Ploidy level and caffeine content in leaves of Coffea

Ploidia e conteúdo de cafeína em folhas de Coffea

Ploidy level and caffeine content in leaves of Coffea

Maria Bernadete Silvarolla¹; Paulo Mazzafera^2,3*; Marinez Muraro Alves de Lima^1,3; Herculano Penna Medina Filho^1,3; Luiz Carlos Fazuoli^1,3

¹Centro de Café e Plantas Tropicais - IAC, C.P. 28 - CEP: 13001-970 - Campinas, SP.

²Depto. de Fisiologia Vegetal - IB/UNICAMP, C.P. 6109 - CEP: 13087-970 - Campinas, SP.

³Bolsista do CNPq.

*e-mail: [e-mail]

ABSTRACT: Several species of Coffea and varieties of C. arabica differing in chromosome number had their caffeine content determined in the leaves. For Coffea arabica var. "Angustifolia", var. "Caturra" and var. "Icatu", it was observed caffeine decrease from the haploid (2n = 22) to tetraploid leaves (2n = 44). Caffeine in the tetraploid "Angustifolia" was decreased by 50% when compared to the haploid. Caffeine reduction was also observed in leaves of C. canephora var. "Kouilou" as chromosome number was increased (2n = 22 ® 2n = 44). In this case, caffeine in the leaves of the diploid genotype was close to 4 times higher than in the tetraploid. On the other hand it was observed an increase of the alkaloid when the chromosome number was doubled in the C. canephora var. "Robusta".

Key words: caffeine, Coffea, leaves, ploidy

Ploidia e conteúdo de cafeína em folhas de Coffea

RESUMO: Variedades e espécies de café (Coffea) que diferem quanto ao número de cromossomos tiveram o conteúdo de cafeína determinado em suas folhas. Para as variedades "Angustifolia", "Caturra" e "Icatu" de Coffea arabica foi observada redução no teor de cafeína de folhas haplóides (2n = 22) em relação a tetraplóides (2n = 44). Cafeína na forma tetraplóide da var. "Angustifolia" foi reduzida em 50% quando comparada com a forma haplóide. Redução de cafeína também foi observada em folhas de C. canephora var. "Kouilou" quando o número de cromossomos aumentou (2n = 22 ® 2n = 44). Neste caso, a cafeína nas folhas do genótipo diplóide foi cerca de quatro vezes maior que no tetraplóide. Por outro lado, foi observado aumento desse alcalóide quando o número de cromossomos foi duplicado em C. canephora var. "Robusta". Palavras-chave: cafeína, Coffea, folhas, ploidia

INTRODUCTION

Coffea arabica, the most economically important species of Coffea, is tetraploid (2n=44), while all other species of the genus are diploid (2n=22). Such diploid species are frequently involved in the breeding of C. arabica, therefore, interespecific hybridization is usually preceded by colchicine-mediated chromosome doubling. The chromosome doubling is also experimentally performed in C. arabica di-haploid individuals for the production of completely homozygous plants. Indeed, one of the most successful breeding program of coffee in Brazil, that culminated with the release of "Icatu" lines of C. arabica, started with the artificially doubled di-haploid C. arabica cv. "Bourbon" (2n = 22 ® 2n = 44) crossed with a doubled normal plant of Coffea canephora (2n = 22 ® 4n = 44).

Along the years, many other individuals of several coffee species, varying in chromosome number, were studied and maintained in a living collection in the Genetics Department of the Agronomic Institute in Campinas.

There exists considerable variation for caffeine content in seeds and leaves of Coffea species (Anthony et al., 1993; Charrier & Berthaud, 1975; D'Ornano et al., 1967; Mazzafera & Magalhães, 1991; Mazzafera & Carvalho, 1992; Mazzafera et al., 1997; Rakotomalala et al., 1989). However, little or no information is available about the influence of chromosome number or ploidy levels on the alkaloid content in coffee. Therefore, determinations were carried out in order to expand the knowledge about the subject, aiming to obtain additional information to assist ongoing breeding programs for coffee quality. The analysis were carried out only in leaves since, depending on the ploidy level, plants of several species do not produce seeds, therefore, not allowing any sort of comparison.

MATERIAL AND METHODS

Leaves of the third and fourth pair from the tip of lateral branches of adult plants were collected, dried at 80^oC, and ground in mortar with a pestle.

Caffeine was extracted from 50 mg ground tissue, with 3 ml of 0,01 N H₂SO₄ containing 100 mg MgO in tap sealed tubes (Suzuki & Waller, 1984). The tubes were boiled for 1h with occasional agitation. After cooling 1 ml aliquots were taken, transferred to Eppendorf vials and centrifuged for 15 minutes at 15,000 rpm in a micro centrifuge (Model 5415C, Brinkmann Instruments, Inc., USA). The supernatant was stored at -20^oC until analysis by reversed-phase high-performance liquid chromatography (RP-HPLC).

Caffeine was determined in a Shimadzu HPLC system. Caffeine was separated in a reversed phase Supelcosil-C18 column, 4.5 mm x 150 mm, 5 mm (Supelco Inc., USA), using 15% methanol in 0,5% aqueous acetic acid, with a flow of 1 ml/min. The solvent was delivered by a pump model LC-10AS and the caffeine eluting from the column was monitored using ultra violet detector model SPD-10A, operating at 280 nm. Signals from the detector were integrated in a Shimadzu C-R6A Chromatopac recorder and were compared with those of pure caffeine (Sigma grade).

RESULTS AND DISCUSSION

TABLE 1 shows the caffeine content in leaves of coffee plants differing for chromosome number. More C. arabica plants were available and among them, only in "Angustifolia", "Caturra" and "Icatu" it was observed caffeine decrease from the haploid to tetraploid leaves. Caffeine content in the tetraploid "Angustifolia" was 50% lower compared to the haploid.

Caffeine reduction was also observed in leaves of C. canephora var. "Kouilou" as chromosome number increased. In the case of "Kouilou 66-1", caffeine in the leaves of the diploid genotype was about 4 times higher (0.968%) than in the tetraploid (0.264%). On the other hand, it was observed an increase of the alkaloid when the chromosome number was doubled in "Robusta". Among three tetraploids of C. congensis var. "Uganda", one plant showed, on average, four times more caffeine than the diploids. In addition, the caffeine content of C. racemosa tetraploid doubled compared to the diploid form.

Several reports showed an increase of alkaloids after polyploidization with colchicine. In Papaver somniferum, Andreev (1963) observed that the morphine content in capsules of tetraploid plants had a marked increase. In Solanum khasianum Bhatt & Heble (1978) detected an increase of 35-50% of solasodine in tetraploid genotypes. Wold et al. (1983) and Milo et al. (1987) observed that the content of the alkaloid thebaine was increased in the capsule of triploid and tetraploid genotypes originated from seeds of Papaver bracteatum treated with colchicine.

However, increase of alkaloids due to polyploidization is not a rule. Dynansagar & Sudhakaran (1970) and Kulkarni et al. (1984) observed that tetraploidy did not affect the content of the alkaloids present in Vinca rosea and Catharanthus roseus, respectively.

CONCLUSION

It is concluded that although not so frequently, variations in the ploidy level may change caffeine content in coffee leaves.

ACKNOWLEDGMENT

The authors thank Denise Cristina da Silva for technical assistance in the caffeine analysis.

Received January 23, 1998

Accepted April 02, 1998

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Publication Dates

• Publication in this collection
  17 Sept 1999
• Date of issue
  July 1999

History

• Accepted
  02 Apr 1998
• Received
  23 Jan 1998