Genetic transformation via particle bombardment of Catharanthus ...

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Key words: β-glucuronidase, Catharanthus roseus, genetic transformation, ... In vitro propagation of Catharanthus roseus was achieved using nodal explants.
Biotechnology Letters 21: 997–1002, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Genetic transformation via particle bombardment of Catharanthus roseus plants through adventitious organogenesis of buds Rafael Z´arate1,∗ , Johan Memelink2, Robert van der Heijden1 & Robert Verpoorte1 1 Division

of Pharmacognosy, Gorlaeus laboratories, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands 2 Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands ∗ Author for correspondence (Fax: +31 71 5274511; E-mail: [email protected]) Received 12 August 1999; Revisions requested 9 September 1999; Revisions received 23 September 1999; Accepted 27 September 1999

Key words: β-glucuronidase, Catharanthus roseus, genetic transformation, green fluorescent protein, particle bombardment Abstract In vitro propagation of Catharanthus roseus was achieved using nodal explants. Bud induction was best on medium containing 1.0 mg benzyl aminopurine l−1 . Hardening of rooted shoots to soil was very successful with 98% survival. Genetically transformed C. roseus plantlets were obtained after bombardment of nodal explants, which were then micropropagated, with DNA coated particles with green fluorescent protein (GFP) or β-glucuronidase (GUS) reporter genes. Histological studies showed that the gene insertion method proved effective with many cells and different tissues displaying the reporter gene signals, showing that gene expressions were rather stable.

Introduction Catharanthus roseus (L.) G. Don. (Apocynaceae) is well known for its terpenoid indole alkaloid content; the bisindole alkaloids, vincristine and vinblastine, are strong antitumor agents and ajmalicine is used to improve cerebral circulation. These compounds are synthesised following two metabolic branches: the indole moiety, tryptamine, originates from tryptophan produced from the shikimate pathway, and the terpenoid building block, secologanin, originates either from the novel triose phosphate/pyruvate pathway (Contin et al. 1998) or from mevalonate (Banthorpe et al. 1972). A large body of research has been devoted to the study of the biosynthetic pathway leading to the formation of these secondary metabolites chiefly in cell and tissue cultures, and attempts to boost their production have been reported (Verpoorte et al. 1991). In this regard, several regulatory parameters, e.g., cell development, culture conditions, elicitation, have been investigated and some important enzymes involved in the biosynthesis of terpenoid indole alkaloids (trypto-

phan decarboxylase, strictosidine synthase, strictosidine β-D-glucosidase, geraniol 10-hydroxylase) have been characterised (Meijer et al. 1993) and some have also been cloned (de Luca et al. 1989, McKnight et al. 1990). In our laboratory, different C. roseus cell lines have been genetically transformed using tryptophan decarboxylase and strictosidine synthase genes (Canel et al. 1998). However, the increase in the production of these metabolites in culture has not been optimised for economical exploitation although considerable improvements have been achieved. It has also been reported that the biosynthesis of iridoids represents a limiting factor in the accumulation of alkaloids in some cell cultures of C. roseus (Verpoorte et al. 1997). Consequently, it appears that the establishment of genetically transformed C. roseus plants with different genes encoding major enzymes of the metabolic pathway could show attractive insights in the metabolic behaviour of transformed plants. These transformed plants may then overcome the bottlenecks of precursor shortage, enzyme inactivation or cell compartmentation, often encountered in suspension

998 culture, a metabolic system showing a lower degree of differentiation. Genetic manipulation of plants has been mainly conducted with species of agronomic interest to improve specific crop traits. Nevertheless, more recently this approach has also been applied for the manipulation of metabolic pathways leading to the formation of important secondary metabolites with pharmaceutical interest (Yun et al. 1992). In this regard, only a few reports on the in vitro regeneration of C. roseus have been published (Möllers et al. 1989, Kaur et al. 1996), chiefly aimed at the production of disease-free plant material. Several reports exist on the transformation of callus and suspension cultures via Agrobacterium tumefaciens mediated transformation (Canel et al. 1998) or particle bombardment (van der Fits & Memelink 1997, Hilliou et al. 1999). To the best of our knowledge, no regeneration protocol using differentiated plant material has been established for the genetic transformation of C. roseus plants and attempts to regenerate C. roseus plants from transgenic suspensions or calli have always failed, indicating their recalcitrant nature. Herewith, the in vitro propagation of this species and a novel approach to genetically transform C. roseus plants by means of particle bombardment, employing the plant reporter genes sGFP and GUS, are described. Moreover, some conclusions on the applications of this technique for the genetic transformation of C. roseus plants are also drawn.

poured in glass test tubes (15 × 2.5 cm) covered with a translucent plastic cap. After seed inoculation, the tubes were placed in a culture suite at 25 ± 2 ◦ C, under a photoperiod of 16 h light (1000 lux) (Philips TL 40W/33 RS). Two months after seed inoculation and germination, when plantlets showed two to three nodes and apical buds, the plantlets were taken out of the tube. Nodal explants, comprising two axillary buds, were excised and transferred to bud induction media, composed as above but supplemented with 0.5, 0.75, 1.0 mg l−1 BAP (benzyl aminopurine), namely 1/2MS0.5, 1/2MS0.75 and 1/2MS1.0 respectively, contained in small Petri dishes (90 mm diameter). After inoculation the Petri dishes were sealed with parafilm and placed under the same culture conditions. Induced shoots from the above three media were excised and individually transferred for rooting on two different media, R1 and R2, contained in test tubes. The initial explant was placed back to the bud induction media for further bud/shoot induction and bombardment. R1 medium was identical to germination medium (lacking plant growth regulators) whereas R2 was the same but supplemented with 0.125 mg l−1 NAA (naphthalene acetic acid). Finally, rooted plantlets were taken out of the tubes and hardened in soil for further plant growth by potting them in gardening peat contained in a 30 × 45 × 25 cm plastic box covered with a translucent lid. The chamber was sprayed with water to provide a high humidity environment and soil was watered regularly.

Materials and methods

Particle treatment and bombardment

Plant material

Tungsten particles of