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Interspecific hybridization and hybrid seed yield of winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) lines for rootstock breeding
Scientia Horticulturae 149 (2013) 9–12
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Interspecific hybridization and hybrid seed yield of winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) lines for rootstock breeding Onur Karaa˘gac¸ a,1 , Ahmet Balkaya b,∗ a b
Black Sea Agricultural Research Institute, Samsun, Turkey Ondokuz Mayıs University, Faculty of Agriculture, Department of Horticulture, Samsun, Turkey
a r t i c l e
i n f o
Article history: Received 21 October 2011 Received in revised form 23 October 2012 Accepted 24 October 2012 Keywords: Interspecific hybridization Pumpkins Rootstock Breeding Cucurbita
a b s t r a c t The use of grafted seedlings in Cucurbits has increased in recent years as interspecific hybrids between Cucurbita maxima Duch. and Cucurbita moschata Duch. have become the preferred rootstock for watermelon, melon and cucumber. The interspecific hybrid seed production of C. maxima × C. moschata mainly depends on genotype compatibility. In this study, different interspecific hybridization combinations were evaluated in order to obtain C. maxima × C. moschata rootstocks. The field experiment of this study was carried out in the C¸ars¸amba district of Samsun Province in 2009. The initial genetic materials were inbred and purified up to the S5 generation. These genotypes were selected based on plant vigor, hypocotyls characteristics and seed yields. A total of 234 pollinations of different combinations between twelve C. maxima lines and eleven C. moschata lines were performed. 79 interspecific hybrid fruits were obtained from these hybridizations. Crossing incompatibility was found to be highest in the MO8 lines (C. moschata) in all combinations. The MA4, MA9 and MA12 winter squash (C. maxima) lines were determined to be promising ones for obtaining hybrid seed yield. In conclusion, the MA9 × MO8, MA12 × MO2 and MA4 × MO8 hybrid combinations were the most promising candidates for rootstock breeding. As a result of this study, these selected combinations will be used in the development of promising new rootstock cultivars. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The production of grafted plants first began in Japan and Korea in the late 1920s with watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) grafted onto gourd rootstock (Davis et al., 2008). The use of grafted seedlings in Cucurbits has increased greatly in recent years in many of the major vegetable producing regions of the world. More than 700 million grafted seedlings were estimated to have been produced in 2008 in Korea and Japan alone (Lee et al., 2010). The use of grafted seedlings is expected to increase rapidly throughout the world during the next few decades (Davis et al., 2008; Lee et al., 2010). Cucurbit plants are grafted onto various rootstock species and varieties using a range of grafting methods. Cucurbit crops that are commonly grafted include watermelon, melon and cucumber. The most common rootstocks for watermelon are bottle gourd, interspecific hybrids between Cucurbita maxima 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: [email protected] (O. Karaa˘gac¸), [email protected] (A. Balkaya). 1 Tel.: +90 362 256 05 14; fax: +90 362 256 05 16. 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.10.021
var. citroides) (Davis et al., 2008). The compatibility of watermelon with any of these rootstocks is generally high, although there is variability within the species (Yamamuro and Marukawa, 1974). The most 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 the scion through the resistance to soil pathogens and tolerance to low soil temperatures and or salinity (Ruiz et al., 1997). The practice of breeding to combine traits from different germplasms into desirable rootstock genotypes is increasing in the private sector (King et al., 2010). Breeders have long been interested in interspecific crosses between major Cucurbita species (Baggett, 1979). Interspecific crosses are an effective way to create new germplasms. In the genus Cucurbita, several attempts have been made to produce interspecific 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 interspecific hybridization in ten species within the genus Cucurbita. They reported that 43 interspecific crosses were successful at setting
10
O. Karaa˘gac¸, A. Balkaya / Scientia Horticulturae 149 (2013) 9–12
fruit, but that seed development was not uniform in said fruit, and only 23 of the interspecific crosses were successful at producing first-generation hybrid plants. Yongan et al. (2002b) found that C. moschata × C. maxima and C. argyrosperma × C. maxima were cross-compatible, and that C. maxima may be used as a bridge for interspecific crosses. Similar results were found by Bingdong (1996). In other studies, Yongan et al. (2002a) determined that the number of normal seeds per fruit was what best defined the compatibility between C. maxima and C. moschata crosses. Korakot et al. (2010) developed a novel inbred squash line from interspecific crosses between C. maxima and C. moschata. They did not produce viable plants, although several F1 seeds were obtained when C. moschata was the female and C. maxima was the male parent in all combinations. Most of the cucurbit rootstock breeding work has been done in China, Japan and Korea. These countries have a long history of rootstock breeding. While the most current rootstock cultivars are old releases from 15 years ago, these rootstock breeding studies for cucurbits are a new topic in Turkey. Unfortunately, there has been no comprehensive program for Cucurbita spp. rootstock breeding in Turkey and there are no reported studies. The main objective of this study is to develop an inbred line from interspecific crosses between C. maxima and C. moschata using hybridization. The developed inbred lines may be used for rootstock breeding in the future.
