Author: Rakha, M T; Metwally, E I; Moustafa, S A; Etman, A A; Dewir, Y H
Date published: January 1, 2012
Doubled haploidy is the fastest route to homozygosity in plants. Resulting doubled haploids are useful in fixing traits rapidly in a line/variety and facilitate hybrid breeding. Haploid plants can be generated from microspores through direct embryogenesis or via a callus phase in both anther and isolated microspore culture (Wang et al., 2000). Chromosome doubling, either spontaneous or induced, converts haploids into doubled haploid plants. In a further step, homozygous plants can be obtained in a single generation, facilitating breeding programs tremendously (Forster and Thomas, 2005). In addition to the short period of time required to obtain inbred lines for F^sub -1^-hybrid, haploid technology facilitates selection for recessive and polygenic traits and revealed to be of benefit in genetic transformation and mutation studies as well (Ferrie et al., 2008).
Pure lines are considered as the first step in genetic improvement of vegetable crops as the lines may be the source of superior hybrids new cultivars in self pollinated crops, and high yielding hybrids (Veilluex, 1994; Metwally et al., 1998a). The production of pure lines in vegetable crops especially from open pollinated plants such as Cucurbita requires several years of conventional plant breeding program (Bajaj, 1990; Metwally et al., 1998a). On the other hand, the morphogenesis of the gametophytic cells is remarkably propice for the rapid production of homozygous lines and the detection of mutation. Thus, in vitro anther and/or ovule culture may be used to generate homozygous lines through the production of haploid plants. Although anther culture has been reported to be more efficient for haploid induction compared to ovary/ovule culture (Bajaj, 1990; Ferrie et al., 1995), one of its drawbacks consist of the female plants and male sterile plants that may result from (Doctrinal et al., 1989). Squash (Cucurbita pepo L.) is the most widely grown and polymorphic among the Cucurbita species. The production of haploids in squash has been reported through anther culture (Metwally et al., 1998a), unpollinated ovules (Metwally et al., 1998b) and irradiated pollen technique (Kurtar et al., 2002). Recently, Shalaby (2007) studied some factors affecting in vitro gynogenic embryo yield. The objectives of the present study were to evaluate the potential for in vitro regeneration of interspecific Cucurbita hybrids through anther and ovule cultures, to produce double haploid plants and to assess the vegetative and reproductive traits of the regenerated genotypes in the field.
Results and discussion
Effect of sucrose and 2,4-Dichlorophenoxyacetic acid (2,4-D) concentrations and hybrids on anther culture efficiency
Sucrose and 2,4-D concentrations had a significant impact on androgenesis of Cucurbita hybrids. The combination of 90 g l^sup -1^ sucrose and 1 mg l^sup -1^ 2,4-D was optimal for callus formation and plantlets regeneration for each hybrid. There was a trend towards a decrease in callus formation and plantlets regeneration with increasing either sucrose or 2,4- D concentrations. Callus regeneration was null when the medium was amended with 150 g l^sup -1^ sucrose and 1 or 5 mg l^sup -1^ 2,4-D with the exception for C. pepo L. (Eskandarani cv.) × C. moschata L. Significant differences were observed between the various interspecific hybrids in terms of percentage of callus giving plantlets and number of plantlets per callus (Table 1). Twenty one treatments out of 36 formed neither callus nor plantlets. The anthers resulting from C. pepo L. (Queen F^sub -1^) × C. moschata L. gave the highest percentage (40%) of fertile callus and an average of 7.5 plantlets per callus when the culture medium was supplemented with 90 g l^sup -1^ sucrose and 2,4-D at 1 mg l^sup -1^ . No plantlets were regenerated from the anthers of C. pepo L. (MHTC77 F^sub -1^) × C. martinezii L. Genotypes and medium composition are key factors influencing embryo/callus induction and subsequent plant regeneration. Androgenic response in Cucurbita pepo L. (Shail and Robinson, 1987) and haploid induction in carrot (Gorecka et al., 2005) have been reported to vary according to the genotype. In addition of being a source of carbon and energy, sugars are known to act as an osmotic regulator in the induction medium. The presence of sucrose in the induction medium has been found to influence androgenesis and embryogenesis in many species (Ferrie et al. 1995) However, its concentration in the medium seems to have a differential effect on different species. For example, concentrations of 30, 60, 100 and 150 g l^sup -1^ sucrose were found to be optimal for embryogenesis in Quercus suber (Bueno et al., 1997), Oryza sativa (Afza et al., 2000), Avena sativa and A. sterilis (Kiviharju and Pehu, 1998) and Cucurbita pepo (Metwally et al., 1998a), respectively. The negative effect of high sucrose concentration on plantlets regeneration may be due the rapid hydrolysis of sucrose into fructose and glucose which in turn increases the medium osmosis as reported previously in wheat (Navarro-Alvarez et al., 1994).
