Anthemideae: advances in tissue culture, genetics and transgenic biotechnology

Members of the Anthemideae include important floricultural (cut-flower) and ornamental (pot and garden) crops, as well as plants of medicinal and ethno-pharmacological interest. Despite the use of many of these plants (over 1400 species) in the extraction of important secondary metabolites and essential oils, the greatest emphasis has been on their in vitro tissue culture and micropropagation. Few studies have been conducted on genetic transformation, with those primarily focused on increasing yield of compounds in plants. This review, the first and only available for plants within this Family, highlights all the available literature that exists on Anthemideae (excluding ornamental chrysanthemums) in vitro cell, tissue and organ culture, micropropagation and transformation.


INTRODUCTION
Members of the Anthemideae top over 1400 species (the most common known by different names globally, Table 1) and consist of one of the most important global cut flower and pot plants, Dendranthema grandiflora, as well as important medicinal and aromatic plants from which many important secondary metabolites and essential oils are extracted.Despite this, the number of studies conducted on the tissue culture and micropropagation of its members are few, focusing only on one or two individual species that produce compounds of high economic value.Furthermore, in these same species the scarce genetic transformation studies have been primarily conducted to increase yields of those compounds or oils.A summary of these research findings is presented in this review.
Members of the Anthemideae have occupied an important place in the cultural practices the world over.A review on ornamental chrysanthemum biotechnology is discussed elsewhere (Teixeira da Silva, 2003).Garland chrysanthemum, Chrysanthemum coronarium and C. segetum are widely distributed in the Mediterranean, western Africa and Asia.C. coronarium, cultivated in Japan, China and Southeast Asia, is closely related to lettuce, and is a valuable edible species (Oka et al., 1999).C. coronarium var.coronarium is an ornamental, often found as a common weed, while C. coronarium var.spatiosum is used as a Chinese vegetable (chop-suey).Green leaves and stems of C. segetum are also consumed as vegetables.Chrysanthemum is a source of various valuable metabolites (Schwinn et al., 1994).
Tripleuspermum, among others (Figure 1; Khallouki et al., 2000).Numerous species contain medicinally and cosmetically important compounds and essential oils, some of which (e.g.flavonoids) have been used to differentiate members of the Asteraceae-Anthemideae including genera Achillea, Artemisia and Tanacetum (Williams et al., 1999).

Manipulation of morphogenesis
In vitro induction of roots has been achieved in Chrysanthemum-complex species.In Achillea millefolium, hairy root cultures (Agrobacterium rhizogenes-induced root production) were established for the biosynthetic production of terpenes in a bioreactor system (Lourenço et al., 1999).Hairy root cultures and cell suspension cultures of A. millefolium have been established to biotransform terpenes and to produce essential oils in a controlled environment, the biggest drawback being the low yield (Figueiredo et al., 1995).The A4-Y strain of A. rhizogenes induced hairy roots in Matricaria recutita (Máday et al., 1999) while in adventitious root cultures of Anthemis nobilis, geranyl isovalerate was accumulated (Omoto et al., 1998).

Effect of additives and other factors on morphogenesis
A number of tissue culture studies have been conducted with the aim of inducing various target organs from a number of explant sources.This has been achieved in many primary species of the Anthemideae (Table 2).Numerous studies have recently been completed on the effect of a number of factors and media additives on chrysanthemum thin cell layer (TCL) morphogenesis.To further enhance the medium-dependence of explants, TCLs were used in the experiments.TCLs, derived from cells, tissues or organs, are of a small size, excised either a) longitudinally (lTCL), being thus composed of a few tissue types or b) transversally (tTCL), thus composed of several tissue types, but which are normally too small to separate, as in the case of chrysanthemum.In the TCL system, the morphogenic and developmental pathways of specific organs may be clearly directed and controlled (Nhut et al., 2003).
Most aminoglycoside antibiotics, frequently used in Anthemideae transformation (Table 3), negatively affect in vitro growth and morphogenesis (shoot and root formation) of chrysanthemum tTCLs (Teixeira da Silva, 2002).In tansy (Tanacetum vulgare), cefotaxime, rifampicin and gentamycin (antibiotics commonly used to eliminate Gram-negative bacteria in in vitro shoot cultures)    all had pronounced negative effects on shoot growth and development (Keskitalo et al., 1998).In both studies, the effect of the antibiotic concentration on plant morphogenesis and explant survival depended on the size of the explant, the choice of explant source, the timing of infection by A. tumefaciens and selection pressure in genetic transformation.In separate experiments on the effect of other antibiotics on shoot regeneration, a gradient of phytotoxicity has been shown: bialaphos ® >chloramphenicol>rifampicin>streptomycin>mi nomycin>ampicillin>penicillin G = penicillin V (Teixeira da Silva et al., 2003).Another study (Teixeira da Silva and Fukai, 2001) showed the importance that Agrobacterium selective agent (carbenicillin, cefotaxime or vancomycin) has on maximizing chrysanthemum shoot regeneration capacity, while minimizing phytotoxicity and explant mortality, in one case (cefotaxime up to 250 mg/l) stimulating shoot formation.Contrasting results were found in in vitro cultures of Argyranthemum frutescens, where aureomycin, vancomycin, cefotaxime, carbenicillin and augmentin all inhibited root and shoot formation ≥40 mg/l, differentially controlling Agrobacterium growth (Seyring, 1999).

