Hybridization between Lycopersicon esculentum Mill. and Lycopersicon pennellii Cor.: Pollen Fertility and Viability in F 1.

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1 767 Bulgarian Journal of Agricultural Science, 12 (2006), National Centre for Agrarian Sciences Hybridization between Lycopersicon esculentum Mill. and Lycopersicon pennellii Cor.: Pollen Fertility and Viability in and F 2 Hybrids V. NIKOLOVA and V. RODEVA Maritsa Vegetable Crops Research Institute, BG Plovdiv, Bulgaria Abstract NIKOLOVA, V., and V. RODEVA, Hybridization between Lycopersicon esculentum Mill. and Lycopersicon pennellii Cor.: Pollen fertility and viability in and F 2 hybrids. Bulg. J. Agric. Sci., 12: In result of this investigation it was confirmed very high level of incompatibility between L. esculentum genotypes - line N and Balka and self-incompatibility L. pennellii accession N Different preventing answers to the pollen germination and penetration in the stigma, style and ovary tissue and in the ovules in the reciprocal crosses of the both studied species were observed. In self pollination of an F 2 hybrids and also in backcrossing with the both parents were registered the same type of strong expressed incompatibility. Compared to the plants with high pollen fertility and semi-sterility lower level of incompatibility after self pollination was registered in F 2 generation in a plant with 10% fertility. Successful pollen germination and entering the tubes in the ovules was established only in pollination of mother plants L. esculentum (Balka ) with pollen from hybrid N 336. Key words: incompatibility, in vivo pollen viability, pistil reaction, hybrid and backcross progeny Introduction Genetic variability, the most important base in tomato breeding process, is very limited in self-pollinating Lycopersicon species compared to the self-incompatible representatives of this genus. Crosses between cultivated tomato and most wild relatives are possible when L.esculentum serves as female parent (Causse et al., 2000). Such unilateral incompatibility due to the inhibition of pollen germination and pollen tube growth on stigma and in style or ovaries often occurs in case that selfcompatible species are used as male parent in crosses with self-incompatible species. In tomato this occurs mostly between red-fruited self-compatible and greenfruited self-incompatible species (Hogenboom, 1972a). Lycopersicon pennellii is a green fruited wild specious, which was proven to bear drought and pests resis-

2 768 V. Nikolova and V. Rodeva tance. It is classified as a member of the group of the facultative species with selfcompatible and self-incompatible accessions (Kalloo, 1991). The research reported herein was aimed to studying the ability of the self compatible L.esculentum genotypes to hybridize with the self-incompatible accessions of L. pennellii and establishing the level of gametophytic compatibility between them. Material and Methods The genotypes used in this experiment were L.esculentum (cat N obtained from IPGR, Sadovo, Bulgaria and Balka ) and > L.pennellii - self-incompatible accessions (cat N , obtained from Botanical Garden, Nijmegen, Holland). Standard techniques for emasculation and pollination were applied for obtaining of hybrids and backcross progeny in greenhouse., F 2 and ВС 1 Р 1 plants were obtained from seeds or in result of embryo culture. The pollen fertility in, F 2 and in the parent genotypes was determined by staining with 4% acetocarmine and glycerin (1:1), and the viability in vitro, by pollen germination on nutrient medium after 6 hours incubation. The pollen tube length (µm) was measured by micrometer under the microscope. To investigate the pollen grains - stigma interaction, pollen viability was studied in vivo by fluorescence microscopy. The pistils were excised 24 hours after pollination of open flowers and buds and stained in Naether solution (containing aniline blue), then squashed in drop of glycerol. Since non germinated pollen grains were washed during the staining, the percentage of germinated pollen grains on the stigma cannot be given. The next variants of pollination and examination of pollen viability in vivo were studied: L.escul. (line N ) x L.escul. (line N ) L.penn.(accession N ) x L.penn. (accession N ) L.escul. (line N ) x L.penn.(accessionN ) - (N221 ) L.escul. (Balka ) x L.penn. (N ) - (N336 ) N221 x N221 F 2 N221 x F 2 N221 BC 1 ( N221 x L.escul. line N ) BC 1 P2 ( N221 x L.penn. N ) N336 x N336 BC 1 ( N336 x L.escul. Balka ) Results For transfer a tolerance to biotic and abiotic stress from L.pennellii species in the genome of the L.esculentum varieties a hybridization between both species was performed. The crosses that were made confirmed the existence of incompatibility barriers between species L.esculentum (line N and Balka ) and L.pennellii (accession N ). This forced the performance of preliminary in-

Hybridization between Lycopersicon pennellii... 769 Table 1 Pollen fertility and viability of L. esculentum and L.