Effect of cold treatment and 2,4-D on ovule culture efficiency
The type of hybrid significantly affected plantlet and callus regeneration from ovules and the number of plantlets obtained per ovule and responded ovule rates as well (Table 2). Overall, 75% (27) of the treatment combinations did not generate plantlets. Ovules from the hybrid C. pepo L. (MHTC77 F^sub -1^) × C. moschata L. produced the highest percentage of regenerated plantlets (25.33%), the highest percentage of responded ovules (18.67%), and the highest average number of plantlets per ovule (3.37) in opposite to C. pepo L. (Queen F^sub -1^) × C. ficifolia L. and C. pepo L. (Jedida F^sub -1^) × C. moschata L.. Among treatments that produced calli, C. pepo L. (MHTC77 F^sub -1^) × C. moschata L. generated the highest percent 9% versus 5 and 3% from C. pepo L. (Queen F^sub -1^) × C. ficifolia and C. pepo L. (Queen F^sub -1^) × C. moschata L. (Table 2). Ovules from the interspecific hybrid C. pepo L. (MHTC77 F^sub -1^) × C. martinezii L. did not respond to in vitro culture (data not shown). These results are consistent with previous repports on Cucurbita (Dumas de Vaulx and Chambonnet, 1986; Kwack and Fujieda, 1988; Shail and Robinson, 1987). Arafeh (2006) reported that the genotype proved to be one of the most important factors affecting in gynogenesis in summer squash. In sugar beet, the yield of haploid plants depended on the genotype, and the effect of genotype was significant only for certain media (Lux et al., 1990) as shown in the present study. These differences indicated that genotype specific results can be obtained depending on the genetic constitution of the individual used hybrids. In the present study, cold pretreatment had deleterious effects on gynogenesis except for the interspecific hybrid C. pepo L. (MHTC77 F^sub -1^) × C. moschata L. Temperature shocks are believed to improve gynogenesis by diverting normal gametophytic development into a saprophytic pathway leading to the formation of haploid embryos. Lux et al. (1990) reported that cold pretreatment of sugar beet flower buds at 4 oC for 4 - 5 days led to increased embryo yield in consistence with other reports on Cucurbita moschata L (Kwack and Fujieda, 1988), onion (Hanna, 1994) and Cucurbita pepo (Shalaby, 2007), On the other hand, in consistence with our results, a deleterious effect of cold pre-treatment was reported by Metwally et al. (1998b) on squash and by Arefeh (2006) on flower buds. The combination of 2,4-D with cold pretreatment had a significant effect on gynogenesis in Cucurbita interspecific hybrids. The highest percentage of regenerated plantlets (25.33%) and highest number per ovule (3.37) were obtained when the ovules of C. pepo L. (MHTC77 F^sub -1^) × C. moschata L. were cultured on media supplemented with 5 mg l^sup -1^ and cold pretreated for 7 and 14 days, respectively. Kwack and Fujieda (1988) indicated that plant growth regulators (PGRs) are important for embryogenesis of Cucurbita moschata. Bridgen (1994) found that somatic embryo induction was inhibited by high concentrations of exogenous auxin in the medium and stimulated by low concentrations. According to Evans et al. (1981), the primary medium for induction of somatic embryogenesis includes 2,4-D for the majority of crop species. Also, Campion et al. (1992) found that 2,4-D gave good results in both ovary and flower culture of onion.
