Tropical Agricultural Research Vol. 12:50-55 (2000) Department of Crop Science, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka. Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Peradeniya, Sri Lanka. Transient Expression of uidA. Reporter Gene in Regenerable Callus Tissues of Anthurium andraeanum Lind. by Agrobacterium Mediated Transformation G. Yakandawala, S.E. Peiris' and Y.L.N. Yakandawala2 Postgraduate Institute of Agriculture University of Peradeniya Peradeniya, Sri Lanka ABSTRACT. A tissue culture scheme for plant regeneration via callus, and a protocol for gene transfer to regenerable callus are described for Anthurium andraeanum variety A vo Nette. Callus was induced from shoot bases and from leaf blades of Anthurium andraeanum on modified MS media containing 0.3 mg t' 2,4-D and 0.5 mg t' BAP. Shoot bases, leaf blades with a single cut at the base, and sectioned leaf blades produced callus at frequencies of 90%, 20% and 8%, respectively. Plant regeneration was obtained at a high frequency in all callus types, irrespective of their origin, in modified MS media containing 0.5 mg t' BAP. Callus was tested for gene transfer efficiency by 2 Agrobacterium tumefaciens strains, C58 andLB4404, carrying binary vector pCAMBIAi}oi containing hpt and uidA reporter genes under plant expression signals. Transient expression of uidA gene monitored by GUS histochemical assay was observed only in callus inoculated with Agrobacterium strain LB4404 at a frequency of 3.33%. INTRODUCTION Anthurium andraeanum, a perennial herbaceous plant, is a highly priced ornamental for its attractive, long lasting flowers and for exotic foliage. It is being traditionally propagated by off shoots, nodal cuttings and seeds. The uniformity of flowers is an important character, and hence clonal propagation has become an important practice in Anthurium cultivation. In vitro clonal propagation is widely used for commercial cultivation of ornamental plants. One of the major disadvantage of growing clones in any plant species is the danger of devastation by pest and disease epidemics. The biggest threat for Anthurium cultivation worldwide is the disease 'bacterial blight' caused by Xanthomonas compastris pv. dicambacia. Lack of resistant genotypes for certain pests and diseases of Anthurium led to initiate genetic engineering approaches to bring resistant characteristics from other organisms into Anthurium. The capacity to introduce and express diverse foreign genes in plants, first described for tobacco in 1984 (De Block et al., 1984; Horsch et al., 1984; Paszkowski et Transient Expression or uidA Reporter Gene in Regenerable Callus Tissues MATERIALS AND METHODS Plant material; callus induction and regeneration Immature 'leaf blades and shoot bases were collected from in vitro grown Anthurium plahtlets. Fully expandedtender leaves cut into rectangular pieces of 1-2 cm'2, whole leaf blade with a single cut at the base, and shoot bases of approximately 1 cm long were incubated on callus induction medium (CIM) in the dark at 28°C. Callus developed was subcultured twice and transferred onto shoot induction medium (SIM) and incubated under continuous fluorescent light at 28°C. CIM and SIM media contained half-strength macro nutrients, full-strength micro nutrients and vitamins of MS medium (Murashige and Skoog, 1962), 100 mg l'1 myoinisitol, 3% (W/V) sucrose, and 0.7% (W/V) agar, pH 5.8. CIM media contained 0.33 mg l'12,4-D and 0.5 mg I'1 BAP, whereas SIM media contained only 0.5 mg l'1 BAP. Bacterial strains Disarmed Agrobacterium tumefaciens strains C58 and LB4404 carrying the oinary vector p C A M B I A » 0 , (Picambia, Australia) were used for transformation experiments. Overnight cultures of Agrobacterium, prepared by inoculation with a saturated preculture at 1:10 ratio, were induced by adding acetosyringone at a final concentration of 200 pm 1 h before inoculation to explants, .fjhe induced bacterial cells were pelleted and resuspended in plant transformation media.(PTM) containing salts, vitamins and hormones of CIM, 10% (W/V) sucrose, 1 % (W/V) glucose, 5 mM MgCl2 and 200 pm acetosyringone. St al, 1984), has been extended to over 150 species in at least 35 families (Dale, 1995; Birch, 1997). Among the different types of gene transfer techniques, .the presence of Agrobacterium mediated gene delivery is not only for historical and economic reasons, but also for the fact that insertion of transgenes in the host genome, is a very precise technique. Gene transfer to Anthurium has been achieved by inoculation with- Agrobacterium to embryogenic callusetiolated intemodes (Chen and Kuehnle, 1996), and roots (Chen.et al., 1997). The choice of explant for co-cultivation with Agrobacterium is one of the most important factors in rice transformation (Hiei et al, 1994). Tissue culture is not a theoretical prerequisite for plant transformation, but it is employed in almost all current practical transformation systems to achieve a workable efficiency of gene transfer, selection and regeneration of transformants. Plant regeneration via somatic