3 Hybridization between Lycopersicon pennellii Table 1 Pollen fertility and viability of L. esculentum and L. pennellii genotypes participated in hybridization Genotype Fertility, In vitro, Pollen tube s length, % viability % µ m Line N L. esculentum Balka L. pennellii Accession N (N 221) Line N x N (N 336) Balka x N Fig.1. The pollen behavior in vitro in self-pollination of L. esculentum vestigations in order to establish the pollen fertility and in vitro viability in the studied genotypes. The data from Table 1 show that both studied genotypes of the cultivated species possess high pollen fertility ( %) and in vitro pollen viability (germination % respectively and pollen tube length and µm respectively). Normal germination and penetration of pollen was established in vivo in self pollination of L.esculentum plants (Figure 1). The elongation of the pollen tubes was normal in all investigated pistils and it was registered invasion of the tubes in the ovules (Figure 2). In the other parent L.pennellii N the pollen fertility and in vitro viability were 58.7 and 38.4% respectively. The pollen tube's length was shorter (158.8µm). It is known that in vivo mechanisms exist preventing pollen tube penetration in the stigmas, styles, ovaries and ovules in self pollination of self-incompatible L.pennellii. In the period 2000/2001 year in vivo behavior of the own L. pennellii pollen was studied in accession N h after its laying on the stigma large differences in the pollen germination and the elongation of pollen tubes among the studied pistils were observed. In most of them the pollen did not germinated on the stigma after controlled self

770 Fig. 2. Invasion of the pollen tubes in the ovules in self-pollination of L. esculentum pollination and washed out easily in the staining procedure.

4 770 Fig. 2. Invasion of the pollen tubes in the ovules in self-pollination of L. esculentum pollination and washed out easily in the staining procedure. There were also pistils with a small number of pollen grains growing on the stigma with very short pollen tubes often curved in back or apart direction. Wavy folded pollen tubes entering in the styles, where arrested their growth were also observed. It was very rarely in single pistils to find a few pollen tubes to fluorescent around the ovules, but penetration in them was not established. Regardless of the established values of the pollen fertility and in vitro viability, which allow fertilization, obtaining of hybrids between the both studied species was difficult. This is the reason in vivo pollen behavior on the stigmas and in the styles and ovaries after reciprocal crosses to be followed. In the result of our fluorescent microscopy strongly expressed barriers of incompatibility were observed in hybridization of the cultivated genotypes of L.esculentum - line N and Balka V. Nikolova and V. Rodeva with L.pennellii pollen from N Pollen germination on the stigma in the most of the studied pistils was not observed. In a small number of the pistils part of the pollen grains germinated with deformed tubes, frequently wavy folded and not reached to the ovules. This strong incompatibility reflected on preventing of fertilization and lack of seeds in the small amount of obtained fruits. In the combination L.esc. line N х L.penn. N one single fruit was developed with two undeveloped embryos and in the combination L.esc. Balka x L.penn. N were received three seeds and two undeveloped embryos. In progeny in both studied crosses six plants (1 and 5 plants, respectively) were grown. This occasionally obtaining of hybrid plants is probably due to random overcoming of strongly expressed barriers of incompatibility in the studied parent combinations. The flowers dropped off after pollination in the reciprocal crosses which confirmed the unilateral incompatibility established by Hardon (1967) and Rick (1969) and impossibility to obtain of hybrid plants in the case when L.pennellii (N ) participated as a mother parent. In order segregating progenies to be obtained, the pollen fertility and in vitro viability were studied in F1 hybrids N221 (L.esc. linen x L.penn. N ) and N336 (L.esc Balka x L.penn. N ) (Table 1). The data from the Table 1 show, that in hybrid N221 the pollen fertility is comparatively high %, but the pollen grain viability is very low. The percentage of the pollen grains, germinated after sowing on the medium in vitro was only The length of pollen tubes was shorter µm, compared to this of the studied par-

Parallel with studying of pollen fertility and in vitro viability in progeny the behavior of pollen in vivo was also studied in controlled self-pollination of the hybrid plants.

