Sunkifolias and Buxisunkis: Sexually obtained reciprocal hybrids of Citrus sunki x Severinia buxifolia

Citrus sunki x Severinia buxifolia

^*

Herculano P. Medina-Filho, Rita Bordignon and Rosa M.L. Ballvé

Seção de Genética, Instituto Agronômico de Campinas, Caixa Postal 28, 13001-970 Campinas, SP, Brasil. Send correspondence to H.P.M-F.

ABSTRACT

The present investigation reports reciprocal hybrids between

Citrus sunki, belonging to the "True Citrus" taxonomic group, and

Severinia buxifolia of the "Primitive Citrus" group. This represents the first case of sexual hybridization between genera of distinct taxonomic groups of the subtribe Citrinae, which have several complementary agronomic and phytosanitary attributes. Thus, there exists considerable interest in determining the potential of such hybrids as rootstocks, as well as a possible genetic bridge to combine, by breeding at diploid level, nuclear and cytoplasmic genes of

Severinia with

Citrus and other citrus intergeneric hybrids. INTRODUCTION

Citrus and related genera are members of the Rutaceae, subfamily Aurantioideae, subtribe Citrinae. Citrinae is subdivided into three affinity groups (

Figure 1). The wide cross-compatibility among

Citrus species and related genera was evident since the early breeding work with citrus at the begining of the century (Frost, 1926; Swingle and Robinson, 1927; Traub and Robinson, 1937; Cameron and Frost, 1968). Nevertheless, it is a consensual tenet that the genera belonging to subtribe Citrinae are cross-incompatible (Swingle and Reece, 1967; Grosser

et al., 1988; Iwamasa

et al., 1988; Grosser and Gmitter Jr., 1990; Louzada and Grosser, 1994; Grosser

et al., 1995, 1996). This is seemingly corroborated by the fact that the existing sexual hybrids, though numerous are all confined to the genus

Citrus or between genera belonging to the "True Citrus" taxonomic group (

Figure 1) (Swingle and Reece, 1967; Hodgson, 1967; Grosser

et al., 1988, 1992).

Figure 1

The present investigation reports the pioneer success of three attempts to sexually hybridize Citrus sunki Hort. ex Tan. (group of True Citrus) and Severinia buxifolia (Poir.) Tenore (group of Primitive Citrus). The hybrids were named Sunkifolias (C. sunki x S. buxifolia) and the reciprocals, Buxisunkis.

MATERIAL AND METHODS Emasculations were performed in 156 flower buds of

C. sunki at various developmental stages and stigmatic fluid of ripe flowers of

S. buxifolia rubbed onto the

C. sunki stigma surface (Pandey, 1963; Shivanna and Rangaswamy, 1969; Yamashita, 1981; Van Tuyl

et al., 1982). Pollinations with

S. buxifolia pollen were conducted immediately and the twigs bagged for 10 days. In a second attempt, 219 immature buds of

S. buxifolia were emasculated four days before anthesis, bagged, and regular pollinations performed according to Bordignon

et al. (1990). A third attempt, similar to the second one, was realized with 141 flowers of

C. sunki pollinated with

S. buxifolia. Seeds were extracted from ripe fruits, washed, testa and tegmen removed, germinated in Petri dishes and transplanted to plastic trays.

Two-month-old seedlings grown in a green-house were morphologically scored, and a piece of leave was isozymically analyzed for Pgi-1, Pgm-1, Got-1, Got-2 and Prxa-1 loci according to Ballvé et al. (1991, 1995).

RESULTS AND DISCUSSION

The 156 flowers of

C. sunki pollinated with

S. buxifolia provided six fruits that reached maturity. Eight normal monoembryonic seeds were extracted, which yielded seven seedlings. Three of them were morphologically scored as hybrids and named Sunkifolias. Among these hybrids, two had a general morphology intermediate between the parents. The remaining one had a remarkable resemblance to the staminate parent such as early and sharp spines, dark bronze tips and slender growth. Nevertheless, leaves like the other two hybrids, had winged petioles (

Figure 2). In the second attempt the 219 pollinations in

S. buxifolia resulted in three fruits, each with a single monoembryonic seed. They produced two plants morphologically similar to the Sunkifolias and, as reciprocals, were named Buxisunkis. Another Sunkifolia hybrid was then obtained in the third attempt. Regular emasculation and pollinations of 141 flowers resulted in one fruit bearing a monoembryonic seed.