2. Materials and methods This study was carried out cooperatively by the Black Sea Agricultural Research Institute and the Ondokuz Mayis University Agriculture Faculty. In this experiment, the 12 inbred lines of C. maxima (MA1, MA3, MA4, MA5, MA8, MA9, MA11, MA12, MA13, MA14, MA15 and MA20) at the S5 generation, and 11 inbred lines of C. moschata (MO1, MO2, MO4, MO5, MO6, MO7, MO8, MO10, MO11 and MO13) at the S5 generation were used. These genetic materials, consisting of winter squash and pumpkin populations collected from different parts of Turkey, were characterized and selfed by Balkaya et al. (2009, 2010). The C. maxima plants were used as the female parent and the C. moschata plants were used as the male parent in diallel crossing treatments. The field experiment of this study was carried out in the experiment areas of the Black Sea Agricultural Research Institute in Samsun Province in 2009. The experimental site is located at 41◦ 14 N, 36◦ 29 E. The soil in the experimental area is sandy loam with a pH of 6.5. The seeds were sown in plastic flats (cell volume 150 cm3 and 28 cells per flat) containing a mixture of peat moss:perlite (3:1, v/v) on the 27th of April 2009. Seedlings were raised in an unheated glasshouse, and 40 seedlings from each genotype were planted at the 3–4 leaf stage with spacing of 2.5 m × 3.0 m on the 15th of May 2009. Standard fertilization and weed control practices were applied. Plants were regularly protected with fungicides and insecticides throughout the growing season. All interspecific pollination combinations were carried out at this experimental site from June 29th until July 27th 2010. Female flowers were isolated with white cloth bags (15 × 10 cm) and male flowers were collected around noon on the day before anthesis. Anthers without filaments were excised and mixed equally for each genotype, then placed into small cardboard boxes (5 cm × 7 cm × 2 cm). Female flowers were pollinated using pollen on the morning of the following day between 0600 and 0800 h. Female flowers were then isolated with cloth bags again to avoid undesired pollen contamination. The cloth bags were removed on the 2nd day after pollination. Fruits that developed were harvested 65–70 days after hybridization. The fruit set number and fruit set percentage (%) (the number of normal seeds per fruit and the percentage of abortion seeds/fruit) and seed yields per fruit were
Table 1 Results of interspecies hybridization in genotypes with C. maxima as the female parent in different combinations (mean ± standard deviation). Female parents (C. maxima) MA1 MA3 MA4 MA5 MA8 MA9 MA11 MA12 MA13 MA14 MA15 MA20 Total
Number of cross 18 16 9 15 18 12 36 30 21 17 24 18 234
Number of fruit set 3 0 3 0 0 9 9 24 15 0 1 15
± ± ± ± ± ± ± ± ± ± ± ±
0.38 0.00 0.50 0.00 0.00 0.45 0.44 0.41 0.46 0.00 0.20 0.38
79 ± 7.90
Number of fruit set with normal seeds 3 0 3 0 0 6 9 6 0 0 1 0
± ± ± ± ± ± ± ± ± ± ± ±
0.38 0.00 0.50 0.00 0.00 0.52 0.44 0.41 0.00 0.00 0.20 0.00
28 ± 3.11
measured. In addition, some physical seed traits (seed length (L), width (W) and thickness (T)) were measured on 50 seeds for all combinations with a high seed yield. Seed weight was also determined by weighing air-dried seeds. A standard germination test was composed of three replicates of 100 seeds that have been randomly selected and are representative of the seed lot being tested. The number of seeds less than 100 was germinated for some hybrid genotypes with less number of seeds. Seed germination rate were estimated as the peak germination percent/peak count day (ISTA, 2004). A statistical evaluation for detailed variables was carried with the ANOVA analysis. 3. Results and discussion A total of 234 pollinations of different combinations between the C. maxima and C. moschata species were performed. The crossing affinity between C. maxima and C. moschata varies depending on the crossing ability of self-lines. Only seventy-nine combinations were considered successful and resulted in developing embryos. The fruit development of these combinations was stopped at different stages (Tables 1 and 2). After hybridization treatments, fruit set was determined for each genotype. However, the fruits of some combinations were dried 7–10 days later. The main reason of this situation, somatic cells could not developed at a particular stage as a result parents used in combination with each other were crossing incompatibility. It was found that the fruit-set percentage averaged out at 33.8%. This percentage was different among different selflines (Tables 1 and 2). In the 12 inbred lines with C. maxima as the female parent, the highest value of this parameter was recorded in MA12 (80%), MA9 (75%) and MA13 (71%), while MA3, MA, MA8 and Table 2 Results of interspecies hybridization in genotypes with C. moschata as the male parent in different combinations (mean ± standard deviation). Male parents (C. moschata) MO1 MO2 MO4 MO5 MO6 MO7 MO8 MO10 MO11 MO12 MO13 Total
Number of cross 8 3 12 8 67 9 57 3 7 15 45 234
Number of fruit set 0 3 6 0 15 9 24 1 0 6 15
± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.52 0.00 0.42 0.00 0.50 0.58 0.00 0.51 0.48
79 ± 7.88
Number of fruit set with normal seeds 0 3 0 0 0 0 24 1 0 0 0
± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.50 0.58 0.00 0.00 0.00
28 ± 7.17
O. Karaa˘gac¸, A. Balkaya / Scientia Horticulturae 149 (2013) 9–12
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Table 3 A 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
×
50 Ab
25.0 Ab
×
76.4 ± 10.1 Ab
15.5 Ab
×
18.0 Ab
MO6
0.0 Ab
×
15.0 ± 5.0 Ab
×
24.0 ± 7.5 Ab
8.4 ± 2.2 Ab
×
2.5±2.2 Ab
MO7
×
×
×
×
28.8 ± 18.9 Ab
18.1 ± 10.2 Ab
×
7.0 ± 5.4 Ab
MO8
116 ± 25.4 12.0 ± 8.0
365 ± 41.3 9.6 ± 8.1
207 ± 28.9 0.0 ± 0.0
122±15.1 0.0±0.0
148 ± 20.0 3.0 ± 1.4
54.2 ± 11.4 Ab
×
5.4 ± 2.5 Ab
MO10
×
×
×
×
×
×
125.0 15.2
×
MO12
×
×
36.0 69.1
×
17. ± 4.5 Ab
3.0 Ab
×
5.9 ± 1.1 Ab
MO13
×
×
17.0 ± 6.7 Ab
×
15.3 ± 5.0 Ab
4.5 ± 3.5 Ab
×
6.8 ± 0.7 Ab
x: Absence of fruit set; y: Seed number/fruit; z: Rate of abortive seed; Ab: All seeds were aborted.
