Nodal and inter-nodal explants of 0.5 - 0.7 cm size were excised and used as explants, while soft leaf explants of 0.5 - 0.7 cm² size were excised and used as explants obtained from carnation cultivar (Dianthus caryophyllous L.). Explants were obtained from pot grown plants which were washed thoroughly with tap water and house hold detergent to remove all the traces of dust particles for 20 minutes. The explants were surface sterlised and inoculated according to the method described by Ali et al. [2] . MS medium [10] supplemented with different concentrations of auxin and cytokinin along with 3% sucrose was used and pH of the medium was adjusted to 5.71 ± 0.5 and 10% of Agar-agar was used for solidification of media which was autoclaved at 121˚C for 15 minutes at 15 lbs/in² pressure. For callus induction and proliferation, MS media containing different concentration and combination of auxin and cytokinin was used. Ten explants were cultured in each test tube and were sub-cultured for proliferation. Sub-culturing was carried out after every 4-week interval unless otherwise indicated. The temperature of 27˚C ± 1˚C was maintained in the growth chamber and photoperiod was 16 hours with 8 hours dark period in every 24 hours cycle. 21˚C - 28˚C temperature was maintained in individual smaller growth chamber to check the effect of temperature on callus proliferation. The cultures were maintained under light intensity of 2500 - 3000 lux.

Callogenesis: Callus was induced from three different explants, leaf, node and inter-nodal explants in MS medium supplemented with different concentrations of 2,4-D alone and BAP in combination with NAA.

For callus induction, leaf explants were cultured in MS medium supplemented with growth regulators and incubated under light (35 µmole∙m⁻²∙s⁻¹ and dark conditions (24 h dark) at 27˚C ± 1˚C.

Callus induction frequencies were calculated as the percent explants inducing callus by using following equation and was converted to mean CIF, as described [11] .

Callus induction frequency (%) = number of calli producing explants/total number of explants in the culture × 100.

Total fresh weight of proliferating calli and multiplied shoots were assessed after specific interval. Total fresh weight was determined and growth value (GV) was calculated according to the following equation as described [12] .

Fresh weight (%) = (final weight – initial weight/initial weight) × 100.

Growth value (%) = (final fresh weight – initial fresh weight/initial fresh weight) × 100.

Total dry weight of calli was measured after dehydration treatment at 70˚C ± 1˚C for two days.

To standardize the medium for the regeneration frequencies of somatic embryos, the well proliferated embryogenic calli from leaf explants were transferred to regeneration media comprising MS medium supplemented with different concentration of BAP and NAA.

After initiating germination of somatic embryos, the plantlets were isolated and transferred onto the MS medium for shoot multiplication. These shoots were sub-cultured on MS medium supplemented with NAA for root induction.

Callus regeneration frequencies were calculated as the percent calli inducing shoots, using following equation and was converted to mean CRF.

Callus regeneration frequency (%) = (number of calli inducing shoots/total number of calli in the culture) × 100.

Mass scale production of carnation from shoot tip culture (Apical Meristem) was described by many scientists [13] [14] . Numerous studies have also been made to regenerate carnation in vitro using different explants through callus phase [15] -[17] . In present study callus induction was investigated using MS medium supplemented with different concentrations of 2,4-D ranging from 1 - 5.0 mg/l. leaf explants showed 100 % callus induction response within 6 days of inoculation on MS medium containing 3.0 mg/l 2,4-D (Table 1). Callus induction started from the cut edges of the leaf explants and covered the whole tissue (Figure 1). Calli obtained from leaf explants were friable and granular, well-proliferated, greenish yellow and morphogenic. Increased or decreased concentration of exogenous auxin, not only reduced the rate of callus initiation but also suppressed the proliferation efficiency. The higher concentration of auxin caused necrosis in calli which appeared in the form of dark brownish patches on yellow callus (Figure 1, Table 1). Callus induction response of nodal explants was 90% within 9 days of inoculation on MS medium containing 3.0 mg/l 2,4-D and calli were friable and granular, well-proliferated, yellowish white and morphogenic (Figure 1, Table 2). 90% callogenic response within 10 days of inoculation was obtained by inter-nodal explants of carnation on MS medium supplemented with 3.0 mg/l 2,4-D (Table 3). Calli obtained from inter-nodal explants were also friable and granular, well-proliferated, yellowish white and morphogenic.

Many researchers reported the best callus induction response of carnation on MS medium supplemented with

Abbreviation: ±: standard error of mean; CIF: callus induction frequency.

Abbreviation: ±: standard error of mean; CIF: callus induction frequency.

Abbreviation: ±: standard error of mean; CIF: callus induction frequency.

auxin alone or auxin along with cytokinins. Esmaiel et al. [5] , reported the best callus induction response from leaf base explants MS medium supplemented with 0.5 mg/l of NAA + 0.5 mg/l of BAP and medium supplemented with 0.01 mg/l of NAA + 1.0 mg/l of BAP. To observe the effects of different higher concentration of exogenous auxin in combination with cytokinin, MS medium supplemented with 1 - 5.0 mg/l NAA and 1.0 mg/l BAP was used. Leaf explants showed 20% callogenic response on MS medium supplemented with 2.0 mg/l NAA and 1.0 mg/l BAP within 16 days of inoculation (Table 4). Calli were brownish yellow, compact, less proliferated and non morphogenic. Nodal explants showed 20% callogenic response on MS medium supplemented with 2.0 mg/l and 3.0 mg/l NAA in combination with 1.0 mg/l BAP within 21 and 24 days of inoculation respectively. Calli were brownish yellow, compact, less proliferated and non morphogenic (Table 5). Inter-nodal explants showed 20% and 10% callogenic response in MS medium supplemented with 2.0 mg/l and 3.0 mg/l NAA in combination with 1.0 mg/l BAP within 18 and 23 days of inoculation respectively (Table 6).

