An efficient procedure was developed for in vitro callus induction, proliferation and regeneration of carnation cultivar (Dianthus caryophyllus L.) using leaf, nodal and inter-nodal explants on Murashige and Skoog’s medium (MS) supplemented with exogenous plant growth regulators. For morphogenic callus induction and proliferation from various explants, MS medium supplemented with 3.0 mg/l 2,4-D was highly efficient with 100% callus induction frequency from inter-nodal explants. Leaf explants showed quicker response than nodal and inter-modal explants, for callus initiation within 6 days of inoculation. Best grown callus was obtained from leaf explant. The leaf-derived callus was maintained up to several weeks, which indicated that 8-week incubation period was the most suitable for obtaining well proliferated, morphogenic callus. Temperature variation also affected the growth of in vitro induced morphogenic callus from various explants. Results have shown that 27°C proved to be the best temperature for morphogenic callus induction and proliferation from leaf and inter-nodal explants. Among the auxin-cytokinin combination, MS medium containing 1.0 mg/l N(6)-benzylaminopurin (BAP) and 2.0 mg/l NAA showed the highest efficiency of callus initiation and proliferation from leaf, nodal and inter-nodal explants. Light conditions proved better for callogenesis and proliferation from leaf, nodal and inter-nodal explants. Regeneration response from well grown morphogenic callus was prominent on MS medium supplemented with 3.0 mg/l BAP alone and 1.0 mg/l NAA with 3.0 mg/l BAP.
Carnation, a member of the family Caryophyllous, has 88 genera and 1750 species, which were cultivated over 2000 years ago in Asia and Europe. There are over 300 species, mostly perennials having showy petals of the colors of the rainbow except blue [
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. [
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 [
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 [
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 [
Many researchers reported the best callus induction response of carnation on MS medium supplemented with
Media composition (mg/l) | Days for callus induction | Mean No. of cultures showing callus induction | Types of callus | CIF (%) |
---|---|---|---|---|
1.0 mg/l 2,4-D | 18 | 2 ± 0.247 | Whitish yellow, granular, soft, less proliferated | 20 |
2.0 mg/l 2,4-D | 10 | 4 ± 0.420 | Whitish yellow, granular, friable, morphogenic | 40 |
3.0 mg/l 2,4-D | 6 | 10 ± 0.414 | Greenish yellow, granular, morphogenic, soft | 100 |
4.0 mg/l 2,4-D | 8 | 5 ± 0.414 | Yellowish brown, globular, morphogenic, rough | 50 |
5.0 mg/l 2,4-D | 12 | 2 ± 0.609 | Brown, non-morphogenic, less proliferated | 20 |
Abbreviation: ±: standard error of mean; CIF: callus induction frequency.
Media composition (mg/l) | Days for callus induction | Mean No. of cultures showing callus induction | Types of callus | CIF (%) |
---|---|---|---|---|
1.0 mg/l 2,4-D | 22 | 3 ± 0.346 | Whitish yellow, friable, soft, less proliferated | 30 |
2.0 mg/l 2,4-D | 12 | 5 ± 0.171 | Whitish yellow, soft, granular, less proliferated | 50 |
3.0 mg/l 2,4-D | 9 | 9 ± 0.39 | Whitish yellow, friable, well proliferated | 90 |
4.0 mg/l 2,4-D | 10 | 8 ± 0.579 | Whitish yellow, granular, well proliferated | 80 |
5.0 mg/l 2,4-D | 14 | 4 ± 0.5 | Brownish yellow, compact, non-morphogenic | 40 |
Abbreviation: ±: standard error of mean; CIF: callus induction frequency.
