Abstract: Haploid induction potentials in Lycopersicon esculentum Mill. (2n=2x=24) via unilateral pollination with Solanum sisymbriifolium Lam. (2n=2x=24) pollen were investigated. In April, the cv. Invictus set 12 fruits out of 72 pollinations and, the remaining six cultivars did not set fruit. Whereas in May, from a total of 242 pollinations onto eight varieties, 26 fruits were obtained from only six of the varieties. In addition to the ovules obtained from the cv. Invictus fruits, cv. Sagit 146 fruits contained 65 ovules. While cv. Invictus ovules gave rise to plantlets, ovules from cv. Sagit 146 were lost due to contamination. Chromosome counting of eight in vitro plants of cv. Invictus showed cells with 24, 25 and 26 chromosomes. Haploid chromosome numbers were not observed in any of the cells studied. Potentials of the approach were also discussed.
Introduction
Tomato (Lycopersicon esculentum Mill.) is the most produced vegetable crop in the World (Anonymous, 2002) and variety improvement has been continuous to meet the demands of various crop production and protection practices and needs (Kalloo, 1993). In general, production of new varieties is realized via hybrid breeding methods by which production of F1 hybrid varieties, displaying characteristics of higher performance than their parents, require pure or inbred lines which are produced either by repeated selfings if applicable, or doubling the chromosome number of the haploids.
Anther culture, the commonly practiced method for the haploidy in tomato, has not met breeders’ satisfaction (Chlyah et al., 1990) and, alternative methods of haploid production have not been reported to date.
F1 varieties, containing desired characteristics introgressed via interspecific or intergeneric crosses, are mainly used in the tomato crop production (Kalloo and Banerjee, 1990; Kalloo, 1993; Stevens and Rick, 1994). For the production of F1 varieties Lycopersicon peruvianum, taxonomically most distant to tomato, L. chilense, L. hirsutum, L. pimpinellifolium and Solanum lycopersicoides have been used with varying degrees of success (Rick et al., 1987; Gradziel and Robinson, 1991; Lan Zhuang and Adachi, 1996; Chetelat et al., 1997; Doganlar et al., 1997; Vidavski et al., 1998; Egashira et al., 1999; Foolad and Lin, 2001).
Previous interspecific or –generic hybridization reports dealt only with gene introgression and perhaps overlooked its potential for the induction of haploids of parthenogenetic origin. In general, haploid production through the induction of a haploid cell of the embryo sac can be realized using interspecific or –generic crosses. In this way, either the haploid egg cell is induced to develop and produce the embryo (Hougas et al., 1964) or a complete chromosome set of one of the parents is eliminated from the developing diploid zygotic embryo (Kasha and Kao, 1970) resulting in haploids or doubled haploids.
Use of Solanum sisymbriifolium Lam., a member of the Solanaceae family, has been previously reported for gene introgression purposes in Solanum melongena (Bletsos et al., 1998). In addition, fruit set and plant development were determined when Solanum sisymbriifolium pollen was placed on the Lycopersicon esculentum stigmata, but the report lacked detailed information on pollen germination, pollen tube development in situ and the details of the progeny obtained especially regarding haploidy. Alternatively, however, some cells with the haploid number of 12, in addition to the diploid number of 24, were observed, but all the eight progeny developed into fertile plants (Chambonnet, 1996).
Therefore, bearing in mind the potentiality of haploid production of tomato via distant hybridization, the present work focused on unilateral intergeneric hybridization of Lycopersicon esculentum with Solanum sisymbriifolium Lam.
Materials and Methods
The experiments were conducted at the plastic greenhouses and laboratories of Department of Horticulture, Faculty of Agriculture, Cukurova University, Turkey during March-June 2001.
Growing of plants: Seeds of only one genotype of Solanum sisymbriifolium were obtained from Dr. M.C. Daunay, INRA, Montfavet, France and sown in the previous year in seed trays. Developing seedlings were grown in the field and pollen was obtained from fully matured plants. Similarly, seeds of a total of eight tomato varieties, namely Falcon, Gokce, Elif, Sagit 146, Invictus, SC2121, Y-410-F1 and Y-18-90, were sown in seed trays and seedlings at the 3-4 true leaf stage were transplanted to plastic greenhouses with 0.4 m and 1.25 m in and between row distances, respectively.
Pollinations: Pollinations were carried out twice both in April and May. Flowers, one to two days prior to anthesis, were emasculated by removing petals and others. Stigmas, now exposed to open air, were not further protected before or after pollination. Several pollinations were carried out onto each tomato variety and the S. sisymbriifolium combination and two to three unpollinated flowers were left as the control.