embryogenesis from Anthurium is important and facilitate micropropagation and genetic engineering (Kuehnle era/., 1992). This study was concentrated on developing a plant regeneration system for Anthurium andraeanum variety Avo Nette from different explants via callus phase. A preliminary attempt for transient expression of a foreign gene (i.e. uidA gene) in callus tissues is described. Two Agrobacterium stains, a low virulent type (i.e. LB4404) and a super'virulent type (i.e. C58) were tested for their ability to transform Anthurium. Yakandawala, Peiris & Yakandawala Preparation of explants for inoculation .. One-month-old callus was plasmolyzed for 60 min prior to inoculation by dipping in a liquid CIM media containing 10% sucrose. Plasmolyzed callus was directly transferred .to induced bacterial suspension in PTM, and incubated at room temperature for 20 min. Inoculated callus was transferred onto co-cultivation media, which is similar to PTM but contains only 3% (W/V) sucrose and solidified with 7% (W/V) agar. After 2 days of incubation at room temperature, the calli were subjected to GUS histochemical assay. Histological staining for GUS enzyme activity GUS assay (Jafferson, 1986) was performed according to a modified method of Mendal et al. (1989). The callus co-cultivated with Agrobacterium was incubated overnight at 37°C in darkness in sterile GUS staining solution containing Magenta Gal (Cat. No. B-4657, Sigma Chemical Co., St. Louis, USA). The Magenta coloured loci or sectors were counted under dissecting microscope. RESULTS AND DISCUSSION Before attempting transformation experiments, different types of explant of Anthurium andraeanum Lind. variety Avo Nette were tested for their ability to produce callus and regenerate plants from them. The highest frequency of callus formation was observed in shoot base explant. Other 2 explant types had very low frequency of callus formation (Table 1). Most important factors affecting callus induction and regeneration are the genotype, physiological status of explant, culture media and incubation conditions. Theoretically, for each type of explant, the best media composition and incubation conditions have to be experimentally found. For this experiment the culture media was adopted from previous reports (Kunisaki, 1980; Kuehnle and Sugii, 1991; Pierik et al., 1974; Geier, 1986) and modified accordingly. Table 1. Frequency of callus formation from different explants. Type of Explant Number of Explants Cultured Number of Calli- formed Explants Frequency of Callus Formation Shoot base 100 90 90% Whole leaf blade with a single cut 70 25 20% Sectioned leaf blade 80 10 8% Least number of callus was produced from sectioned leaf blades. Kuehnle et al.. (1992) reported that sectioned leaf blades of Anthurium have not produced any callus under their experimental conditions. Callus derived from different explants did not differ in their regeneration frequency and morphology of the shoots developed. Nearly 100% of the 52 Transient Expression of uidA Reporter Gene in Regenerable Callus Tissues Table 2. Effect of callus type and Agrobacterium strain on transformation frequency. Callus origin Agrobacterium Number of calli Number of GUS Transformation strain co-cultivated positive calli frequency Shoot base LB4404 30 1 3.33% -Shoot base C58 30 0 0% Shoot base - 30 0 0% Leaf blade LB4404 30 0 0% Leaf blade CS8 30 0 0% Leaf blade - 30 0 0% Only 1 magenta sector, corresponding to P-glucuronidase activity of transformed uidA reporter, was observed on a shoot base derived calli inoculated with Agrobacterium, strain LB4404. Lack of GUS activity in negative controls, where calli were not inoculated with Agrobacterium, indicates no detectable endogenous B-glucuronidase activity in Anthurium. 53 callus produced shoot upon transfer onto shoot induction medium (data not shown). It appears that physiological differences of different explants, used for callus induction, do not carry over to the callus they produced. The physiological differences affect when explants of different origin, that are devoted for specialized functions, are forced back to meristematic function and to produce callus. Higher callusing frequency of shoot bases may be attributed to presence of axillary buds, which are of meristematic nature. Transformation was conducted in 2 independent experiments to test the feasibility of 2 types of callus for gene transfer efficiency by 2 Agrobacterium strains (i.e. C58 and LB4404). However, Agrobacterium strain LB4404 showed 33.3% transformation frequency only in the callus originated in the shoot base (Table 2). It has been observed, in various plant species both under natural and laboratory conditions, that the transformation frequencies by Agrobacterium vary according to the genotype, age and physiological status of the explant. Meristematic tissues, young plants and cells undergoing dedifferentiation are the choicest material for Agrobacterium mediated transformation (Mahalakshmi and Khurana, 1997). Another reason for choosing callus in our experiments for gene transfer is the higher probability of transformed cells to regenerate via somatic embryogenesis, which then minimize the number of chimeric plants for the transgene. Further, the relative easiness of callus induction, multiplication, maintenance and ability for aseptic handling have made callus the ideal material for gene transfer experiments. However, callus can cause somaclonal variation. Transformation frequency to Anthurium by different Agrobacterium strains has not yet been documented. In this study, a low virulent strain (LB4404) and a supervirulent strain (C58) of Agrobacterium were evaluated for their ability and frequency to transform regenerable Anthurium callus. Both strains carry binary vector p C A M B 1 A 1 3 0 1 (Picambia, Australia). Yalundawala, Peiris & Yakandawala It is difficult to conclude that those tissues inoculated with Agrobacterium and produced no GUS expression-are completely incompetent for infection by Agrobacterium strains used. Very low frequency of gene delivery may exist due to low competency of target tissue. It could also be due to suboptimal conditions for the infection process to occur. The hidden low frequency of gene transfer could be examined by employing large number of calli for transformation experiment Also, the efficiency of gene transfer could be improved by manipulating the transformation conditions. Immature embryos of maize of different ages were differentially susceptible to Agrobacterium (Mahalakshmi and Khurana, 1997). Results also indicated that there exists a specific window of competence in the explant for gene transfer. Further, the correlation between transient gene expression and stable transformation is not clear (Laparra et al., 1995). CONCLUSIONS In this preliminary study, we described a method for Agrobacterium - mediated gene delivery to regenerate callus of Anthurium andraeanum variety Avo Nette. The results indicate that combination of shoot base derived callus and the low virulent Agrobacterium strain LB4404 is the choice for further experiments. Further improvement "of transformation frequency could be obtained by manipulating the' crucial steps in transformation protocol such as age of the explant, cocultivation and inoculation temperature, media composition and bacterium concentration and growth media. REFERENCES Birch, R.G. (1997). Plant transformation: Problems and strategies for practical application. Annu. Rev. Plant Physiol. 48:297-326. Chen, F.C. and Kuehnle, A .R . (1996). Obtaining transgenic Anthurium through Agrobacterium mediated transformation of etiolated internodes. Hort. Sci. 121(1)4:47-51. • ' ' Chen, F . C , Kuehule, A .R . and Sugii, N . (1997). Anthurium roots for micropropagation and Agrobacterium tumefaciens mediated gene transfer. Plant Cell Tissue and Organ Culture. 49(1): 71-74. Dale, P.J. (1995). R A D regulation and field trial ing of transgenic crops. TrendsBiotechnol. 13:398-403. De Block, M., Henera-Estrella, L., Van Montagu, M., Schell, J. and Zambryski, P. (1984). Expression of foreign genes in regenerated plants and their progeny. E M B O J. 3:1681-1689. Geier, T. (1986). Factors affecting plant regeneration from leaf segments of Anthurium scherzerianum schott (araccae) cultured in vitro. Plant Cell Tissue and Organ Culture. 6(2): 115-120. Hiei, Y„ Ohta, S . , Komari, T. and Kumashiro, T. (1994). Efficient transformation of rice (Oryza saliva L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T -DNA. Plant J. 6:271 -282. Horsch, R.B. , Fraley, R.T., Rogers, S.G., Sanders, P.R. and Lioyd, A- (1984). Inheritance of functional foreign . . . genes in plants. Science. 223:496-498. " Jafierson, R.A. (1986). Assaying chimeric genes in plants: The G U S gene fusion system. Plant Mol. Biol. Rep. 5:387-405. 54 Transient Expression of uidA Reporter Gene in Regenerable Callus Tissues Kuehnle, R.A., Chen, F.C. and Sugii, N. (1992). Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids. Plant Cell Rep. 11:438-442. Kuehnle, R.A. and Sugii, N. (1991). Callus induction and plantlet regeneration in tissue cultures of Hawaiian Anthurium. Hort. Sci. 26(7): 919-921. Kunisaki, A . (1980). In vitro propagation of Anthurium andreaeanum Lind. Hort. Sci. 15(4): 508-509. Laparra, R , Burms, M., Hunold, R., Damm, B . and Bravo-Angle, A . M . (1995). Expression of foreign genes in sunflower (Helianthus annus L.). Euphytica. 85:63-74. Mahalakshimi, A . and Khurana, P. (1997). Agrobactertum-meawSed cereal transformation: A critical appraisal. J. Experimental Biol. 35:416-426. Mendal, R.R., Muller, B., Schnlze, J., Kolensuikov, V . and Zelenin, A . (1989). Delivery of foreign genes to intact barley cells by high velocity microprojectiles. Theor. Appl. Genet 78:31-34. Murashige, J. and Skoog, F. (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant. IS : 473-497. PaszkowskLJ.,Shillito,R.D., Saul, M.,Mandak,V. and Hohn,T. (1984). Direct gene transfer to plants. E M B O , J. 7:4021-4026. Pierik, R.L.M. , Streegmann, H.H.M. and Van Der Meys, J.A.J. (1974). Plantlet formation in callus tissues of Anthurium andraeanum Lind. Scientia Hort 2: 193-198. < 55