Plantlets hardening and mitotic studies of androgenic and gynogenic genotypes
Androgenic and gynogenic plantlets were separated and cultured individually into MS medium without PGRs. Supplementation of MS medium with NaCl was beneficial for establishing a good root system. Plantlets cultured on medium without NaCl developed thin, friable roots and could not survive in greenhouse. Plantlets grown a medium amended with 1 mg l^sup -1^ NaCl developed 2 - 3 thick roots with 1- 2 cm in length (Fig. 1A). The plantlets were hardened in a growth chamber for 2 weeks followed by one week in greenhouse prior to their transplanting in the field (Fig. 1B, C and D). Chromosome count (Fig. 2 D, E) in root tips of the androgenic plantlets revealed that 50% of plants were haploid (n = x = 20) and the other 50% were dihaploid (2n = 2x = 40). The analysis of root tips of gynogenic plantlets revealed 60 % of haploid (2n = x = 20) and 40% of diploid plants (2n = 2x = 40). In summer squash, plants regenerated from anther and ovule culture where 50% and 25% where haploid, respectively, (Metwally et al 1998 b; Shalaby 1996) whereas plants derived from ovule cultures of Rosina F^sub -1^, Gabbla cv., and Eskandarani cv. were 64, 58, and 41% haploid and 36, 42, and 59% dihaploid (Arafeh, 2006) To the contrary, all species from the Cucurbita genus were found to be dihaploid (2n = 2x = 40) (Robinson and Walters, 1997).
Field evaluation of vegetative traits in androgenic regenerated strains
Among 200 dihaploid regenerated plantlets, only 10 androgenic strains produced seeds while all other plants were either weak or abnormal. These 10 strains as well as a local open cultivar (Eskandarani) were evaluated for their traits. Table 3 indicates that the differences among genotypes were highly significant for all the vegetative traits. Strain 5 had the highest stem length, leaf area and fresh and dry weights per plant while strain 7 had the highest number of leaves per plant. Strain 8 had the smallest values of all the vegetative traits whereas Eskandarani showed intermediate values regardless of the trait. These evaluated strains were obtained from anther culture of interspecific hybrids between the cultivated species of C. pepo L. and other cucurbits species viz., C. moschata L., C. ficifolia and C. martinezii L. This indicates that the majority of the obtained strains had intermediate vegetative traits between the cultivated species of C. pepo L. and other Cucurbita species. It has been found that variance of stem length of the short species C. pepo L. was less than the variance of the long species C. moschata L. (Hassan et al. 1984). In summer squash, Eskandarani plants produced the largest values for all the vegetative traits evaluated, compared with its 16 dihaploid lines (Metwally et al.,1998c).
Evaluation of flowering and yield traits in double haploid genotypes
The differences among genotypes were highly significant for all the flowering traits (Table 4). The number of days to the first male flower ranged between 38.6 days for Eskandarani and 55.3 days for strain 1 whereas the number of days to the first female flower ranged between 35.0 days for Eskandarani and 54.3 days for strain 2. Strain 10 and 3 and 5 produced the largest number of male and female flowers per plant respectively, and Eskandarani cv. only produced lower numbers of male and female flowers compared to the induced strains. The lowest and highest sex ratios was observed for strains 5 (0.9) and strain 7 (1.6) (Table 4). Eskandarani cv. produced nearly equal number of male and female flowers per plant and therefore, sex ratio was nearly equal to one. The above results indicate that female flowering occurred earlier than male flowering. It has been reported that female flowering occurs one or two days before male flowering (Metwally, 1985). In summer squash, the number of days from planting to the first male flower differed between dihaploid lines and ranged from 39.8 to 50.6 days (Metwally et al., 1998c). The same authors reported 43.3 day-period between planting to the first female flower and a sex ratio of 1.3 for Eskandarani cv. For fruit characteristics, the highest fruit weight (127 g), fruit length (15.6 cm), fruit diameter (3.5 cm) and early yield per plot (23.9 Kg) were obtained from Eskandarani cv. while strain 3 had the lowest values. The highest number of fruits in terms of early yield (308.4) and total yield (911.2 g) per plot were obtained from strain 6 (Fig. 3 A). Strain 5 recorded the highest total yield per plot (45.1 kg). The lowest values of early and total yield per plot were obtained from strain 1 and 2 (Fig. 3 B). It has been found that fruit shape of F^sub -1^ plants of the cross between C. moschata L. × C. maxima was intermediate (Robinson et al. 1978). In summer squash, fruit traits, i.e., average fruit weight, length and diameter, the differences within dihaploid lines and Eskandarani cultivar were highly significant (Metwally et al. 1998c). In conclusion, the present study investigated androgenesis and gynogenesis of six Cucurbita interspecific hybrids. Genotypes and medium composition influenced the plantlets regeneration from anther and ovule cultures. There was a trend towards decreasing callus formation and plantlets regeneration from anthers with increasing either sucrose or 2,4-D concentrations. In most cases, cold pretreatment had negative effects on gynogenesis. Evaluation of the androgenic regenerated plants revealed that the majority of strains were intermediate in their traits between the cultivated species of C. pepo L. and other Cucurbita species. However, two regenerated strains proved superior to the local cultivar 'Eskandarani' in number of fruits (early and total yield/plot) as well as total fruit yield per plot.
Materials and methods
Six interspecific hybrids of Cucurbita were used to obtain male and female flower buds. The combination consisted of C. pepo L. (Eskandarani cv.) × C. moschata L. (Nigerian local cv.), C. pepo L. (Queen F^sub -1^) × C. moschata L. (Nigerian local cv.), C. pepo L. (Jedida F^sub -1^) × C. moschata L. (Nigerian local cv.), C. pepo L. (MHTC77 F^sub -1^) × C. moschata L. (Nigerian local cv.), C. pepo L. (Queen F^sub -1^) × C. ficifolia L. and C. pepo L. (MHTC77 F^sub -1^) × C. martinezii L. The hybrid C. pepo L. (MHTC77 F^sub -1^) × C . martinezii L. was produced through embryo culture technique and grown in the experimental farm of Kafrelsheikh University using the same technique described by Metwally et al. (1998a).
Sterilization of male flower buds
Cytological observations on anther's microspores were conducted to determine their developmental stages (Fig. 2 A, B and C). Male flower buds having a length of 9 - 10 mm and containing anthers at the mid to late uninucleate microspores were collected in the early morning and exposed to cold temperature at 4 °C for 4 days. The buds were surface sterilized for 60 s in 70% ethanol followed by 20 min in 5.2% sodium hypochlorite solution and 2 - 3 drops of Tween-20 on a gyratory shaker at 150 rpm. Buds were aseptically washed three times with sterile distilled water.
Anthers without filaments from the six aforementioned Cucurbita interspecific hybrids were excised and cultured on MS medium containing 0.8% agar (Murashige and Skoog, 1962) and supplemented with various concentrations and combinations of sucrose at 50, 120 and 150 g l^sup -1^ and 2,4-D at 1.0 and 5.0 mg l^sup -1^. Sucrose and 2,4-D were added to the medium prior to autoclaving at 121 oC and 1.1 kg cm-2 pressure for 20 min and the pH was adjusted to 5.8 ± 0.1 before autoclaving as well. Anthers were cultured in 100 × 15 mm sterile Petri dishes containing 30 ml of MS medium. Four replicate-Petri dishes were cultured with five anthers each and the dishes were incubated in dark for 1 week at 35 oC followed by 9 weeks at 25 ± 1 oC. All cultures were subcultured in the same conditions described above once after 5 weeks of initial culture. For callus differentiation, calli were numbered and transplanted to MS medium supplemented with 0.0494 mg l^sup -1^ kinetin and 0.05 mg l^sup -1^ 1- Naphthalene acetic acid (NAA) and grown for 4 weeks before being re-transplanted to MS medium free of PGRs for 4 additional weeks. The cultures were incubated at 25 ± 1 oC, with a16 h phtoperiod at 40 Ê mol m-2 s-1 photosynthetic photon flux (PPF) provided by cool white fluorescent tubes. After 18 weeks of anthers' culture, the percentage of callus which gave plantlets and number of plantlets per callus were recorded.
Sterilization of ovaries and ovule culture
At flowering stage, ovaries from the six Cucurbita interspecific hybrids were picked a day before anthesis as reported perviously (Metwally et al., 1998b; Dumas de Vaulx and Chambonnet, 1986). The buds were exposed to cold pretreatment at 4 oC for either 0, 7 or 14 days.