MA14 had the lowest values (Table 1). In the 11 inbred lines with C. moschata as the male parent, MO2 and MO7 had the greatest fruitset percentage (100%) (Table 2). It was found that crossing barriers exist between the MO1, MO5 and MO11 pumpkin lines (Table 2). The hybrid fruits were obtained from 30 different combinations (Table 3). The other remaining seeds of the fruits were found to be abortive or were not well filled out. The number of fruits set with normal seeds was determined for 28 fruits at nine different interspecific combinations. In the other fifty-one, the percentage of seeds set was very low, and the embryo did not develop normally. These results showed that C. moschata lines had different rate affinities to C. maxima. These results are similar to the results of the research by Yongan et al. (2002a). Based on these findings, the average seed set was about 12.0% (Tables 1 and 2). The seed traits of 28 interspecific hybrid fruits were evaluated and a high seed yield was obtained in 10 of them. The rate of high seed yield for interspecific fruit was found to be 4.27% (10 fruits/234 cross). Hybrid seed yield was found to be high in five different combinations with MO8 lines (Table 3). This may be because MO8 lines have a higher interspecific compatibility than the other genotypes. Otherwise, the hybrid fruits were not obtained at all combinations that used male parents of MO1, MO5, MO11 and female parents of MA3, MA5, MA8, MA14. MA4 × MO8 had the highest seed width at 15.18 mm, followed by MA1 × MO8 at 14.25 mm. The average seed length for these combinations varied from 17.11 to 23.23 mm. Seed thickness measurements ranged between 3.59 and 5.16 mm (Table 4). Considerable variability for seed dimensions was found
for these combinations. The highest seed weight was obtained with the MA9 × MO8 combination (51.43 g/100 seed weight). Seed number per fruit had an important effect on seed yield, and high seed numbers are required by seed companies. The best combinations for 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 demonstrated that substantial differences in seed germination rate on exist in the promising interspecific hybrid combinations. These combinations showed a range of 18.0% (MA12 × MO2)–72.98% (MA11 × MO8) for seed germination rate (Table 4). Doubtless, a high seed germination rate is a desirable trait. However, the seed germination rates of the promising interspecific combinations were not found desired at suitable levels in this study. Cucurbit breeders have long been interested in interspecific crosses between Cucurbita species. Selecting the best combinations of interspecific crosses is important for vegetable rootstock breeding. Nowadays, the interspecific rootstocks are used largely for 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 parent showed fertilization at different ratios depending on which line was used. Crossing incompatibility was found to be highest for the MO8 lines (C. moschata) in all combinations (Table 2). Reviewing the results of this study, the MA9 × MO8, MA12 × MO2 and MA4 × MO8 hybrid combinations seem to be the most
Table 4 Seed characteristic of the promising interspecific hybrid combinations. Combinations
Width (mm)
Length (mm)
Thickness (mm)
Seed germination rate (%)
100 seed weight (g)
Seed yield (g/fruit)
Number of seeds (n./fruit)
Seed abortion rate (%)
MA1 × MO8 MA11 × MO8 MA9 × MO8 MA12 × MO2 MA12 × MO8 MA4 × MO8 MA15 × MO10 P CV (%)
14.25 d 11.07 a 12.99 cd 11.38 e 12.43 c 15.18 e 12.05 b <0.001 2.7
21.24 b 20.25 c 17.11 d 19.90 c 21.00 b 21.77 b 23.23 a <0.001 2.1
5.16 a 5.10 a 4.24 b 3.59 b 4.96 a 5.00 a 3.67 b <0.01 8.2
25.00 e 72.98 a 63.39 c 18.00 f 62.21 c 56.07 d 65.06 b <0.01 9.4
34.99 ab 21.73 ab 51.43 a 28.32 b 34.35 ab 39.75 ab 31.50* <0.05 13.5
39.46 c 25.59 e 104.92 b 102.20 b 36.04 d 144.93 a 42.36 <0.001 2.4
116.32 d 122.21 d 207.19 b 361.47 a 148.49 c 365.25 a 125.42 <0.001 1.9
12.04 a 0.00 e 0.00 e 6.01 c 3.00 d 9.60 b 15.2 <0.001 11.7
*
These combinations gave only one fruit. For this reason, the statistical analysis was not reported.