Media composition (mg/l) | Days for callus induction | Mean No. of cultures showing callus induction | Types of callus | CIF (%) |
---|---|---|---|---|
1.0 mg/l 2,4-D | 24 | 3 ± 0.269 | Whitish yellow, granular, less proliferated | 30 |
2.0 mg/l 2,4-D | 15 | 4 ± 0.5 | Whitish yellow, granular, less proliferated | 40 |
3.0 mg/l 2,4-D | 10 | 9 ± 0.171 | Whitish yellow, granular, less proliferated | 90 |
4.0 mg/l 2,4-D | 11 | 7 ± 0.577 | Yellow, globular, well proliferated | 70 |
5.0 mg/l 2,4-D | 15 | 3 ± 0.25 | Brownish yellow, compact, less proliferated | 30 |
Abbreviation: ±: standard error of mean; CIF: callus induction frequency.
auxin alone or auxin along with cytokinins. Esmaiel et al. [
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).
Media composition (mg/l) | Callus induction | Mean No. of cultures showing callus induction | Types of callus | CIF (%) |
---|---|---|---|---|
1.0 mg/l BAP + 1.0 mg/l NAA | NI | --- | --- | --- |
1.0 mg/l BAP + 2.0 mg/l NAA | 16 | 2 ± 0.609 | Whitish yellow, compact, prolifrated | 20 |
1.0 mg/l BAP + 3.0 mg/l NAA | 20 | 1 ± 0.216 | Brownish yellow, less proliferated | 10 |
1.0 mg/l BAP + 4.0 mg/l NAA | 20 | 1 ± 0.856 | Brownish yellow, less proliferated | 10 |
1.0 mg/l BAP + 5.0 mg/l NAA | 26 | 2 ± 0.609 | Brownish yellow, less proliferated | 10 |
Abbreviation: ±: standard error of mean; NI: no induction; CIF: callus induction frequency.
Media composition (mg/l) | Days for callus induction | Mean No. of cultures showing callus induction | Types of callus | CIF (%) |
---|---|---|---|---|
1.0 mg/l BAP + 1.0 mg/l NAA | NI | --- | --- | --- |
1.0 mg/l BAP + 2.0 mg/l NAA | 21 | 2 ± 0.5 | Brownish yellow, friable, soft | 20 |
1.0 mg/l BAP + 3.0 mg/l NAA | 24 | 2 ± 0.25 | Brownish yellow, friable, less proliferated | 20 |
1.0 mg/l BAP + 4.0 mg/l NAA | NI | --- | --- | --- |
1.0 mg/l BAP + 5.0 mg/l NAA | NI | --- | --- | --- |
Abbreviation: ±: standard error of mean; NI: no induction; CIF: callus induction frequency.
Media composition (mg/l) | Days for callus induction | Mean No. of cultures showing callus induction | Types of callus | CIF (%) |
---|---|---|---|---|
1.0 mg/l BAP + 1.0 mg/l NAA | NI | --- | --- | --- |
1.0 mg/l BAP + 2.0 mg/l NAA | 18 | 2 ± 0.5 | Yellow, friable, less proliferated, non-morphogenic | 20 |
1.0 mg/l BAP + 3.0 mg/l NAA | 23 | 1 ± 0.25 | Brownish yellow, friable, less proliferatednon-morphogenic | 10 |
1.0 mg/l BAP + 4.0 mg/l NAA | NI | --- | --- | --- |
1.0 mg/l BAP + 5.0 mg/l NAA | NI | --- | --- | --- |
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 (
Media: MS + 3.0 mg/l 2,4-D | ||
---|---|---|
Incubation time (weeks) | Type of callus | Proliferation response |
3 | Whitish yellow, soft, friable, granular, less proliferated | ++ |
5 | Whitish yellow, soft, friable, granular, less proliferated and morphogenic | +++ |
8 | Whitish yellow, soft, friable, granular, well proliferated, morphogenic | ++++ |
10 | Brownish yellow, globular, non-morphogenic, decreased rate of proliferation | + |
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 (
