinations Width (mm) Length(mm)

Thickness(mm)

Seed germinationrate (%)

MA1 × MO8 14.25 d 21.24 b 5.16 a 25.00 e

MA11 × MO8 11.07 a 20.25 c 5.10 a 72.98 a

MA9 × MO8 12.99 cd 17.11 d 4.24 b 63.39 c

MA12 × MO2 11.38 e 19.90 c 3.59 b 18.00 f

MA12 × MO8 12.43 c 21.00 b 4.96 a 62.21 c

MA4 × MO8 15.18 e 21.77 b 5.00 a 56.07 d

MA15 × MO10 12.05 b 23.23 a 3.67 b 65.06 b

P <0.001 <0.001 <0.01 <0.01

CV (%) 2.7 2.1 8.2 9.4

* These combinations gave only one fruit. For this reason, the statistical analysis was n

Ab Ab Ab

aborted.

for these combinations. The highest seed weight was obtained withthe MA9 × MO8 combination (51.43 g/100 seed weight). Seed num-ber per fruit had an important effect on seed yield, and high seednumbers are required by seed companies. The best combinationsfor this trait were the MA4 × MO8 (365 seeds/fruit), MA12 × MO2(361 seeds/fruit) and MA9 × MO8 (207 seeds/fruit) combinations.The highest total seed yield was obtained from the MA4 × MO8(144.93 g/fruit) combination (Table 4). This study demonstratedthat substantial differences in seed germination rate on exist in thepromising interspecific hybrid combinations. These combinationsshowed a range of 18.0% (MA12 × MO2)–72.98% (MA11 × MO8) forseed germination rate (Table 4). Doubtless, a high seed germina-tion rate is a desirable trait. However, the seed germination ratesof the promising interspecific combinations were not found desiredat suitable levels in this study.

Cucurbit breeders have long been interested in interspecificcrosses between Cucurbita species. Selecting the best combina-tions of interspecific crosses is important for vegetable rootstockbreeding. Nowadays, the interspecific rootstocks are used largelyfor grafting watermelon, melon and cucumber (Lee et al., 2010;King et al., 2010). In this study, the combination between C.maxima as the female parent and C. moschata as the male par-ent showed fertilization at different ratios depending on whichline was used. Crossing incompatibility was found to be high-

est for the MO8 lines (C. moschata) in all combinations (Table 2).Reviewing the results of this study, the MA9 × MO8, MA12 × MO2and MA4 × MO8 hybrid combinations seem to be the most

100 seedweight (g)

Seed yield(g/fruit)

Number of seeds(n./fruit)

Seed abortionrate (%)

34.99 ab 39.46 c 116.32 d 12.04 a21.73 ab 25.59 e 122.21 d 0.00 e51.43 a 104.92 b 207.19 b 0.00 e28.32 b 102.20 b 361.47 a 6.01 c34.35 ab 36.04 d 148.49 c 3.00 d39.75 ab 144.93 a 365.25 a 9.60 b31.50* 42.36 125.42 15.2<0.05 <0.001 <0.001 <0.00113.5 2.4 1.9 11.7

ot reported.

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romising rootstock for breeding (Tables 3 and 4). The seed yieldsave been determined in this study. However, the seed yieldsf these combinations were not found desired at suitable lev-ls. Numerous crosses are often required to obtain a few viableeeds. Whitaker and Davis (1962) concluded that C. moschataccupies a central position among the annual species and can berossed with difficulty with C. pepo, C. maxima and C. mixta. Theyxhibit a wide range of crossing barriers at both the presyngamicfailure of pollen tube growth) and postsyngamic (breakdown ofmbryo development) developmental phases. Many techniques,ncluding bridge crosses (Zhang et al., 2012), bud pollinationHayase, 1961), repeated pollination (Yongan et al., 2002b), andhe use of growth regulators (Nascimento et al., 2007), have beenmployed in attempts to improve the success of interspecificybridization (Lebeda et al., 2007). In Cucurbita, the successfululturing of embryos from mature fruits has been reported byany researchers (Kwack and Fujieda, 1987; Sisko et al., 2003;

oy, 2012). Gene transfer between C. maxima and C. moschataas been difficult. Crosses between some cultigens of the twopecies yield neither seed nor fruit. The successes of the pre-ented interspecific hybridizations were, in general, comparableo the published data. In the test of interspecific crosses, differ-nt cultivars within the species performed differently. For thiseason, these interspecific hybrids derived from crosses of C. max-ma × C. moschata also will be utilized as bridge crosses withackcross programmes in future breeding work. Thus, it wille increased both seed yield and seed viability of these com-inations. Hybridization programs with these lines have been

mplemented.

cknowledgements

We gratefully acknowledge the support of fundingy the Ondokuz Mayıs University Research FoundationPYO.ZRT.1901.09.015) and Black Sea Agricultural Researchnstitute (KTAE). We also appreciate comments on this manuscripty Prof. Dr. María Belén Pico of Polytechnic University of Valencia,pain.

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