5 Hybridization between Lycopersicon pennellii... ent genotypes. The pollen fertility of the second studied hybrid N336 was lower (44.2%) compared to the value of this characteristic in hybrid N221, but on the medium the viability of the pollen grains was higher %. Parallel with studying of pollen fertility and in vitro viability in progeny the behavior of pollen in vivo was also studied in controlled self-pollination of the hybrid plants. Strongly expressed incompatibility, typical for the representatives of L. pennellii, was observed. The pollen germinated in the self-pollination of hybrid No 221 in, but its tubes had stopped in the stigmas and did not reach the styles at all. We observed the abnormally growing pollen tubes frequently - irregular direction of growth, enlargement of the tubes, etc. (Figure 3). In result of in vivo investigation we established that the self-incompatibility in is comparatively more slightly expressed in the other hybrid N336. Great amount of pollen on stigmas was observed in the most flowers in self-pollination. Pollen reaction was different - small part of it (4 to 30 grains) germinated and their tubes either 771 reached 1/3 from the style length and stopped their growth or strongly folded and winded as a globe in style tissue. Flowers were observed in which individual pollen tubes fluoresced around the ovules but didn't penetrate in (Figure 4). Five-six pollen tubes penetrating in the ovules were registered only in one pistil. The data from the self-pollination of both hybrids show that the barriers of incompatibility in first hybrid progeny was determined from one side by the lower pollen fertility and viability. From the other side the existence of the mechanisms that inhibited pollen tube growth in the stigma, style and ovary tissue, disturbed their orientation and most often did not allow them to reach the ovules. This incompatibility reflected on total sterility and forming of parthenocarpic fruits of the greater part from the studied hybrid plants. In the next generation of No 221 the pollen fertility in some of the obtained F 2 plants was studied. Segregation by this character was established - the fertility of a part of the plants was close to this of the Fig. 3. Pollen grains-stigma interaction in self-pollination of the hybrid N 221 Fig. 4. Pollen tubes fluoresced around the ovules without penetrating in

6 772 parent genotypes, while in the other part it was strongly reduced. It was studied the in vivo pollen behavior in self-pollination and in backcrossing with both parent components in plant N10 with pollen fertility 90.0%; in plant N1 with low pollen fertility 10.0% and in plants NN3,6,7 and 9 with fertility from 34 to 55%. In controlled selfpollination of the plants from F 2 progeny the pollen did not germinate on the stigmas in greater part from the flowers of the hybrids with semi-sterility and with high fertility, regardless of the comparatively high values of the observed fertility. In another flowers the pollen germinated with very short pollen tubes, which did not penetrate in the styles. Only in inbreeding of plant N 1 (with fertility 10.0%) it was observed pollen tubes penetrating in the styles, but entering in the ovules was observed only in one flower. In this case pollen tubes reaching the ovules might be due to the small amount of fertile pollen fallen and germinated on the stigma which to a certain extent reduced the pistil tissue negative reaction. Described barriers of incompatibility established in self-pollination of second progeny were observed once again in crossing of F 2 (No221) high fertility and semi-sterility plants with L.esc. line N for obtaining of BC 1. In this case a lot of pollen grains in pollinated open flowers germinated and pollen tubes penetrating the stigma papillae and growing into the styles were observed. The elongations of the tubes had stopped in the style tissue and did not reach the ovaries. We observed a few germinated pollen grains, penetrated the stigma papillae in bud pollinated pistils, but their tubes did not enter in the styles. The strong incompatibility established in the parent form L.pennelli influenced V. Nikolova and V. Rodeva on pollination of F 2 hybrids for obtaining of BC 1 P 2 progeny. In backcrossing of F 2 with parent form L.pennellii - (N221 x L.pennellii N ) the incompatibility barriers were more effective than those in obtaining of BC 1. We did not observe pollen grains on the stigmas. This indicated that in this case the L.pennelli pollen grains were not able to germinate and could not remain on the stigma during staining procedure. As a result BC 1 P 2 progeny was not obtained. During backcrossing in of the second studied hybrid (N336) with L. esculentum (Balka ) (ВС 1 ) it was established that in some flowers the incompatibility was strongly expressed and the pollen did not germinate on the stigmas. In the many flowers part of the pollen grains germinated with short tubes which stopped early their growth. Deformation of the tubes or changing of their growth direction was also observed. Frequently the pollen tubes winded at a globule in the top part of the ovaries. Only in case when Balka (mother parent in the hybridization) was pollinated from hybrid N336 barriers of incom- Fig. 5. The pollen behavior in vitro in the cross L. esculentum (Balka ) x N 336