Thereafter, peduncles, petals and styles were removed. The ovaries were surface sterilized for 60 s in 70% ethanol and aseptically washed three times with sterile distilled water. The ovules were excised from ovaries and cultured for 4 weeks on MS medium supplemented with 30 g l^sup -1^ sucrose and 2,4-D at 1 and 5 mg l^sup -1^ followed by 4 weeks on PGRsfree MS medium. The pH of the medium was adjusted to 5.8 ± 0.1 and the medium was autoclaved as explained above. Four-replicates 100 mm Petri dishes were cultured with 100 ovules each and dishes were incubated at 25 ± 1 oC under a 16 h photoperiod. After 8 weeks, the numbers of regenerated plantlets, responded ovules, plantlets per ovule and callus formed were recorded.
Plantlets hardening and mitotic studies
Regenerated plantlets from anthers and/or ovules cultures were cultured individually in jars containing PGRs-free MS medium for 4 weeks after what the root system was cut off and shoots were transplanted into jars containing 50 ml of MS medium supplemented with 0.5 mg l^sup -1^ NaCl and grown for 10 days. The root system was cut off one more time and shoots were cultured for 10 days on fresh medium supplemented with 1 mg l^sup -1^ NaCl. The plantlets were harvested, washed under tap water and cultured in pots containing sterilized peat moss and the pots were covered with clear plastic bags and kept in growth chamber for 2 weeks. The grown plantlets were grown in a greenhouse for 1 week prior to their transplanting to the field. For mitotic studies, root tips from 20 androgenic as well as 20 gynogenic plantlets were observed using a light microscope. The root tips were fixed for 3 h in 0.05% aqueous colchicine, then for 24 h in a solution of ethanol and glacial acetic acid (3:1; v/v), and finally stained with acetocarmine stain as described by Darlington and Lacour (1976).
Evaluation of regenerated strains through androgenesis and gynogenesis
Two hundred dihaploid plants selected from Cucurbita interspecific hybrids were planted in greenhouse. The plants were self pollinated at flowering stage and seeds were obtained from vigorous plants (strains) while abnormal plants were excluded. In total, 10 androgenic strains as well as the open pollinated cultivar (Eskandarani) were evaluated. Three seeds of each strain were sown in hills on the north side of ridges (6 × 1 m) and planting space of 30 cm. The experiment was set up in a randomized complete block design with three replicates. The field was immediately irrigated after sowing. Fertilizers were added to the soil as follows: 1) old cattle manure was added at the rate of 47.6 m-3 per ha before planting. 2) commercial fertilizers were applied as a mixed fertilizer having 1: 2: 1 ratio with a rate of 238 kg per ha. Ammonium sulphate, super phosphate and potassium sulphate were used as sources of N, P and K, respectively. One half of the fertilizers' mixture was applied 14 and 21 days post-sowing. For evaluation of vegetative traits, three plants were up-rooted from each plot after 45 days of planting. Stem length, number of leaves per plant, fresh and dry weights per plant and leaf area per plant were recorded. Leaf area was determined by LI-3100 area meter (LI-COR, Ing. Lincoln, Nebraska, USA) and dry weights were determined after leaves were dried for 48 h at 70 oC. During flowering stage, five plants in each plot were labeled, and total number of staminate and pistillate flowers during the whole flowering period, number of days to opening of the first pistillate and staminate flowers, number of pistillate and staminate flowers per plant and sex ratio (male/female flowers) were recorded on labeled plants. For fruit characteristics and yield assessments, the pistillate flowers were labeled in each plot and the fruits were picked 3 days post-flowering. Fruit weight, fruit length and fruit diameter were recorded. Fresh fruits were picked at 2 days intervals, where the number and weight of fruits were recorded. Early yield (fruits yield of the first five pickings) and total yield (fruits yield of all pickings) in terms of fruit number and weight were recorded.
Data were subjected to ANOVA and Duncan's multiple range test using SAS program (Version 6.12, SAS Institute Inc., Cary, USA).
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M.T. Rakha, E.I. Metwally, S.A. Moustafa, A.A. Etman, Y.H. Dewir*
Horticulture Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt
* Corresponding author: firstname.lastname@example.org