Essential Oil Constituents of Intact Plants and In Vitro Cultures of Tagetes patula L.

Journal of Essential Oil Research: JEOR, Jan/Feb 2007 by Szarka, Szabolcs, Héthelyi, Éva B, Lemberkovics, Éva, Bálványos, István, Et al

Abstract

The occurrence of volatile compounds in flowers and roots of intact plants and in vitro hairy root cultures of Tagetes patula L. was investigated by GC and GC/MS. The flowers oil was dominated by mono- and sesquiterpenes. The main component was the sesquiterpene hydrocarbon β-caryophyllene (50.2%). However, the oils of normal roots and hairy root cultures were found to contain sulfur-containing thiophene structures. The following four compounds were identified: 5-(3-buten-1-ynyl)-2,2'-bithiophene (BBT), 5-(3-penten-1-ynyl)-2,2'-bithiophene (PBT), 2,2':5',2''-terthiophene (α-terthiophene ) and 5-(4-acetoxy-1-butynyl)-2,2'-bithiophene (BBTOAc). The main components were BBT (47.0%) and α-terthiophene (19.8%) in the normal root oil and BBT (25.0%), α-terthiophene (14.3%) and BBTOAc (13.7%) in hairy root oil, respectively.

Key Word Index

Tagetes patula, Asteraceae, essential oil composition, β-caryophyllene, palmitic acid, α-terthiophene, in vitro culture.

Introduction

Tagetes patula L. (Asteraceae), commonly known as French marigold, is native of Mexico and other warmer parts of America. The plant is named after Tages, the Etruscan God of prophecy. He supposedly emerged from the earth, predicted the future to the villagers who saw him and taught them the rites of prophecy then died. His words became the foundation of Etruscan prophecy.

Today, T. patula is awell-known ornamental plant widespread all over the world. The plant is a bushy annual and has yellow or orange flowerheads. This herb biosynthesizes many biologically active products that can be used by the flavor, fragrance and pharmaceutical industries. The essential oil components have antibacterial and antifungal activity (1-8). Lutein dipalmitate, the main component of petal xanthophylls, is a pharmacologically active compound, and is used as ophtalmological agent. These compounds are used as food colorants as well, because of the greater solubility in vegetable oils compared to synthetic carotenoids (1). Tagetes species accumulate a wide range of thiophenes in their roots (2-5). Thiophenes exhibit strong biocidal activity thus environmentally safe, potential alternatives to synthetic pesticides (2). These compounds show impressive UVA-dependent biological activities. The mechanism of action is thought to be a type II mechanism which requires singlet oxygen exclusively. So, in the presence of UVA, the generated free radicals may induce fatty acid peroxidation and hence membrane damage. Thiophenes have extremely potent phototoxic effects on animal viruses with membranes, bacteria, fungi or nematodes (6-8).

In vitro plant cell cultures can beusedto produce high value secondary metabolites. The transgenic hairy root culture is an excellent model system to study the secondary metabolism of biologically active compounds. These cultures are produced by Agrobacterium rhizogenes inoculation of intact plant seedlings. The rhizogenic bacteria strains contain a single copy of a large Ri plasmid. One part of this plasmid, called T-DNA, is transferred to wounded plant cells and it gets stably integrated into the host genome. After the transformation, profuse roots form at the inoculation site (2,4,5,9,10). These hairy roots produce growth hormones, so they growvery intensively on hormone-free media as well. These cultures have a better advantage over callus and dispersed cultures for their genetic and biochemical stability. In many instances, they can synthetize a range of secondary metabolites that are produced by intact plants (5).

The purpose of the present study was to investigate the volatile compound production and composition. Transformed hairy root cultures and different organs (flowers and roots) of intact T. patula were used for GC and GCYMS analyses.

Experimental

Plant material: Tagetes patula L. seeds were collected in Murau (Austria). The harvested intact plants were raised in a garden near Kaposvár (Hungary). The seeds were sown at the beginning of April 2004. For chemical analyses 90 plants were collected during June 2004.

Cultivation ofhairy root cultures: The hairy root cultures were obtained by transformation of T. patula with A. rhizogenes strain R-1601 using the microinjection technique. Hypocotyl regions of seedlings were wounded with a scalpel that was previously immersed in the bacterial culture. The isolated hairy roots were transferred to solid MS medium containing 1 g/L ampicillin and 0.25 g/L cephatoxim. The bacteria-free cultures were maintained in liquid Gamborg's B5 medium, in 500 mL Erlenmeyer flasks filled with 100 mL medium, on a rotary shaker (140 rpm) at 23±2°C in the dark. Thereafter, they were transferred every three or four weeks. The hairy roots were collected for chemical analysis after seven weeks. During the long-term cultivation the medium was replaced by fresh medium after three weeks.

Oil isolation and content determination: Hairy roots (30-40 g fresh weight) were homogenized in distilled water withablender(Homogenizertype302).Air-driedflowers(25g dry weight) and roots (10 g dry weight) were crushed with a grinder. The essential oil was obtained by water distillation using a Clevenger-type apparatus for 3 h. The volatile compounds were accumulated in the organic phase (3 mL hexane) added to the cohobated water. Afterward the hexanoic solution was separated from the aqueous phase and then was evaporated at room temperature. The residual oil was measured gravimetrically. The samples were dissolved in 0.5 or 1.0 mL hexane and were stored in a refrigerator at 5°C until analyzed.