7 Hybridization between Lycopersicon pennellii... patibility were not observed or their expression was very slight. Most frequently pollen grains germinated on the stigmas and their tubes penetrated in the ovaries and ovules (Figure 5). The low incompatibility here in some flowers was manifested as non germination pollen grains or curliness of the tubes in the style tissue. Discussion On the basis of the performed studied was established that very high level of incompatibility exist between genotypes from L.esculentum (line N and Balka ) and self-incompatible accession from L.pennellii (N ). In vitro embryo culture was necessary in order hybrids to be obtained. In F 2 progeny and in the first backcrossing (BC 1 ) single seeds were formed rare in individual fruits. In the study of pollen behavior in vivo at controlled self-pollination of hybrids N221 in F 2 and in backcrossing with line L.esc. N , penetration of very few pollen tubes in the ovules was established only in plant N1 (with very low pollen fertility - 10,0%). In the other F 2 plants with high pollen fertility and in these with semi-sterility, well expressed barriers of incompatibility stopping in different extent penetration of the pollen tubes in the stigma, style and ovary tissues were observed. It might be possible due to the different quantity of L.pennellii genes in the plants from the segregating F 2 progeny. Comparatively normal pollen grain germination, growth of their tubes in the ovary tissues and penetration in the ovules were established only in pollination of the mother L.esculentum parent plants Balka with pollen from hybrid N336. For the successful obtaining of backcross progenies non-typical crosses for the classical breeding is necessary to be performed. For BC 1 instead of the traditional heaping of pollen from parent L.esculentum on the stigmas of hybrid plants more successful was heaping of the pollen from the hybrid on the stigmas of the L.esculentum plants. Conclusions Our investigation confirmed very high level of incompatibility between L. esculentum genotypes - line N and Balka and self-incompatibility L.pennellii accession N It is necessary to use in vitro embryo culture for successful developing of hybrid embryos. It was established different preventing answers to the pollen germination and penetration in the stigma, styles, ovaries and ovules in the reciprocal crosses of the both studied species, as well as in self pollination of an F 2 hybrids and also in back crossing with the both parents. Compared to the plants with high pollen fertility and semi-sterility lower level of incompatibility after self pollination was registered in F 2 generation in plant with 10% fertility. Successful pollen germination and entering the tubes in the ovules was established only in pollination of mother plants L.esculentum (Balka ) with pollen from hybrid N 336. References 773 Causse, M., C. Caranta, V. Saliba-Colombani, A. Morreti, R. Damidaux and P. Rousselle, Enhancement of tomato genetic resources via molecular markers. Agricultures, 9, No 3:

8 774 Hardon, J. J., Unilateral incompatibility between S.pennellii and L.esculentum. Genetics, 57: Hogenboom, N. G. 1972a. Breaking breeding barriers in Lycopersicon. 1. The genus Lyco-persicon, its breeding barriers and the importance of breaking these barriers. V. Nikolova and V. Rodeva Euphytica, 21: Kallo, G. /Ed./, Genetic Improvement of Tomato. Springer-Verlag. Rick, C. M., Controlled introgression of chromosomes of Solanum pennellii into Lycopersicon esculentum: segregation and recombination. Genetics, 62: Received July, 10, 2006; accepted September, 23, 2006.

THE GENETIC CONTROL OF SELF-INCOMPATIBILITY IN THE GENUS GASTERIA (LILIACEAE)

Heredity (1978), 40 (1), 165-169 THE GENETIC CONTROL OF SELF-INCOMPATIBILITY IN THE GENUS GASTERIA (LILIACEAE) p. E. RANDHAM and S. J. OWENS Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey