MATERIALS AND METHODS

Parsi Zira seeds were collected from Kalat at Khorasan province in Iran. Seeds were surface sterilized in 1.5% (w/v) sodium hypochlorite solution for 15 min and rinsed three times with sterile distilled water. Seeds were then cultured on media supplemented with full-strength of macro- and micro-elements, vitamins and sucrose (30 g L^-1 in MS and 20 g L^-1 in B5) for 80 days at 4°C in the dark to germinate After 3-4 days, hypocotyls were cut into segments with the same lengths (5 mm). The different plant growth regulators, NAA (0.1, 1, 2 mg L^-1), 2,4-D (0.1, 1, 2 mg L^-1) only or together with Kin (0, 0.5, 1, 2, 4 mg L^-1) were used. Prior to autoclaving at 121°C for 15 min pH was adjusted to 5.7. The medium were solidified with 8% (w/v) agar (Sigma).

The explants were cultured in sterile dishes (7x12 mm) each containing 15 ml of culture medium, which was sealed with Para film and maintained at 25±2°C under 16 h photoperiod (30 μmoles m^-2 s^-1). After 4-6 weeks, the calli were sub cultured. After eight weeks, the number of explants producing callus and regeneration and the number of shoots regenerated from each replication were counted. Then the frequency of callus induction and shoot regeneration were calculated by dividing the number of calli and shoots to the original number of plated explants. Regenerated plantlets were transferred to basal medium to be rooted. The experiment was conducted as a completely randomized design with 30 treatments and 10 replications per treatment. Mean of replications was used for statistical analysis. ANOVA was used to analyze the frequency of callus formation and shoot regeneration. Mean separation was conducted using Duncan^,s Multiple Range test at 0.05 probability. This study carried out in Khorasan Agriculture and Natural Resources Research Center of Iran in 2005.

RESULTS

Callus initiation and regeneration were observed on B5 medium, 2 and 8 weeks after explant transfer to medium, respectively. They were observed on MS medium, 3 and 8 weeks after explant transfer to medium, respectively (Fig. 1 and 2). The results showed significant effects of treatments on regeneration and callus induction (Table 2).

Fig. 1:	Callus initiation on hypocotyl explant of Bunium persicum after 3 weeks

Fig. 2:	Early regeneration from hypocotyl explant of Bunium persicum after 8 weeks

Fig. 3:	Root induction after transfer of Bunium persicum plantlet to basal medium without PGRs

Table 1:	The effect of PGRs on shoot regeneration and callus induction frequency on B5 and MS media in some treatments
Different letter(s) within each column indicate significant differences

Table 2:	Variance analyze of shoot regeneration and callus induction frequency on B5 and MS media.
*Significant difference at 0.05 probability level, **Significant difference at 0.01 probability level

Due to PGR treatments the size of callus was different. Generally, the amount of callus in treatments without cytokinin was lower. The highest callus frequency was observed on MS medium containing 0.1 mg L^-1 2,4-D or 1 mg L^-1 2,4-D as well as 2 mg L^-1 2,4-D and 0.5 mg L^-1 Kin (Table 1). The best response for shoot regeneration was observed on MS medium containing 1 mg L^-1 2,4-D. MS medium supplemented with 1 mg L^-1 2,4-D was the best for callus induction and shoot regeneration simultaneously (Table 1). Generally, regeneration and callus induction frequency on MS medium and treatments containing 2,4-D auxin were higher than B5 and treatments with NAA (Table 1).

The above results were obtained eight weeks after explant transfer to media. Callus proliferation and stem elongation significantly increased by sub culturing the regenerated calli. The regenerated plantlets were rooted after transfer to basal medium without PGRs (Fig. 3).

DISCUSSION

Somatic embryogenesis during callus induction is not unusual in Umbeliferae family specially carrot and fennel (Hunault et al., 1989). 2,4-D and NAA only or in combination with Kin are essential for continuity of callus induction. Auxin reduction leads to organogenesis and adventitious embryo formation in Parsi Zira. Regeneration occurred in some treatments without kinetin, showing that kinetin is not essential for Parsi Zira regeneration like other Umbeliferae species such as carrot and fennel (Hunault et al., 1989), but it is essential for cumin regeneration (Ebrahimie et al., 2003). The previous studies suggest that callus transfer to media supplemented with 1 mg L^-1 2,4-D (Wakhlu, 1990) or 0.5 mg L^-1 2,4-D (Bonianpoor, 1995) leads to embryogenesis. However suitable combination of auxins and cytokinins are important for embryogenesis and organogenesis (Guohua, 1998). In some other species, induced somatic embryos might need a little cytokinin or other plant growth regulators to grow (Kumar et al., 1988). Subsequent callus proliferation and differentiation on basal medium without PGRs depend on 2,4-D and NAA amount in callus induction medium. Also in the previous studies about cumin, callus proliferation and stem elongation occurred after transfer to basal medium without PGRs (Tawfik and Noga 2002; Wakhlu et al., 1990). Such an embryogenesis development is not unusual in other Umbeliferae species (Hunault et al., 1989). However 2,4-D prevents the induced somatic embryos to germinate. Embryonic callus transfer to basal medium decreases the 2,4-D and solves this problem. With regard to the results, it seems that MS medium and 2,4-D auxin are more suitable for Bunium persicum regeneration and callus induction.

As mentioned above, Parsi Zira production is highly limited because of seed dormancy and fungal diseases. By application of this method and transduction of disease resistant genes, a large number of desirable and pathogen free genotypes can be propagated.

ABBREVIATIONS

BAP- 6-benzylaminopurine; 2,4-D - 2,4-dichlorophenoxyacetic a cid; IAA- indol-3-acetic acid; Kin - kinetin; MS - Murashige and Skoog (1962); NAA - α-Naphthalene acetic acid; PGRs - plant growth regulators.