Calli were brownish yellow, compact, less proliferated and non morphogenic. Higher and lower concentration of cytokinin along with BAP showed a reduction in callus induction efficiency (Tables 4-6).

Abbreviation: ±: standard error of mean; NI: no induction; CIF: callus induction frequency.

Abbreviation: ±: standard error of mean; NI: no induction; CIF: callus induction frequency.

Abbreviation: ±: standard error of mean; NI: no induction; CIF: callus induction frequency.

To observe the effects of environmental factors on callus proliferation response, we incubated in vitro grown well-induced calli by leaf, nodal and inter-nodal explants on MS media supplemented with 3.0 mg/l 2,4-D under the temperature of 21˚C, 23˚C, 25˚C, 27˚C, and 30˚C for the period upto 3, 5, 8 and 10 weeks. The observation has shown that 8 weeks proved to be the excellent incubation period to obtain highly proliferated, in vitro grown whitish yellow, friable, soft granular and morphogenic callus. After 8-week of incubation the rate of proliferation was lost as callus started to lose the vigor and appeared brownish yellow (Figure 2, Table 7). Fresh weight of these calli proliferated at various temperatures was measured which indicated the highest fresh weight gain in callus mass proliferated at 27˚C temperature. Dry weight of these calli obtained after dehydration at 70˚C for 2 days indicated the highest dry mass of calli proliferated at 27˚C of incubation temperature. Our experiment proved that 27˚C is most optimal incubation temperature for attaining fully proliferated callus (Figure 3). Under

Key: --- = no induction; + = slow response of proliferation; ++ = moderate; +++ = good; ++++ = excellent.

dark condition callus induction frequency was very low in leaf, nodal and inter-nodal explants. All calli were brownish yellow with strong necrotic signs. Leaf explants proved to be the best with 100% callus induction efficiency within 6-days of callus initiation when exposed to white light. Morphologically these calli were embryogenic, greenish yellow, compact and smooth (Table 8).

Abbreviation: CIF: callus induction frequency.

Callus differentiation on MS medium supplemented with 0.2 mg/l NAA and 1.0 mg/l BAP have been reported by Esmaiel et al., [5] while regeneration protocols for in vitro propagation of carnation via somatic embryogenesis have also been reported [2] [18] . However, Shibat and Mii [19] , reported difficulty in regenerating plants from callus due to their highly dedifferentiated nature. Shoot regeneration in carnation is influenced by genotype, explant source and the balance of plant growth regulators [20] . Our research showed that best grown calli were obtained from leaf explant and transferred to regeneration medium to study regeneration potential. Somatic embryos from morphogenic calli gave rise to shoots with the frequency of 100 % on MS medium supplemented with 3.0 mg/l BAP alone and 1.0 mg/l NAA in combination with 3.0 mg/l BAP. Initially originated shoots were sub-cultured for shoot multiplication onto 3.0 mg/l BAP supplemented medium to assess growth value of in vitro multiplied shoots (Table 9). Healthy shoots were further separated and transferred onto the medium supplemented with root induction growth regulator 1.0 mg/l NAA alone and 1.0 mg/l NAA in combination with 3.0 mg/l BAP which showed 70% root regeneration efficiency in both. Fully grown plantlets were sub-cultured for maintenance in growth chamber in controlled conditions (Figure 4, Table 10). Mangal et al. [21] , and Onamu et al. [22] , used MS medium supplemented with combination of NAA and Kinetin for shoot induction from meristem. Liquid MS medium was found to be more effective for efficient shoot multiplication than MS medium solidified with agar or phytagel [2] . Kanwar and Kumar [23] , reported the rooting response from in vitro multiplied shoots regenerated from callus, on half strength MS medium supplemented with 2.0 mg/l indole butyric acid (IBA) and 0.2% activated charcoal. In current study, well developed multiple shoots when attained considerable heights (~3 cm) were shifted to MS medium containing growth regulator 1.0 mg/l NAA alone and 1.0 mg/l NAA in combination with 3.0 mg/l BAP which showed best in vitro rooting response. The callus mediated regeneration not only improves genetic variability of otherwise limited germplasm of carnation but may be utilized in carnation improvement program via in vitro cell selection, somaclonal variations, genetic transformation and for various biotic and abiotic stresses in this crop. In the present study tissue culture methodology was used in three steps to optimize and standardized the in vitro growth conditions for efficient carnation cultivation i.e., callus induction, callus proliferation and callus regeneration to induce organogenesis directly from somatic embryogenesis. Additionally, variable responses are observed depending on the kind of exogenous-hormone, which are largely related to the source of explants and micro-environment such as incubation period, temperature and light conditions.

Abbreviation: CRF_(S): callus regeneration frequencies of shoots; CRF_(R): callus regeneration frequencies of roots.