Figure 2 Sunkifolias and Buxisunkis: reciprocal hybrids of Citrus sunki x Severinia buxifolia. A) Branch and fruits of C. sunki and S. buxifolia (right). B) Branch of S. buxifolia showing fruits and spines. C) From right to left, wingless leaf petiole of S. buxifolia, Sunkifolia and C. sunki (winged). D) Banding patterns of Got-1 and Got-2 isozyme loci of S. buxifolia (first four samples), C. sunki (following three samples) and Sunkifolias (arrows). E) Branch of Buxisunkis showing vigorous leaves and spines. F) Sunkifolias and Buxisunkis grafted onto Rangpur lime (C. limonia).

Analysis of Pgi-1, Prxa-1, Got-1, Got-2 and Pgm-1 isozyme loci confirmed the six aforementioned hybrids (Figure 2). C. sunki is FF for Pgi-1. The S. buxifolia clone used in these crosses is heterozygous BS, with the allele S also common to several citrus species, and B a new allele for this locus. Similarly to C. sunki, S. buxifolia is heterozygous FM for Prxa-1. Despite the great taxonomic distance and the striking morphological differences between Citrus and Severinia, there is a reasonable homology between the two genera provided the polypeptides of the Prxa-1 and Pgi-1 alleles of each species are able to combine and produce active heterodimers in the tissues of the hybrids between them.

The GOT isozymic system in citrus presents two loci: Got-1 and Got-2, the latter being a duplicated locus (Ballvé et al., 1991). The genetic interpretation of this system in Severinia is not yet established. However, the banding patterns of Sunkifolias and Buxisunkis complement the parental bands, providing good evidence of their hybrid nature.

C. sunki is homozygous FF for Pgm-1 while S. buxifolia is homozygous for the P allele that occurs in Poncirus trifoliata. Sunkifolias and Buxisunkis are seen in the gels as heterozygous FP.

Based on the previously published information about the impossibility of effecting crosses of Citrus with genera outside its taxonomic group, no customary pollinations in C. sunki were tried in the first attempted cross with Severinia. Therefore, after securing the first three Sunkifolia hybrids, it was not certain whether the success of this cross was due to the bud pollinations, to the use of stigmatic fluid of S. buxifolia rubbed onto the C. sunki stigma, or to a fortuitous choice of parents. The explanation awaited the ensuing attempts.

The Buxisunkis and the additional Sunkifolia hybrids were obtained after regular pollinations of ripe stigmas, without the use of any special methodology. Therefore, the success in obtaining such wide hybrids must be credited to an unexpected and hitherto unknown partial cross-compatibility between these two species.

According to the literature, previous attempts to sexually hybridize these and other species of different taxonomic groups had been unsuccessful (Cameron and Frost, 1968; Iwamasa et al., 1988), reason why somatic hybridization has been used as an alternative strategy for obtaining wide hybrids (Ohgawara et al., 1985; Grosser et al., 1988; Ohgawara et al., 1989; Grosser, 1991; Grosser et al., 1992; Louzada et al., 1992). Notwithstanding the value of the somatic hybridization, the results herein obtained indicate that sexual hybridization is a powerful tool for obtaining wide hybrids of citrus. Slight differences in the genotypes of clones or specific environmental conditions where plants are grown may play an important role in the mechanisms affecting cross-compatibility and the fertilization process. These could account for the failure of the previous attempts of hybridization reported in the literature.

The six individuals of Sunkifolias and Buxisunkis are, in general terms, phenotypically similar; however they display conspicuous differences in morphology, vigor and isozyme genotypes. This variability is quite interesting from a breeding standpoint since it reflects the diversity in the combinations of Citrus and Severinia genes. In addition, because these sexual hybrids are reciprocals, there exists the opportunity of exploiting nucleo-cytoplasmic interactions of these various genotypes with cytoplasms of Citrus and of Severinia. A further stimulating aspect is that, unlike in the case of somatic hybridization, these possibilities of exploiting nucleo-cytoplasmic interactions are at the diploid level, since 2n = 18 in both species (Krug, 1943; Santos and Guerra, 1993) and probably also in the sexual hybrids between them. At least four of the six hybrids produced were quite vigorous, cuttings rooted easily without hormones in commercial potting mixture and buds were graft-compatible onto Rangpur lime (C. limonia) (Figure 2).