Observation of in situ pollen tube development: In order to observe pollen germination on the stigmas, pollen tube development in the style and entry of tubes into ovules, 24 and 48 hours following pollination, at least three samples of each combination of pollination were removed and fixed in 3:1 glacial acetic and ethanol. Following a washing of 12 h under running tap water, the samples were softened in 8 N NaOH for 6-8 hours and washed once more for 12 hours, then transferred to anilyn blue dye. Samples left in the dye for at least 24 h in the dark were then examined microscopically under UV light. The stock of anilyn blue dye was prepared by mixing of 1 g of anilyin blue and 11.28 g tripotassium phosphate in 250 ml of distilled water. A working solution was prepared by diluting one part of the stock solution in three parts of distilled water.
Rescue and culture of ovules: To overcome the risk of embryo abortion, fruits were harvested early in development. Ovules containing embryos were cultured following dissection for which fruits were removed 24 and 36 days after pollination and, immediately after, sterilized in 10% NaOCl solution containing 2-3 drops of Tween 80. Following sterilization, the fruits were washed three times in distilled water and cut open. Ovules observed were dissected out and cultured in jars containing basic MS medium (Murashige and Skoog, 1962) without growth regulators.
Chromosome counting from in vitro growing root tips: Tips of actively growing root tips, ca. one cm in length, were removed from the in vitro plantlets of 5-10 cm length. The root tips were collected at 10-11 o’clock in the morning and following a brief washing in tap water, transferred to 8-hydroxyquinoline solution for mitotic arrest at 4oC for 24 hours, then fixed overnight in a mixture of 3:1 ethanol and glacial acetic acid at room temperature. Before staining, the root tips were hydrolized in 1 N HCl at 60 oC for 15-20 minutes. The hydrolyzed root tips were stained in Feulgen for at least 2 hours in the dark and magenta coloured, i.e. ca. 2 mm long tips, were macerated on slides in a drop of acetocarmine making temporary slides.
Results and Discussion
Crosses were carried out twice, both in April and May 2001 and in the earlier pollination only 12 fruit sets (32.4%) were obtained from the cv. Invictus, being the only cultivar, among the seven, resulting in fruit set, out of a total of 237 pollinations (Table 1). In May pollinations, using two more cultivars, a total of 242 flowers were pollinated and fruit set was obtained in five of the eight cultivars (Table 2). While the highest fruit set was 25% from the cultivar Y-18-90, the lowest was 6.8 % from the cv. Elif. Regarding the cv. Invictus, the rate of fruit set decreased from 32.4% in April to 17.9% in May.
To confirm S. sisymbriifolium pollen tube growth and its entry into ovules, in situ pollen tube development was researched in flowers from May pollinations. Pollen tubes developed in situ through the style and entered the ovules within 24 hour of the pollination in all the cultivars except for the cultivars Y-410-F1 and Y-18-90 in which pollen tubes developed only to the halfway of the style. Pollen tube development in 48 hour samples was similar to the 24 hour samples in all the cultivars tested (Table 2; Fig. 1). Although pollen tube entry in the ovules was readily visible in six of the eight varieties, it is uncertain whether the fertilization took place. In the cv. Falcon, despite the observation of the pollen tube entry into the ovule, fruit set was not observed.
A total of 12 fruits obtained were from the cv. Invictus in the April pollinations and 10 of the fruits were cut open to rescue the embryos before a probable abortion. Of the 12 fruits obtained from the cv. Invictus in the April pollinations, 10 were cut open and seven of the fruits were empty without developing ovules. From the remaining three fruits a total of 14 ovules were dissected out and cultured. Ovules cultured were of three types, i.e. 10 were light green and larger then all, three medium and one small. Four large ovules and one medium, germinated approximately two weeks after the initial culture.
| Fig. 1: | Pollen tube entry into ovules of the cv. Invictus, 24 hours after pollination |
| Fig. 2: | A cell carrying 26 chromosomes in the plant number four |
Although seedling development was rapid following germination and despite being similar to ordinary tomato seedling development, morphology of the developing seedlings differed from the characteristics of ordinary tomato seedling growth in that stem and cotyledon leaves were thicker and lighter green and true leaves had more lobes.
Fruit set and ovule development varied for the cv. Invictus from 0, 17.9 to 32.4% for the three different pollination dates. This was similar to the results obtained from Lycopersicon esculentum x Solanum lycopersicoides crosses when the fruit set varied from the first year to the second. Flowers pollinated with Solanum lycopersicoides pollen set fruit 19.9 to 38 % for the first and second years, respectively. Similarly, in the first year, while 97 embryos from 38 fruits were obtained, 397 embryos were obtained from a total of 76 fruits in the second year (Chetelat et al., 1997).
| Table 1: | Fruit set and ovule culture from the cultivars pollinated with Solanum sisymbriifolium Lam. (Pollination date April 2001) |
| *: day/month of the year 2001 | |
Regarding the number of ovules and developing fruits, a similar instability was observed in L. peruvianum crosses (Doganlar et al., 1997; Egashira et al., 1999).