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O. Karaa˘gac¸, A. Balkaya / Scientia Horticulturae 149 (2013) 9–12
promising rootstock for breeding (Tables 3 and 4). The seed yields have been determined in this study. However, the seed yields of these combinations were not found desired at suitable levels. Numerous crosses are often required to obtain a few viable seeds. Whitaker and Davis (1962) concluded that C. moschata occupies a central position among the annual species and can be crossed with difficulty with C. pepo, C. maxima and C. mixta. They exhibit a wide range of crossing barriers at both the presyngamic (failure of pollen tube growth) and postsyngamic (breakdown of embryo development) developmental phases. Many techniques, including bridge crosses (Zhang et al., 2012), bud pollination (Hayase, 1961), repeated pollination (Yongan et al., 2002b), and the use of growth regulators (Nascimento et al., 2007), have been employed in attempts to improve the success of interspecific hybridization (Lebeda et al., 2007). In Cucurbita, the successful culturing of embryos from mature fruits has been reported by ˇ sko et al., 2003; many researchers (Kwack and Fujieda, 1987; Siˇ Loy, 2012). Gene transfer between C. maxima and C. moschata has been difficult. Crosses between some cultigens of the two species yield neither seed nor fruit. The successes of the presented interspecific hybridizations were, in general, comparable to the published data. In the test of interspecific crosses, different cultivars within the species performed differently. For this reason, these interspecific hybrids derived from crosses of C. maxima × C. moschata also will be utilized as bridge crosses with backcross programmes in future breeding work. Thus, it will be increased both seed yield and seed viability of these combinations. Hybridization programs with these lines have been implemented. Acknowledgements We gratefully acknowledge the support of funding by the Ondokuz Mayıs University Research Foundation (PYO.ZRT.1901.09.015) and Black Sea Agricultural Research Institute (KTAE). We also appreciate comments on this manuscript by Prof. Dr. María Belén Pico of Polytechnic University of Valencia, Spain. References Baggett, J.R., 1979. Attempts to cross Cucurbita moschata (Duch.) Poir. ‘Butternut’ and C. pepo L. ‘Delicata’. Cucurbit Genet. Cooperat. Rep. 2, 32–34. Balkaya, A., Kurtar, E., Yanmaz, R., 2009. Evaluation and selection of suitable pumpkin (Cucurbita moschata Duchense) types from Turkey. Proceedings of the IVth Balkan Symposium on Vegetables and Potatoes, Plovdiv, Bulgaria, 6–10 September. Acta Hortic. 830, 55–62.
Balkaya, A., Özbakır, M., Kurtar, E.S., 2010. The determination of diversity and fruit characterization of winter squash populations from the Black Sea region of Turkey. Afr. J. Biotechnol. 9 (2), 152–162. Bemis, W.P., Nelson, J.M., 1963. Interspecific hybridization within the genus Cucurbita I, Fruit set, seed and embryo development. J. Ariz. Acad. Sci. 2 (3), 104–107. Bingdong, L., 1996. Biology researches and breeding progress of gourd crops (Cucurbita L.). Chin. Veg. 6, 48–50. Colla, G., Rouphael, Y., Leonardi, C., Bie, Z., 2010. Role of grafting in vegetable crops grown under saline conditions. Sci. Hortic. 127 (2), 147–155. Davis, A.R., Perkins-Veazie, P., Sakata, Y., López-Galarza, S., Maroto, J.V., Lee, S.G., Huh, Y.C., Sun, Z., Miguel, A., King, S.R., Cohen, R., Lee, J.M., 2008. Cucurbit grafting. Crit. Rev. Plant Sci. 27, 50–74. Depei, L., 2000. Inheritance gene and interspecies cross of squash plant. Chin. Watermelon Muskmelon 3, 41–44. Hayase, H., 1961. Cucurbita-crosses, XIII. Utilization of bud pollination in obtaining interspesific hybrids of C. pepo × C. maxima. Jpn. J. Genet. 25, 181–190. ISTA, 2004. International Rules for Seed Testing. International Seed Testing Association. Edition 2004, Switzerland. King, S.R., Davis, A.R., Zhang, X., Crosby, K., 2010. Genetics, breeding and selection of rootstocks for Solanaceae and Cucurbitaceae. Sci. Hortic. 127 (2), 106–111. Korakot, N., Yang, J., Zhang, M., Ye, F., Lin, Y., 2010. A novel inbred squash line developed from interspecific crosses between Cucurbita maxima and Cucurbita moschata. In: Cucurbitaceae 2010, Charleston, South Carolina, 14–18 November 2010, pp. 129–131. Kwack, N.C., Fujieda, K., 1987. Seed abortion and techniques for obtaining hybrids in interspecific crosses of Cucurbita. J. Jpn. Soc. Hortic. Sci. 55 (4), 455–460. Lebeda, A., Widrlechner, M.P., Staub, J., Ezura, H., Zalapa, J., Kˇrístková, E., 2007. Cucurbits (Cucurbitaceae; Cucumis spp., Cucurbita spp., Citrullus spp.). In: Singh, R.J. (Ed.), Genetic Resources, Chromosome Engineering, and Crop Improvement. Vegetable Crops, vol. 3. CRC Press, Boca Raton, FL, USA, pp. 271–376 (Chapter 8). Lee, J.M., Kubota, C., Tsao, S.J., Hoyos Echevarria, P., Morra, L., Oda, M., 2010. Current status of Vegetable grafting: diffusion grafting techniques, automation. Sci. Hortic. 127, 93–105. Loy, B., 2012. Breeding squash and pumpkins. In: Wang, Y.H., Behera, T.K., Kole, C. (Eds.), Genetics, Genomics and Breeding of Cucurbits. Science Publishers, SC, USA, pp. 107–110. Nascimento, W.M., Pinheiro, F., Freitas, R.A., 2007. Utilizac¸ão do ethephon para a produc¸ão de sementes de híbrido de abóbora tipo tetsukabuto. Rev. Bras. Sement. 29 (2), 10–14. Ruiz, J.M., Belakbir, A., Lopez-Cantarero, I., Romero, L., 1997. Leaf macronutrient content and yield in grafted melon plants. A model to evaluate the influence of rootstock genotype. Sci. Hortic. 71, 227–234. ˇ sko, M., Ivanˇciˇc, A., Bohanec, B., 2003. Genome size analysis in the genus Cucurbita Siˇ and its use for determination of interspecific hybrids obtained using the embryorescue technique. Plant Sci. 165, 663–669. Whitaker, T.W., Davis, G.N., 1962. Cucurbits. Interscience Publishers, Inc., New York. Yamamuro, K., Marukawa, S., 1974. Studies on the selection of Cucurbita spp. as watermelon rootstocks. 3. Compatibility and yield in relation to differences in cultivation. Bull. Ibaraki-ken Hortic. Exp. Station 5, 123–149. Yongan, C., Bingkui, Z., Enhui, Z., Zunlian, Z., 2002a. Germplasm innovation by interspecific crosses in pumpkin. Cucurbit Genet. Cooperat. Rep. 25, 56–57. Yongan, C., Bingkui, Z., Enhui, Z., Zunlian, Z., 2002b. Study on affinity of sexual hybridization between Cucurbita maxima D. and Cucurbita moschata D. Cucurbit Genet. Cooperat. Rep. 25, 54–55. Zhang, Q., Yu, E., Medina, A., 2012. Development of advanced interspecific-bridge lines among Cucurbita pepo, C. maxima, and C moschata. Hortic. Sci. 47 (4), 452–458.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Interspecific hybridization and hybrid seed yield of winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) lines for rootstock breeding Onur Karaa˘gac¸ a,1 , Ahmet Balkaya b,∗ a b
Black Sea Agricultural Research Institute, Samsun, Turkey Ondokuz Mayıs University, Faculty of Agriculture, Department of Horticulture, Samsun, Turkey
a r t i c l e
i n f o
Article history: Received 21 October 2011 Received in revised form 23 October 2012 Accepted 24 October 2012 Keywords: Interspecific hybridization Pumpkins Rootstock Breeding Cucurbita
a b s t r a c t The use of grafted seedlings in Cucurbits has increased in recent years as interspecific hybrids between Cucurbita maxima Duch. and Cucurbita moschata Duch. have become the preferred rootstock for watermelon, melon and cucumber. The interspecific hybrid seed production of C. maxima × C. moschata mainly depends on genotype compatibility. In this study, different interspecific hybridization combinations were evaluated in order to obtain C. maxima × C. moschata rootstocks. The field experiment of this study was carried out in the C¸ars¸amba district of Samsun Province in 2009. The initial genetic materials were inbred and purified up to the S5 generation. These genotypes were selected based on plant vigor, hypocotyls characteristics and seed yields. A total of 234 pollinations of different combinations between twelve C. maxima lines and eleven C. moschata lines were performed. 79 interspecific hybrid fruits were obtained from these hybridizations. Crossing incompatibility was found to be highest in the MO8 lines (C. moschata) in all combinations. The MA4, MA9 and MA12 winter squash (C. maxima) lines were determined to be promising ones for obtaining hybrid seed yield. In conclusion, the MA9 × MO8, MA12 × MO2 and MA4 × MO8 hybrid combinations were the most promising candidates for rootstock breeding. As a result of this study, these selected combinations will be used in the development of promising new rootstock cultivars. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The production of grafted plants first began in Japan and Korea in the late 1920s with watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) grafted onto gourd rootstock (Davis et al., 2008). The use of grafted seedlings in Cucurbits has increased greatly in recent years in many of the major vegetable producing regions of the world. More than 700 million grafted seedlings were estimated to have been produced in 2008 in Korea and Japan alone (Lee et al., 2010). The use of grafted seedlings is expected to increase rapidly throughout the world during the next few decades (Davis et al., 2008; Lee et al., 2010). Cucurbit plants are grafted onto various rootstock species and varieties using a range of grafting methods. Cucurbit crops that are commonly grafted include watermelon, melon and cucumber. The most common rootstocks for watermelon are bottle gourd, interspecific hybrids between Cucurbita maxima 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: [email protected] (O. Karaa˘gac¸), [email protected] (A. Balkaya). 1 Tel.: +90 362 256 05 14; fax: +90 362 256 05 16. 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.10.021