The Sunkifolias and Buxisunkis are scientifically and agronomically of great interest. As far as we know, they represent the first case of wide sexual hybridization between genera of distinct groups of subtribe Citrinae as shown in Figure 1. They may provide new rootstocks for citrus, and if fertile be useful as a genetic bridge to combine, at the diploid level, nuclear and cytoplasmic genes of Severinia with the Citrus genome and its intergeneric hybrids.

Among several rootstocks extensively tested in Brazil, C. sunki mandarin is the only one resistant to blight (Pompeu Jr., 1990). Currently, it is not widely planted because of susceptibility to Phytophthora root rot (Feichtemberger, 1990). S. buxifolia, on the other hand, is considered a good source of resistance to this fungus (Hutchison and Grimm, 1973) and also to the citrus nematode (Phillips, 1969), salinity (Cooper, 1961) and presumably produces small oranges of excellent quality when grafted onto it. According to Grosser et al. (1996), tetraploid somatic hybrids of sweet orange (C. sinensis) with S. buxifolia lack vigor and the scion show nutrition deficiency. The use of S. buxifolia as a rootstock per se is hindered by the slow growth in the nursery and by the susceptibility to the citrus tristeza virus (Bitters et al., 1969) to which Sunki is highly resistant (Pompeu Jr., 1990). For these and other reasons there is considerable interest in the agronomic evaluation and in the determination of the full potential of such hybrids for citrus rootstock improvement.

ACKNOWLEDGMENTS

The authors thank the Centro de Citricultura Sylvio Moreira of Instituto Agronômico for providing the parental plants, for rooting and grafting the hybrids.

H.P. Medina-Filho and R. Bordignon were recipients of CNPq and CAPES fellowships, respectively.

RESUMO Este trabalho relata a obtenção inédita de híbridos entre

Citrus sunki (grupo dos Citros Verdadeiros) e

Severinia buxifolia (Citros Primitivos) bem como seus recíprocos. Eles representam o primeiro caso de hibridações sexuais entre gêneros de grupos taxonômicos distintos da subtribo Citrinae.

C. sunki e

S. buxifolia possuem diversos atributos agronômicos e fitossanitários complementares. Existe, pois, considerável interesse na avaliação de tais híbridos como porta-enxertos e também como possível ponte genética para combinar, através de melhoramento ao nível diplóide, genes nucleares e citoplasmáticos de

Severinia com o genoma de

Citrus e de seus híbridos intergenéricos. REFERENCES

Ballvé, R.M.L., Bordignon, R., Medina Filho, H.P., Siqueira, W.J., Teófilo Sobrinho, J. and

Pompeu Júnior, J. (1991). Isoenzimas na identificação precoce de híbridos e clones nucelares no melhoramento de citros.

Bragantia 50: 57-76.

Ballvé, R.M.L., Medina Filho, H.P., Bordignon, R. and Lima, M.M.A. (1995). Methodology for starch gel electrophoresis and protocols for isozymes of 32 plant genera. Rev. Bras. Genet. 18: 491-502.

Bitters, W.P., Cole, D.A. and Brusca, J.A. (1969). The citrus relatives as citrus rootstocks. Proceedings of First International Citrus Symposium, Riverside, 1: 411-415.

Bordignon, R., Medina-Filho, H.P. and Ballvé, R.M.L. (1990). Melhoramento genético de citros no Instituto Agronômico. Laranja 11: 167-176.

Cameron, J.W. and Frost, H.B. (1968). Genetics, breeding and nucellar embryony. In: The Citrus Industry (Reuther, W., Batchelor, L.D. and Webber, H.J., eds.). Vol. II. University of California Press, Berkeley, pp. 325-370.