CONVENTIONAL BREEDING
Nature has played a role in inducing popyploidy in chrysanthemum through evolution, giving rise to tetra-, hexa-, octa-and decaploids, but humans too have contributed, through artificial interference, to changes in chrysanthemum.Many techniques are still employed by chrysanthemum breeders to improve varieties such as chromosome-doubling.GISH (genomic in situ hybridization) was used to confirm the successful intergeneric hybrid between Dendranthema lavandulifolia and Ajania remotipinna (El-Twab et al., 1999) and the use of FISH (fluorescence in situ hybridization) and GISH to confirm hybrids between Leucanthemella and Nipponanthemum (Ogura and Kondo, 1998;El-Twab and Kondo, 2001).
nrDNA internal transcriber spacer (ITS) and cpDNA trnL/trnF intergenic spacers were used to analyze the phylogeny of the Anthemideae (Oberprieler, 2002).ITS of nuclear ribosomal DNA were sequenced, and morphological cladistic analyses, cytology and isozyme analysis were conducted to differentiate 52 species from 32 genera and 8 subtribes of the Anthemideae (Francisco-Ortega et al., 1997).In separate studies, oligonucleotide fingerprinting and RAPD analysis were used as markers of stability during Achillea spp.micropropagation (Wallner et al., 1996).
Chromosome studies still continue to be important in separating chrysanthemum species (Kondo et al., 1998) while, due to high ploidy, isozymes/allozymes are effective in differentiating cultivars (Roxas et al., 1993).In the case of the Chrysantheminae, the geographic origin of genera and species within it could be desciphered by the use of PAGE (polyacrylamide gel electrophoresis) for a number of enzyme systems (Francisco-Ortega et al., 1995).Allele frequency data for polymorphic loci could be obtained when nine allozyme profiles were used to differentiate different populations of Achillea (Purdy and Bayer, 1996).

TRANSFORMATION
Few transformation studies have been conducted on members of the Anthemideae (Tables 3-5).The use of A. rhizogenes in the production of transgenic hairy roots allows for the mass production of secondary metabolites through a bioreactor system.In addition the use of A. tumefaciens, biolistics or any other gene transfer technique would confer the ability to transform economically important medicinal and aromatic varieties to modify characteristics such as compound yield, plant shape, height and growth morphology, longevity, horticultural traits, insect and disease resistance, and resistance to environmental stresses.In the Anthemideae, the main focus has been the use of hairy roots in bioreactor systems to improve the yield of economically and pharmacologically important compounds such as artemisinin (100 mg = approx.70 USD) in Artemisia annua, parthenolide (100 mg = approx.50 USD) in Tanacetum parthenium or pyrethrins in Tanacetum (syn.Chrysanthemum) cinerariaefolium.Artemisinin is one of the most important commercial antimalarials, and this compound also shows antitumor and antivial activities, among others, while parthenolide is primarily used in pesticides but also shows many biological activities, including antibacterial, anticancer, anti-inflammatory and fungicidal activities (USDA-ARS-NGRL, 2003).In the case of Artemisia absinthium, genetic modification of the plant was done to increase essential oil yield (Table 5).No genetic transformants have been obtained by biolistics.

CRYOPRESERVATION AND GERMPLASM PRESERVATION
Cryopreservation, an important method for the conservation of plant genetic resources (Engelmann, 2000) uses freeze preservation in liquid N 2 to immobilize metabolic activity, thus suspending changes that may arise in the plant cell genome.Storage of ornamental chrysanthemum (and to a limited extent other members of the Anthemideae) genetic resources has been achieved through cryopreservation, low temperature preservation, and room temperature preservation (Fukai, 1995) in which the successful cryopreservation of shoot tips involves the ability to regenerate thawed shoots as well as maintaining their genetic composition.Cryopreservation of C. cinerariaefolium, or Dalmatian pyrethrum, was achieved by a 3 day pre-culture period in sucrose-enriched medium, and using a 7.5% DMSO cryoprotectant, with an average cryopreservability rate at 62% (Hitmi et al., 1998a(Hitmi et al., , 1998b(Hitmi et al., , 1999a)).Cryopreservation, however did not affect the biosynthetic properties, the composition or the amount of pyrethrins (Hitmi et al., 2000a).Sucrose was shown to be an effective cryoprotectant to confer freezing tolerance to pyrethrin cell cultures (Hitmi et al., 1999a(Hitmi et al., , 1999c(Hitmi et al., , 2000b)).
Root tips of A. rhizogenes-transformed hairy roots in Artemisia annua resulted in a 65% regrowth rate following liquid N 2 immersion (Teoh et al., 1996).A. annua callus could be cryopreserved in a cryoprotectant containing 15% ethylene glycol, 15% dimethyl sulfoxide, 30% glycerol and 13.6% sucrose, a simplified and effective method for long-term storage of callus without an effect on regeneration (Chenshu et al., 2003).Artemisia pallens encapsulated shoot buds could regenerate well, especially in the presence of ABA (Sharief et al., 1997).

CONCLUSIONS
Members of the Anthemideae comprise a large number of species, many of which have economic medicinal and aromatic value, which can be increased with the exploration of in vitro culture techniques (tissue culture, cryopreservation) to increase yield and standardize quality, and molecular methodologies to improve growth characteristics and maximize yield.

Table 1 .
Some common names of main species within the Anthemideae.

Table 5 .
Details of Anthemideae transformation studies.