Although cross compatibility can be considered as the main factor for the instability, pollen viability may have also played an important role. The instability may have been the result of environmental factors or mechanical damages to the style during emasculation or pollination. A tomato flower’s style is tender in texture and can easily be damaged or broken from its base at its attachment point to the ovary. During pollination, even a little extra pressure on the stigma may result in style breakage. Such styles do not permit pollen tube development into the ovary and soon wither, contributing to lowering the rates of fruit set.
In our study, the fact that pollen tubes developed down the style, entered the ovary and fruits developed in most parental combinations, ovules developed only in the cvs. Invictus and Sagit 146 which can be attributed either to the induction of parthenocarpy by Solanum sisymbriifolium Lam. pollination or embryo breakdown early in the development in the probable intergeneric background. Stevens and Rick (1994) observed parthenocarpic fruit set in crosses between Lycopersicon esculentum x Lycopersicon pimpinellifolium. Also, cv. Red Cherry x cv. Bonny Best cross combination resulted in parthenocarpic fruit development in higher temperatures, whereas developing fruits set seeds only in temperate growing conditions. In Lycopersicon esculentum x Lycopersicon pimpinellifolium, crosses, however, despite the abortion of the embryos within 2-4 weeks of pollination, fruits continued developing to the full size (Ho and Hewit, 1994). The above suggests that in Lycopersicon esculentum x Solanum sisymbriifolium Lam. crosses, in order to obtain a progeny, whether haploid, dihaploid or hybrid, early abortion of the embryos must be taken into account and an earlier embryo rescue schedule should be considered. In addition, availability of temperate growing temperatures before and after fruit set may be significant and high daytime temperatures may have also been a factor in our experiments in the development of parthenocarpic fruits as the city of Adana, where the experiments were carried out, is located in a considerably warmer area.
| Table 2: | In situ pollen tube development and fruit set in cultivars pollinated with Solanumsisym briifolium Lam. (Pollination date: May 2001) |
| 1: | data not available |
| 2: | pollen tubes developed through to the middle of the style in both cases |
| 3: | ovules to be cultured were not available both for the cv. Falcon for which fruit development did not occur and for the other pollination combinations in which fruits did not contain ovules |
| 4: | All the cultured ovules were lost due to contamination following four days in culture. |
In the chromosome counts of five plants a total of 12 cells with 24, 25 and 26 chromosomes were determined. The chromosome numbers remained stable for each root tip and the plant studied. The progeny produced by Chambonnet (1996) between Lycoperscion esculentum x Solanum sisymbriifolium were fertile and carried the same chromosome number as the parents, i.e. 24. The author also observed cells with 12 chromosomes which is unlike our study where not a single haploid chromosome cell was observed. The chromosome numbers determined were 24, 25 and 26 (Fig. 2). It may be that the progeny carrying the exact and complete number of chromosomes of tomato, i.e. 24, are doubled haploids. The chromosome number doubling may have been induced by the alien pollen, i.e. of Solanum sisymbriifolium, determination of which requires further work.
The progeny with the extra chromosomes, however, suggests that the plants may have been trisomic or tetrasomic. Although previous reports on Solanum melongena and Solanum sisymbriifolium and Lycopersicon esculentum and Lycopersicon peruvianum pollinations resulted in a progeny with 24 chromosomes (LanZhuang and Adachi, 1996; Bletsos et al., 1998), in a different Lycopersicon esculentum and Lycopersicon peruvianum hybridization experiment the progeny obtained resulted in chromosome numbers varying from diploid to triploid. It was thought that the extra chromosomes could be tolerated by the hybrid background of the progeny and tomato plants could tolerate up to three extra chromosomes and the number tolerated could rarely be four (Quiros, 1991).
Genotypic effect is significant in the haploid production via wide hybridization as in the cases of Triticum aestivum with Zea mays (Bitsch et al., 1998) and Penisetum americanum (Matzk and Mahn, 1994) and triticale with Zea mays (Wedzony et al., 1998). It is already known that pollinations onto Lycopersicon results in varying degrees of fruit set, ovule and embryo development depending on the parental combinations. The fact that only one Solanum sisymbriifolium genotype as the male parent in our study may provide limited information regarding the effects on the induction of Lycopersicon haploids. Therefore, the performance of other Solanum sisymbriifolium genotypes in combination with the varying genotypes of Lycopersicon esculentum remains to be tested.
Results, in general, indicate that Lycopersicon esculentum can be crossed with Solanum sisymbriifolium with a certain degree of success and further investigation is needed in order to determine full potentials of this approach in tomato breeding.