Cooper, W.C. (1961). Toxicity and accumulation of salt in citrus trees on various rootstocks in Texas. Proceed ings of Florida State Horticultural Society, Tallahassee, 74: 95-104.

Feichtemberger, E. (1990). Gomose de Phytophthora dos citros. Laranja 11: 97-122.

Frost, H.B. (1926). Summary of the work of the research divisions. Citrus Expt. Sta. Plant Breeding. Univ. Calif. Agric. Exp. Sta. Rep. 1925-26: 55.

Grosser, J.W. (1991). Hybrid rootstocks from cell-fusion offer great potential. Citrus Ind. 12: 41-42.

Grosser, J.W. and Gmitter Jr., F.G. (1990). Somatic hybridization of Citrus with wild relatives for germplasm enhancement and cultivar development. Hortscience 25: 147-151.

Grosser, J.W., Gmitter Jr., F.G. and Chandler, J.L. (1988). Intergeneric somatic hybrid plants from sexually incompatible woody species: Citrus sinensis and Severinia disticha. Theor. Appl. Genet. 75: 397-401.

Grosser, J.W., Gmitter Jr., F.G., Sesto, F., Deng, X.X. and Chandler, J.L. (1992). Six new somatic citrus hybrids and their potential for cultivar improvement. J. Am. Soc. Hort. Sci. 117: 169-173.

Grosser, J.W., Gmitter Jr., F.G., Castle, W.S. and Chandler, J.L. (1995). Production and evaluation of citrus somatic hybrid rootstocks: progress report. Proceedings of Florida State Horticultural Society, Tallahassee, 108: 140-143.

Grosser, J.W., Mourão Filho, F.A.A., Gmitter Jr., F.G., Louzada, E.S., Jiang, J., Baergen, K., Quiros, A., Cabasson, C., Schell, J.L. and Chandler, J.L. (1996). Allotetraploid hybrids between Citrus and seven related genera produced by somatic hybridization. Theor. Appl. Genet. 92: 577-582.

Hodgson, R.W. (1967). Horticultural varieties of citrus. In: The Citrus Industry (Reuther, W., Webber, H.J. and Batchelor, L.D., eds). Vol. I. University of California Press, Berkeley, pp. 431-591.

Hutchison, D.J. and Grimm, G.R. (1973). Citrus clones resistant to Phytophthora parasitica: 1973 screening results. Proceedings of Florida State Horticultural Society, Tallahassee, 86: 88-91.

Iwamasa, M., Nito, N. and Ling, J.T. (1988). Intra and intergeneric hybridization in the orange subfamily, Aurantioideae. Proceedings of International Citrus Congress, Tel Aviv, Israel, pp. 123-130.

Krug, C.A. (1943). Chromosome numbers in the subfamily Aurantioideae with special reference to the genus Citrus. Bot. Gaz. 104: 602-611.

Louzada, E.S. and Grosser, J.W. (1994). Somatic hybridization of Citrus with sexually incompatible wild relatives. In: Biotechnology in Agriculture and Forestry (Bajaj, Y.P.S., ed.). Vol. 27. Springer-Verlag, Berlin, Heidelberg, pp. 427-438.

Louzada, E.S., Grosser, J.W., Gmitter Jr., F.G., Nielsen, B., Chandler, J.L., Deng, X.X. and Tusa, N. (1992). Eight new somatic hybrid citrus rootstocks with potential for improved disease resistance. Hort. Sci. 27: 1033-1036.

Ohgawara, T., Kobayashi, S., Ohgawara, E., Uchimiya, H. and Ishii, S. (1985). Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata. Theor. Appl. Genet. 71: 1-4.

Ohgawara, T., Kobayashi, S., Ishii, S., Yoshinaga, K. and Oiyama, I. (1989). Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.). Theor. Appl. Genet. 78: 609-612.

Pandey, K.K. (1963). Stigmatic secretion and bud-pollination in self and cross-incompatible plants. Naturwissenschaften 50: 408-409.

Phillips, R.L. (1969). Dwarfing rootstocks for citrus. Proceedings of First International Citrus Symposium, Riverside, I: 401-406.

Pompeu Jr., J. (1990). Situação do uso de porta-enxertos no Brasil. In: Seminário Internacional de Citros. Bebedouro, Brasil. Anais, pp. 1-10.