var. citroides) (Davis et al., 2008). The compatibility of watermelon with any of these rootstocks is generally high, although there is variability within the species (Yamamuro and Marukawa, 1974). The most 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 the scion through the resistance to soil pathogens and tolerance to low soil temperatures and or salinity (Ruiz et al., 1997). The practice of breeding to combine traits from different germplasms into desirable rootstock genotypes is increasing in the private sector (King et al., 2010). Breeders have long been interested in interspecific crosses between major Cucurbita species (Baggett, 1979). Interspecific crosses are an effective way to create new germplasms. In the genus Cucurbita, several attempts have been made to produce interspecific 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 interspecific hybridization in ten species within the genus Cucurbita. They reported that 43 interspecific crosses were successful at setting
10
O. Karaa˘gac¸, A. Balkaya / Scientia Horticulturae 149 (2013) 9–12
fruit, but that seed development was not uniform in said fruit, and only 23 of the interspecific crosses were successful at producing first-generation hybrid plants. Yongan et al. (2002b) found that C. moschata × C. maxima and C. argyrosperma × C. maxima were cross-compatible, and that C. maxima may be used as a bridge for interspecific crosses. Similar results were found by Bingdong (1996). In other studies, Yongan et al. (2002a) determined that the number of normal seeds per fruit was what best defined the compatibility between C. maxima and C. moschata crosses. Korakot et al. (2010) developed a novel inbred squash line from interspecific crosses between C. maxima and C. moschata. They did not produce viable plants, although several F1 seeds were obtained when C. moschata was the female and C. maxima was the male parent in all combinations. Most of the cucurbit rootstock breeding work has been done in China, Japan and Korea. These countries have a long history of rootstock breeding. While the most current rootstock cultivars are old releases from 15 years ago, these rootstock breeding studies for cucurbits are a new topic in Turkey. Unfortunately, there has been no comprehensive program for Cucurbita spp. rootstock breeding in Turkey and there are no reported studies. The main objective of this study is to develop an inbred line from interspecific crosses between C. maxima and C. moschata using hybridization. The developed inbred lines may be used for rootstock breeding in the future.
2. Materials and methods This study was carried out cooperatively by the Black Sea Agricultural Research Institute and the Ondokuz Mayis University Agriculture Faculty. In this experiment, the 12 inbred lines of C. maxima (MA1, MA3, MA4, MA5, MA8, MA9, MA11, MA12, MA13, MA14, MA15 and MA20) at the S5 generation, and 11 inbred lines of C. moschata (MO1, MO2, MO4, MO5, MO6, MO7, MO8, MO10, MO11 and MO13) at the S5 generation were used. These genetic materials, consisting of winter squash and pumpkin populations collected from different parts of Turkey, were characterized and selfed by Balkaya et al. (2009, 2010). The C. maxima plants were used as the female parent and the C. moschata plants were used as the male parent in diallel crossing treatments. The field experiment of this study was carried out in the experiment areas of the Black Sea Agricultural Research Institute in Samsun Province in 2009. The experimental site is located at 41◦ 14 N, 36◦ 29 E. The soil in the experimental area is sandy loam with a pH of 6.5. The seeds were sown in plastic flats (cell volume 150 cm3 and 28 cells per flat) containing a mixture of peat moss:perlite (3:1, v/v) on the 27th of April 2009. Seedlings were raised in an unheated glasshouse, and 40 seedlings from each genotype were planted at the 3–4 leaf stage with spacing of 2.5 m × 3.0 m on the 15th of May 2009. Standard fertilization and weed control practices