Leaf explant | |||||||
---|---|---|---|---|---|---|---|
Treatment | Media | CIF (%) | Days for callus induction | Callus morphology | |||
Light | MS + 2,4-D 3.0 mg/l | 100 | 6 | Greenish yellow, morphogenic, proliferated | |||
MS + 2,4-D 1.0 mg/l + BAP 3.0 mg/l | 80 | 9 | Whitish yellow, morphogenic, proliferated | ||||
Dark | MS + 2,4-D 3.0 mg/l | 30 | 16 | Brownish yellow, necrotic, non proliferated | |||
MS + 2,4-D 1.0 mg/l + BAP 3.0 mg/l | 20 | 18 | Brownish yellow, necrotic, non proliferated | ||||
Nodal explant | |||||||
Light | MS + 2,4-D 3.0 mg/l | 30 | 9 | Whitish yellow, morphogenic, proliferated | |||
MS + 2,4-D 1.0 mg/l + BAP 3.0 mg/l | 20 | 15 | Brownish yellow, necrotic, non proliferated | ||||
Dark | MS + 2,4-D 3.0 mg/l | 10 | 20 | Brownish yellow, necrotic, non proliferated | |||
MS + 2,4-D 1.0 mg/l + BAP 3.0 mg/l | 10 | 24 | Brownish yellow, necrotic, non proliferated | ||||
Inter-nodal explant | |||||||
Light | MS + 2,4-D 3.0 mg/l | 20 | 10 | Whitish yellow, morphogenic, proliferated | |||
MS + 2,4-D 1.0 mg/l + BAP 3.0 mg/l | 10 | 16 | Brownish yellow, necrotic, non proliferated | ||||
Dark | MS + 2,4-D 3.0 mg/l | 10 | 24 | Brownish yellow, necrotic, non proliferated | |||
MS + 2,4-D 1.0 mg/l + BAP 3.0 mg/l | 10 | 25 | Brownish yellow, necrotic, non proliferated | ||||
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., [
MS Media | Shoot induction (%) | No. of shoots/culture | Average length of shoots (cm) | Growth value |
---|---|---|---|---|
3.0 mg/l BAP | 100 | 15 | 3.5 cm | 79.56 |
MS media | Leaf explant | MS + NAA (mg/l) | Inter-nodal explant | ||
---|---|---|---|---|---|
CRF(S) (%) | CRF(R) (%) | CRF(S) (%) | CRF(R) (%) | ||
Basal medium (control) | 0 | 0 | Basal medium (control) | 0 | 0 |
1.0 mg/l BAP | 10 | 20 | 1.0 mg/l NAA | 10 | 30 |
2.0 mg/l BAP | 50 | 40 | 2.0 mg/l NAA | 20 | 50 |
3.0 mg/l BAP | 100 | 70 | 3.0 mg/l NAA | 70 | 100 |
4.0 mg/l BAP | 60 | 50 | 4.0 mg/l NAA | 30 | 40 |
5.0 mg/l BAP | 10 | 10 | 5.0 mg/l NAA | 10 | 20 |
1.0 mg/l BAP + 1.0 mg/l NAA | 10 | 10 | 1.0 mg/l BAP + 1.0 mg/l NAA | 30 | 20 |
2.0 mg/l BAP + 1.0 mg/l NAA | 50 | 40 | 2.0 mg/l BAP + 1.0 mg/l NAA | 50 | 40 |
3.0 mg/l BAP + 1.0 mg/l NAA | 100 | 70 | 3.0 mg/l BAP + 1.0 mg/l NAA | 80 | 90 |
4.0 mg/l BAP + 1.0 mg/l NAA | 70 | 40 | 4.0 mg/l BAP + 1.0 mg/l NAA | 50 | 40 |
5.0 mg/l BAP + 1.0 mg/l NAA | 20 | 10 | 5.0 mg/l BAP + 1.0 mg/l NAA | 10 | 20 |
Abbreviation: CRF(S): callus regeneration frequencies of shoots; CRF(R): callus regeneration frequencies of roots.
From the present study it was concluded that although the callus can be produced under several growth conditions using leaf, nodal and inter-modal explants but the differentiation of callus induced from nodal and inter- nodal explants was difficult. Thus the efficient protocol is developed to elevate the efficiency of callogenesis us- ing leaf explants source, by optimizing hormonal concentrations suitable for high frequency of callus induction and regeneration via somatic embryogenesis that can facilitate mass propagation of carnation predominantly.