Santos, K. and Guerra, M. (1993). Padrão de bandas CMA em representantes de alguns gêneros próximos a Citrus. I. Tribo Clauseneae. Rev. Bras. Genet. 16: 183 (Abstract).

Shivanna, K.R. and Rangaswamy, N.S. (1969). Overcoming self-incompatibility in Petunia axillaris (Lam.). Delayed pollination, pollination with stored pollen and bud pollination. Phytomorphology 19: 372-380.

Swingle, W.T. and Reece, P.C. (1967). The botany of Citrus and its wild relatives. In: The Citrus Industry (Reuther, W., Webber H.J. and Batchelor, L.D., eds.). Vol. I. University of California Press, Berkeley, pp. 190-430.

Swingle, W.T. and Robinson, T.R. (1927). Citranges and some related hybrid fruits. U.S. Dept. Agr. Yearbook 1926: 223-225.

Traub, H.P. and Robinson, T.R. (1937). Improvement of subtropical fruit crops: citrus. U.S. Dept. Agr. Yearbook 1937: 749-826.

Van Tuyl, J.M., Marcucci, M.C. and Visser, T. (1982). Pollen and pollination experiments VII. The effect of pollen treatment and application method on incompatibility and incongruity in Lilium. Euphitica 31: 613-619.

Yamashita, K. (1981). Stigma exudate application for over-coming self-incompatibility of hyuganatsu and hassaku. Proceedings of International Society of Citriculture, Tokyo, 1: 48-51.

(Received July 11, 1996)