were applied. Plants were regularly protected with fungicides and insecticides throughout the growing season. All interspecific pollination combinations were carried out at this experimental site from June 29th until July 27th 2010. Female flowers were isolated with white cloth bags (15 × 10 cm) and male flowers were collected around noon on the day before anthesis. Anthers without filaments were excised and mixed equally for each genotype, then placed into small cardboard boxes (5 cm × 7 cm × 2 cm). Female flowers were pollinated using pollen on the morning of the following day between 0600 and 0800 h. Female flowers were then isolated with cloth bags again to avoid undesired pollen contamination. The cloth bags were removed on the 2nd day after pollination. Fruits that developed were harvested 65–70 days after hybridization. The fruit set number and fruit set percentage (%) (the number of normal seeds per fruit and the percentage of abortion seeds/fruit) and seed yields per fruit were
Table 1 Results of interspecies hybridization in genotypes with C. maxima as the female parent in different combinations (mean ± standard deviation). Female parents (C. maxima) MA1 MA3 MA4 MA5 MA8 MA9 MA11 MA12 MA13 MA14 MA15 MA20 Total
Number of cross 18 16 9 15 18 12 36 30 21 17 24 18 234
Number of fruit set 3 0 3 0 0 9 9 24 15 0 1 15
± ± ± ± ± ± ± ± ± ± ± ±
0.38 0.00 0.50 0.00 0.00 0.45 0.44 0.41 0.46 0.00 0.20 0.38
79 ± 7.90
Number of fruit set with normal seeds 3 0 3 0 0 6 9 6 0 0 1 0
± ± ± ± ± ± ± ± ± ± ± ±
0.38 0.00 0.50 0.00 0.00 0.52 0.44 0.41 0.00 0.00 0.20 0.00
28 ± 3.11
measured. In addition, some physical seed traits (seed length (L), width (W) and thickness (T)) were measured on 50 seeds for all combinations with a high seed yield. Seed weight was also determined by weighing air-dried seeds. A standard germination test was composed of three replicates of 100 seeds that have been randomly selected and are representative of the seed lot being tested. The number of seeds less than 100 was germinated for some hybrid genotypes with less number of seeds. Seed germination rate were estimated as the peak germination percent/peak count day (ISTA, 2004). A statistical evaluation for detailed variables was carried with the ANOVA analysis. 3. Results and discussion A total of 234 pollinations of different combinations between the C. maxima and C. moschata species were performed. The crossing affinity between C. maxima and C. moschata varies depending on the crossing ability of self-lines. Only seventy-nine combinations were considered successful and resulted in developing embryos. The fruit development of these combinations was stopped at different stages (Tables 1 and 2). After hybridization treatments, fruit set was determined for each genotype. However, the fruits of some combinations were dried 7–10 days later. The main reason of this situation, somatic cells could not developed at a particular stage as a result parents used in combination with each other were crossing incompatibility. It was found that the fruit-set percentage averaged out at 33.8%. This percentage was different among different selflines (Tables 1 and 2). In the 12 inbred lines with C. maxima as the female parent, the highest value of this parameter was recorded in MA12 (80%), MA9 (75%) and MA13 (71%), while MA3, MA, MA8 and Table 2 Results of interspecies hybridization in genotypes with C. moschata as the male parent in different combinations (mean ± standard deviation). Male parents (C. moschata) MO1 MO2 MO4 MO5 MO6 MO7 MO8 MO10 MO11 MO12 MO13 Total
Number of cross 8 3 12 8 67 9 57 3 7 15 45 234
Number of fruit set 0 3 6 0 15 9 24 1 0 6 15
± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.52 0.00 0.42 0.00 0.50 0.58 0.00 0.51 0.48
79 ± 7.88
Number of fruit set with normal seeds 0 3 0 0 0 0 24 1 0 0 0
± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.50 0.58 0.00 0.00 0.00
28 ± 7.17
O. Karaa˘gac¸, A. Balkaya / Scientia Horticulturae 149 (2013) 9–12
11
Table 3 A 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
×
50 Ab
25.0 Ab
×
76.4 ± 10.1 Ab
15.5 Ab
×
18.0 Ab
MO6
0.0 Ab
×
15.0 ± 5.0 Ab
×
24.0 ± 7.5 Ab
8.4 ± 2.2 Ab
×
2.5±2.2 Ab
MO7
×
×
×
×
28.8 ± 18.9 Ab
18.1 ± 10.2 Ab
×
7.0 ± 5.4 Ab
MO8
116 ± 25.4 12.0 ± 8.0
365 ± 41.3 9.6 ± 8.1
207 ± 28.9 0.0 ± 0.0
122±15.1 0.0±0.0
148 ± 20.0 3.0 ± 1.4
54.2 ± 11.4 Ab
×
5.4 ± 2.5 Ab
MO10
×
×
×
×
×
×
125.0 15.2
×
MO12
×
×
36.0 69.1
×
17. ± 4.5 Ab
3.0 Ab
×
5.9 ± 1.1 Ab
MO13
×
×
17.0 ± 6.7 Ab
×
15.3 ± 5.0 Ab
4.5 ± 3.5 Ab
×
6.8 ± 0.7 Ab
x: Absence of fruit set; y: Seed number/fruit; z: Rate of abortive seed; Ab: All seeds were aborted.
