Abstract: A new simple method was developed for regeneration of Parsi Zira. This method yielded a large number of shoots within short period of time (30-40 days) without any sub culturing. The effect of various combinations of Plant Growth Regulators (PGRs) on callus formation and shoot regeneration were investigated on MS medium. Simultaneous callus and root formation and shoot regeneration were obtained. The experiment was conducted in a completely randomized design with 30 treatments and 10 replications per treatment. The best treatment for regeneration was the medium supplemented with 0.1 mg L-1 α-Naphthalene Acetic Acid (NAA) and 2 mg L-1 kinetin (Kin). The highest somatic embryogenesis was obtained in the treatment containing 0.1 mg L-1 NAA and 0.5 mg L-1 Kin. The medium containing 2 mg L-1 NAA and 2 mg L-1 Kin was the best treatment for callus and root induction and regeneration simultaneously.
INTRODUCTION
Parsi Zira, Bunium persicum (Boiss.) B. Fedtsch., is a native plant of limited zones of the west Asia and grows in northern areas of Khorasan, Kerman, and east of Zagros to Bandar Abbas and south of Alborz in Iran. Production of this plant is limited due to seed dormancy and several biotic stresses of which wilt diseases are the most serious (Khosravi, 1994). Only cold treatments are effective in seed germination. Other treatments such as gyberlic acid, cytokinin, potassium nitrate, washing and light treatments are not useful (Bonianpoor, 1995).
Generally Umbeliferae species including Bunium persicum have antimicrobial properties (Shetty et al., 1994). Also, potential genetic variability for conventional breeding is limited in Bunium persicum (Hunault et al., 1989). Genetic transformation may enable the development of transgenic plants with enhanced resistance to wilt diseases. However, an efficient micro propagation system with high regeneration frequency is required.
Wakhlu et al . (1990) obtained callus from mericarps of Bunium persicum on MS (Murashige and Skoog, 1962) medium supplemented with 2 mg L-1 2,4-D and 4 mg L-1 Kin. In this report, small white clumps of compactly packed cells developed on the callus on a medium containing 1.0 mg L-1 2,4-D and 0 mg L-1 Kin. These cell clumps differentiated into numerous globular embryos on the same medium. Embryo maturation was achieved on the basal as well as on 1 mg L-1 Kin supplemented medium.
Sharifi (1995) used hypocotyl and cotyledon explants in Bunium persicum tissue culture. The callus growth was faster on B5 (Gamborg et al., 1968) medium containing 2 mg L-1 NAA and 2 mg L-1 Kin. Plumule and shoot formation from hypocotyls and somatic embryogenesis were higher on medium supplemented with 0.1 mg L-1 NAA and 2 mg L-1 Kin and MS medium containing 0.5 mg L-1 2,4-D, respectively.
Wakhlu and Sharma (1998) obtained callus on MS medium containing 0.5 mg L-1 2,4-D and 0.5 mg L-1 BAP from leaf base of Heracleum candicans Wall. Many globular embryos differentiated on callus after transfer to MS medium enriched with 2,4-D without cytokinin. Somatic embryos were matured to plantlet after transfer to medium supplemented with 0.01 mg L-1 BAP and 0.01 mg L-1 IBA.
Ebrahimi et al. (2003) used embryo explants for cumin tissue culture yielding a large number of shoots within short period of time without any sub culturing. In this report, the best treatments were B5 medium containing 0.2 mg L-1 IAA and 1 mg L-1 BAP or 0.2 mg L-1 NAA and 0.2 mg L-1 BAP.
Martin (2004) used stem internodes and leaf explants for regeneration of plantlet from Eryngium foetidum L. (Apiaceae). The callus formation occurred on MS medium supplemented with 5.37-10.74μM NAA and 2.32 or 4.65 μM Kin. Then, somatic embryogenesis occurred on calli after transfer to half- strength liquid MS with 2.69 μM NAA and 1.16 μM Kin.
To obtain plant material in reported methods for Parsi Zira regeneration, seeds have to be germinated in vitro and then hypocotyls and cotyledon leaflets of seedlings used as explant. The reported methods were also laborious and time consuming due to the long callus phase which is associated with using hypocotyl and cotyledon leaflet as explants. Plant regeneration from explants in a short period of time bypassing a long intermediate callus phase could reduce somaclonal variation (Skirvin et al., 1994). Availability of a tissue culture protocol with such criteria would be an important step towards the establishment of a genetic transformation system in this species.
The objective of this study was twofold: first, to determine the possibility of using younger explants like embryo for simultaneous callus and root formation and shoot regeneration without any sub culturing; secondly, to optimize growth regulators for increasing regeneration efficiency and reducing the time.