• Ballvé, R.M.L., Bordignon, R., Medina Filho, H.P., Siqueira, W.J., Teófilo Sobrinho, J. and Pompeu Júnior, J. (1991). Isoenzimas na identificaçăo precoce de híbridos e clones nucelares no melhoramento de citros. Bragantia 50: 57-76.
• Ballvé, R.M.L., Medina Filho, H.P., Bordignon, R. and Lima, M.M.A. (1995). Methodology for starch gel electrophoresis and protocols for isozymes of 32 plant genera. Rev. Bras. Genet. 18: 491-502.
• Bitters, W.P., Cole, D.A. and Brusca, J.A. (1969). The citrus relatives as citrus rootstocks. Proceedings of First International Citrus Symposium, Riverside, 1: 411-415.
• Bordignon, R., Medina-Filho, H.P. and Ballvé, R.M.L. (1990). Melhoramento genético de citros no Instituto Agronômico. Laranja 11: 167-176.
• Cameron, J.W. and Frost, H.B. (1968). Genetics, breeding and nucellar embryony. In: The Citrus Industry (Reuther, W., Batchelor, L.D. and Webber, H.J., eds.). Vol. II. University of California Press, Berkeley, pp. 325-370.
• Feichtemberger, E. (1990). Gomose de Phytophthora dos citros. Laranja 11: 97-122.
• Frost, H.B. (1926). Summary of the work of the research divisions. Citrus Expt. Sta. Plant Breeding. Univ. Calif. Agric. Exp. Sta. Rep. 1925-26: 55.
• Grosser, J.W. (1991). Hybrid rootstocks from cell-fusion offer great potential. Citrus Ind. 12: 41-42.
• Grosser, J.W. and Gmitter Jr., F.G. (1990). Somatic hybridization of Citrus with wild relatives for germplasm enhancement and cultivar development. Hortscience 25: 147-151.
• Grosser, J.W., Gmitter Jr., F.G. and Chandler, J.L. (1988). Intergeneric somatic hybrid plants from sexually incompatible woody species: Citrus sinensis and Severinia disticha Theor. Appl. Genet. 75: 397-401.
• Grosser, J.W., Gmitter Jr., F.G., Sesto, F., Deng, X.X. and Chandler, J.L. (1992). Six new somatic citrus hybrids and their potential for cultivar improvement. J. Am. Soc. Hort. Sci. 117: 169-173.
• Grosser, J.W., Gmitter Jr., F.G., Castle, W.S. and Chandler, J.L. (1995). Production and evaluation of citrus somatic hybrid rootstocks: progress report. Proceedings of Florida State Horticultural Society, Tallahassee, 108: 140-143.
• Grosser, J.W., Mourăo Filho, F.A.A., Gmitter Jr., F.G., Louzada, E.S., Jiang, J., Baergen, K., Quiros, A., Cabasson, C., Schell, J.L. and Chandler, J.L. (1996). Allotetraploid hybrids between Citrus and seven related genera produced by somatic hybridization. Theor. Appl. Genet. 92: 577-582.
• Hodgson, R.W. (1967). Horticultural varieties of citrus. In: The Citrus Industry (Reuther, W., Webber, H.J. and Batchelor, L.D., eds). Vol. I. University of California Press, Berkeley, pp. 431-591.
• Hutchison, D.J. and Grimm, G.R. (1973). Citrus clones resistant to Phytophthora parasitica: 1973 screening results. Proceedings of Florida State Horticultural Society, Tallahassee, 86: 88-91.
• Iwamasa, M., Nito, N. and Ling, J.T. (1988). Intra and intergeneric hybridization in the orange subfamily, Aurantioideae. Proceedings of International Citrus Congress, Tel Aviv, Israel, pp. 123-130.
• Krug, C.A. (1943). Chromosome numbers in the subfamily Aurantioideae with special reference to the genus Citrus. Bot. Gaz. 104: 602-611.
• Louzada, E.S., Grosser, J.W., Gmitter Jr., F.G., Nielsen, B., Chandler, J.L., Deng, X.X. and Tusa, N. (1992). Eight new somatic hybrid citrus rootstocks with potential for improved disease resistance. Hort. Sci. 27: 1033-1036.
• Ohgawara, T., Kobayashi, S., Ohgawara, E., Uchimiya, H. and Ishii, S. (1985). Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata Theor. Appl. Genet. 71: 1-4.
• Ohgawara, T., Kobayashi, S., Ishii, S., Yoshinaga, K. and Oiyama, I. (1989). Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.). Theor. Appl. Genet. 78: 609-612.
• Pandey, K.K. (1963). Stigmatic secretion and bud-pollination in self and cross-incompatible plants. Naturwissenschaften 50: 408-409.
• Phillips, R.L. (1969). Dwarfing rootstocks for citrus. Proceedings of First International Citrus Symposium, Riverside, I: 401-406.
• Pompeu Jr., J. (1990). Situaçăo do uso de porta-enxertos no Brasil. In: Seminário Internacional de Citros. Bebedouro, Brasil. Anais, pp. 1-10.
• Santos, K. and Guerra, M. (1993). Padrăo de bandas CMA em representantes de alguns gęneros próximos a Citrus I. Tribo Clauseneae. Rev. Bras. Genet. 16: 183 (Abstract).
• Shivanna, K.R. and Rangaswamy, N.S. (1969). Overcoming self-incompatibility in Petunia axillaris (Lam.). Delayed pollination, pollination with stored pollen and bud pollination. Phytomorphology 19: 372-380.
• Swingle, W.T. and Reece, P.C. (1967). The botany of Citrus and its wild relatives. In: The Citrus Industry (Reuther, W., Webber H.J. and Batchelor, L.D., eds.). Vol. I. University of California Press, Berkeley, pp. 190-430.
• Swingle, W.T. and Robinson, T.R. (1927). Citranges and some related hybrid fruits. U.S. Dept. Agr. Yearbook 1926: 223-225.
• Traub, H.P. and Robinson, T.R. (1937). Improvement of subtropical fruit crops: citrus. U.S. Dept. Agr. Yearbook 1937: 749-826.
• Van Tuyl, J.M., Marcucci, M.C. and Visser, T. (1982). Pollen and pollination experiments VII. The effect of pollen treatment and application method on incompatibility and incongruity in Lilium Euphitica 31: 613-619.
• Yamashita, K. (1981). Stigma exudate application for over-coming self-incompatibility of hyuganatsu and hassaku. Proceedings of International Society of Citriculture, Tokyo, 1: 48-51.

^*

Part of a thesis presented by R.B. to the Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), 1995, in partial fulfillment of the requirements for the Master’s degree.

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  11 July 1996