MA14 had the lowest values (Table 1). In the 11 inbred lines with C. moschata as the male parent, MO2 and MO7 had the greatest fruitset percentage (100%) (Table 2). It was found that crossing barriers exist between the MO1, MO5 and MO11 pumpkin lines (Table 2). The hybrid fruits were obtained from 30 different combinations (Table 3). The other remaining seeds of the fruits were found to be abortive or were not well filled out. The number of fruits set with normal seeds was determined for 28 fruits at nine different interspecific combinations. In the other fifty-one, the percentage of seeds set was very low, and the embryo did not develop normally. These results showed that C. moschata lines had different rate affinities to C. maxima. These results are similar to the results of the research by Yongan et al. (2002a). Based on these findings, the average seed set was about 12.0% (Tables 1 and 2). The seed traits of 28 interspecific hybrid fruits were evaluated and a high seed yield was obtained in 10 of them. The rate of high seed yield for interspecific fruit was found to be 4.27% (10 fruits/234 cross). Hybrid seed yield was found to be high in five different combinations with MO8 lines (Table 3). This may be because MO8 lines have a higher interspecific compatibility than the other genotypes. Otherwise, the hybrid fruits were not obtained at all combinations that used male parents of MO1, MO5, MO11 and female parents of MA3, MA5, MA8, MA14. MA4 × MO8 had the highest seed width at 15.18 mm, followed by MA1 × MO8 at 14.25 mm. The average seed length for these combinations varied from 17.11 to 23.23 mm. Seed thickness measurements ranged between 3.59 and 5.16 mm (Table 4). Considerable variability for seed dimensions was found
for these combinations. The highest seed weight was obtained with the MA9 × MO8 combination (51.43 g/100 seed weight). Seed number per fruit had an important effect on seed yield, and high seed numbers are required by seed companies. The best combinations for 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 demonstrated that substantial differences in seed germination rate on exist in the promising interspecific hybrid combinations. These combinations showed a range of 18.0% (MA12 × MO2)–72.98% (MA11 × MO8) for seed germination rate (Table 4). Doubtless, a high seed germination rate is a desirable trait. However, the seed germination rates of the promising interspecific combinations were not found desired at suitable levels in this study. Cucurbit breeders have long been interested in interspecific crosses between Cucurbita species. Selecting the best combinations of interspecific crosses is important for vegetable rootstock breeding. Nowadays, the interspecific rootstocks are used largely for 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 parent showed fertilization at different ratios depending on which line was used. Crossing incompatibility was found to be highest for the MO8 lines (C. moschata) in all combinations (Table 2). Reviewing the results of this study, the MA9 × MO8, MA12 × MO2 and MA4 × MO8 hybrid combinations seem to be the most
Table 4 Seed characteristic of the promising interspecific hybrid combinations. Combinations
Width (mm)
Length (mm)
Thickness (mm)
Seed germination rate (%)
100 seed weight (g)
Seed yield (g/fruit)
Number of seeds (n./fruit)
Seed abortion rate (%)
MA1 × MO8 MA11 × MO8 MA9 × MO8 MA12 × MO2 MA12 × MO8 MA4 × MO8 MA15 × MO10 P CV (%)
14.25 d 11.07 a 12.99 cd 11.38 e 12.43 c 15.18 e 12.05 b <0.001 2.7
21.24 b 20.25 c 17.11 d 19.90 c 21.00 b 21.77 b 23.23 a <0.001 2.1
5.16 a 5.10 a 4.24 b 3.59 b 4.96 a 5.00 a 3.67 b <0.01 8.2
25.00 e 72.98 a 63.39 c 18.00 f 62.21 c 56.07 d 65.06 b <0.01 9.4
34.99 ab 21.73 ab 51.43 a 28.32 b 34.35 ab 39.75 ab 31.50* <0.05 13.5
39.46 c 25.59 e 104.92 b 102.20 b 36.04 d 144.93 a 42.36 <0.001 2.4
116.32 d 122.21 d 207.19 b 361.47 a 148.49 c 365.25 a 125.42 <0.001 1.9
12.04 a 0.00 e 0.00 e 6.01 c 3.00 d 9.60 b 15.2 <0.001 11.7
*
These combinations gave only one fruit. For this reason, the statistical analysis was not reported.
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O. Karaa˘gac¸, A. Balkaya / Scientia Horticulturae 149 (2013) 9–12
promising rootstock for breeding (Tables 3 and 4). The seed yields have been determined in this study. However, the seed yields of these combinations were not found desired at suitable levels. Numerous crosses are often required to obtain a few viable seeds. Whitaker and Davis (1962) concluded that C. moschata occupies a central position among the annual species and can be crossed with difficulty with C. pepo, C. maxima and C. mixta. They exhibit a wide range of crossing barriers at both the presyngamic (failure of pollen tube growth) and postsyngamic (breakdown of embryo development) developmental phases. Many techniques, including bridge crosses (Zhang et al., 2012), bud pollination (Hayase, 1961), repeated pollination (Yongan et al., 2002b), and the use of growth regulators (Nascimento et al., 2007), have been employed in attempts to improve the success of interspecific hybridization (Lebeda et al., 2007). In Cucurbita, the successful culturing of embryos from mature fruits has been reported by ˇ sko et al., 2003; many researchers (Kwack and Fujieda, 1987; Siˇ Loy, 2012). Gene transfer between C. maxima and C. moschata has been difficult. Crosses between some cultigens of the two species yield neither seed nor fruit. The successes of the presented interspecific hybridizations were, in general, comparable to the published data. In the test of interspecific crosses, different cultivars within the species performed differently. For this reason, these interspecific hybrids derived from crosses of C. maxima × C. moschata also will be utilized as bridge crosses with backcross programmes in future breeding work. Thus, it will be increased both seed yield and seed viability of these combinations. Hybridization programs with these lines have been implemented. Acknowledgements We gratefully acknowledge the support of funding by the Ondokuz Mayıs University Research Foundation (PYO.ZRT.1901.09.015) and Black Sea Agricultural Research Institute (KTAE). We also appreciate comments on this manuscript by Prof. Dr. María Belén Pico of Polytechnic University of Valencia, Spain. References Baggett, J.R., 1979. Attempts to cross Cucurbita moschata (Duch.) Poir. ‘Butternut’ and C. pepo L. ‘Delicata’. Cucurbit Genet. Cooperat. Rep. 2, 32–34. Balkaya, A., Kurtar, E., Yanmaz, R., 2009. Evaluation and selection of suitable pumpkin (Cucurbita moschata Duchense) types from Turkey. Proceedings of the IVth Balkan Symposium on Vegetables and Potatoes, Plovdiv, Bulgaria, 6–10 September. Acta Hortic. 830, 55–62.
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