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 soaked for 20 days in sterile distilled water at 4°C in the dark. Embryos were extracted from the turgid seeds by cutting the end of the seeds and pressing the middle of them. The embryos were cut and only the hypocotyls were used. MS (Murashige and Skoog, 1962) medium containing full-strength of macro- and micro-elements, vitamins and sucrose (30 g l-1) were also used. The different plant growth regulators, NAA (0, 0.1-1 and 2 mg L-1), 2,4-D (0, 0.1, 1, 2 mg L-1) only or together with Kin (0, 0.5, 1 ,2 and 4 mg L-1) were added to the medium.
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 (one explant per each dish or replication) 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 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. The experiment was conducted in 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 10 days and 4 weeks after
explant transfer to medium, respectively. The results showed significant effects
of treatments (Table 2). Due to PGR treatments the size of
callus was different. Generally, the amount of callus in treatments without
cytokinin was lower. The best treatment for regeneration was the medium supplemented
with 0.1 mg L-1 NAA and 2 mg L-1 Kin (Table
1). The medium containing 2 mg L-1 NAA and 2 mg L-1
Kin was the best treatment for callus and root induction and regeneration simultaneously
(Table 1 and Fig. 2). The highest root induction
was observed on treatment with 2 mg L-1 NAA and 2 mg L-1
Kin (Table 1). The highest somatic embryogenesis was observed
on the medium containing 0.1 mg L-1 NAA and 0.5 mg L-1
Kin (Table 1). Somatic embryo growth and stem elongation occurred
after transfer to medium without PGRs (Fig. 1a). In some treatments,
simultaneous regeneration, root and callus induction occurred in the same medium
(Fig. 1b). The above results were obtained eight weeks after
explant transfer to media. The number of regenerated plantlets per explant can
be significantly increased by sub culturing the regenerated calli.
| Table 1: | The effect of PGRs on the frequency of shoot regeneration, root induction and shoot, root mean number |
| Different letters within each column indicate significant differences | |
| Table 2: | Variance analyse of somatic embryogenesis, shoot and root induction frequency, shoot and root mean number |
| * Significant difference at 0.05 probability level,** Significant difference at 0.01 probability level | |
| Fig.1: | a):Somatic embryo growth after transfer to medium without PGRs in Bunium persicum (b): Simultaneous regeneration, callus and root (arrow) induction in Bunium persicum and(c):Organogenesis and shoot regeneration from embryo explant of Bunium persicum |
| Fig. 2: | Effect of PGRs on callus induction frequency in Bunium persicum |
DISCUSSION
With regard to the results, it seems that NAA auxin is more suitable for Bunium persicum regeneration, root and callus induction. But 2,4-D auxin is superior for somatic embryogenesis. 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). The method which has been practiced here revealed some advantages in comparison with others including higher regeneration frequency and regenerated plantlet number, simultaneous regeneration, root and callus induction in the same medium, shortening tissue culture time, less infection, no sub culturing and less chemical consumption. The higher frequency of shoot regeneration from embryo explants compared with previously reports (hypocotyl segments) may be explained by the fact that the former explants are too old to respond to growth regulators rapidly and efficiently, but the later is younger and more sensitive, responding more quickly to PGRs. In the previous studies, simultaneous regeneration and root induction did not occur and regenerated plantlets were rooted after transfer to medium without PGRs (Wakhlu et al., 1990) or half-strength medium with 0.5 mg L-1 IBA (Tawfik and Noga, 2001). Embryo culture has been used for olive and iris. (Canas et al., 1992). In other plant species, embryo explants have been used for micro propagation and genetic transformation (Yong et al., 1999).
This method yielded a large number of shoots within short period of time (30-40 days) without any sub culturing. The total period is 50-60 days, considering cold treatment. Previously reported methods are consisted of in vitro seed germination, obtaining seedling, cutting hypocotyls and culturing hypocotyls in callus formation medium and transferring callus to regeneration medium. These processes are time consuming (210 days considering 90 day-cold treatment) and laborious to be used as an efficient and economical micro propagation system. The above results show the importance of explant type. The presented method may also be useful for future studies on other spices like Cuminum setifulium. As mentioned above, Parsi Zira production is highly limited because of seed dormancy and fungal diseases. By application of this method, a large number of desirable and pathogen free genotypes can be propagated. A high frequency of plantlet regeneration increases the chance of obtaining transformed plant on genetic transformation procedures and short tissue culture time is helpful in reducing the time and cost. This method could also be used to produce essence and raw materials for pharmaceutical industry.