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Effect of nitrogen sources and 2, 4-D treatment on indirect regeneration of ginger (Zingiber officinale Rosc.) using leaf base explants
J Plant Biotechnol 2019;46:17-21
Published online March 31, 2019
© 2019 The Korean Society for Plant Biotechnology.

Valiyaparambath Musfir Mehaboob, Kunnampalli Faizal, Palusamy Raja, Ganesan Thiagu, Abubakker Aslam, Appakan Shajahan

Plant Molecular Biology Laboratory, Department of Botany, Jamal Mohamed College, Tiruchirappalli 620020, India
Correspondence to: e-mail: shajahan.jmc@gmail.com
Received February 1, 2019; Revised March 2, 2019; Accepted March 2, 2019.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Ginger is an important monocotyledonous plant belonging to the family Zingiberaceae. The objective of this study was to investigate the regeneration potential of ginger using leaf base explants. Auxins such as 2, 4-D and NAA in combination with BA were used for initiation of callus. Different combinations of both ammonium (NH4+) and nitrate (NO3-) were also studied for efficient callus production. High frequency of white friable calli was observed on modified Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2, 4-D, 0.5 mg/L NAA and 0.5 mg/L BA. The highest shoot induction (92.33%), shootlets number (7.33± 0.33) and length (88.33±4.40) mm were achieved on MS media containing 0.5 mg/L BA. Regenerated shoots were transferred to in vitro rooting media containing 1.0 mg/L IBA. Afterwards, plantlets with well-developed root and shoot system were subjected to a twostep hardening process. 71% of plantlets survived after secondary hardening without any abnormal morphology.

Keywords : Ginger, Callus, Ammonium, Nitrate, BA, Hardening
Introduction

Zingiber officinale Rosc., commonly known as ginger, is an essential spice crop of tropical south Asian countries and a common condiment for various foods and beverages as it gives taste and flavour. Gingerols, shagols, monoterpenes (geranial and neral) and sesquiterpenes (β-bisabolene and zingiberene) are the major bioactive compounds present in the ginger (Zuraida et al. 2016). These compounds have been used for their antioxidant, anti-inflammatory and cancer preventive properties (Shukla and Singh 2007).

Ginger is propagated vegetatively by underground rhizome at a low proliferation rate. The absence of natural seed set and lack of genetic variability have hampered the development of disease-resistant ginger by conventional breeding (Kackar et al. 1993). Hence, applying non-conventional method is necessary for the production and propagation of this important crop plant. Plant tissue culture techniques will help in the production of the improved disease-free cultivar with a rapid multiplication rate. Methods used for micropropagation of ginger include organ culture (Sharma and Singh 1997), protoplast culture (Geetha et al. 2000), somatic embryogenesis (Guo and Zhang 2005) and synthetic seed production (Nirmal Babu et al. 2016).

The chemical composition of culture media and optimization of mineral nutrients are very much important for the successful in vitro micropropogation. Nitrogen is an essential macronutrient for the synthesis of nucleic acids, proteins and secondary metabolites (Wada et al. 2015). In tissue culture, Optimum level of nitrogen is essential for callus production, shoot and root multiplication and embryogenesis (Shanjani 2003; Utsumi et al. 2017). Ammonium and nitrate are the two major sources of nitrogen in MS medium (Murashige and Skoog 1962). In our study, we tested the different levels of nitrogen and plant growth regulators for efficient callus production and propagation. There are several investigations on callus induction in ginger using shoot tip explants (Ibrahim et al. 2015; El-Nabarawy et al. 2015; Ali et al. 2016). Unlike shoot tips, leaf explants are easy to collect and need not to kill the mother plant and their availability is not limited to any seasons like inflorescence explant (Chugh et al. 2009). Nirmal Babu et al. (1992) reported the callus mediated regeneration of ginger using young leaves. But production of calli and the percentage of response were meager in these studies. The present study reveals the development of an efficient culture system from leaf base callus as well as transplantation of plantlets to natural field condition.

Materials and Methods

Plant material

The leaf base explants (1 ∼ 2 cm) of Z. officinale plants were excised and disinfected with 2% (v/v) Tween-20 for 5 min and 1% (w/v) mercuric chloride (HgCl2) for 2 min, subsequently washed six times with sterilized distilled water, then transferred to MS medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. The pH of the medium was adjusted to 5.6 ∼ 5.9 before autoclaving at 121°C and 104 K pa pressure for 20 min. The cultures were maintained in an air-conditioned room at 22±2°C with photoperiod of 16 hr light and 8 hr dark with a light intensity of 40 µmol m-2 s-1.

Callus induction

Leaf base explants were inoculated on MS solid medium with different plant growth regulators (PGRs) either alone or in combination to induce callus. Modified MS media were tested for the callus induction by using different levels of nitrogen sources ranges from 2280 to 973 mg/L KNO3 and 1650 to 845 mg/L NH4NO3 (Table 1). Each experimental unit consisted of 12 tubes, and the explants were incubated under standard culture conditions. Calli were subcultured within an interval of 3 weeks. The calli formation frequency in different media were recorded.

Composition of MS macronutrients used for callus formation in ginger

MS MacronutrientsStandard MS medium (mg/L)Modified MS medium (mg/L)

MS1MS2MS3MS4
KNO31900228015201216973
NH4NO31650198013201056845
CaCl2.2H2O440440440440440
KH2PO4170170170170170
MgSO4.7H2O370370370370370

Shoot organogenesis from callus culture

After leaf base was cultured for a total of 3 ∼ 4 weeks, proliferated calli were separated from leaf base explant and cut into pieces with a weight of approximately 100 mg, then transferred to a petri dish containing MS medium with various concentrations of BA and 3% sucrose. The shoots and leaves elongated subsequently, so the number of calli forming shoots were recorded after 4 weeks of culture period and subcultured every 3 weeks for regeneration of plantlets. Regenerated shoots (3 ∼ 4 cm) were transferred to MS medium containing IAA and IBA (0.5 to 1.5 mg/L) for in vitro rooting

Acclimatization

Well grown plantlets were removed from the culture jars and washed under tap water to remove agar. Then the plantlets were transferred to small paper cups containing the autoclaved mixture of the vermiculite, soil and sand (1:2:1) and kept at temperature 28±2°C and 70 ∼ 80% relative humidity. Randomly selected primary hardened plants were transferred to earthen pots containing cattle manure, soil and sand (1:2:1) for secondary hardening and acclimatization.

Statistical analysis

All experiments were repeated three times with at least 12 explants. All data were subjected to one way ANOVA with a statistical significance test.

Results and Discussion

Callus induction

The presence of auxin along with cytokinin is a potent hormonal combination for indirect shoot induction as reported by Skoog and Miller (1957). Sterilized young leaf base explants were cultured on MS medium supplemented with different combinations of 2,4-D, NAA, and BA. After 3 ∼ 4 weeks of culture, explants showed leaf folding, bulging and decolouration. Callus growth started from the surface of the leaf base explant, and the nature of calli were varied from compact to friable and brownish to white in the reduced nitrogen medium. Maximum number of friable white calli were observed in MS Macronutrients containing 1216 mg/L KNO3 and 1056 mg/L NH4NO3 (Table 1). The effect of nitrogen on callus production is studied on different plants like Sorghum (Elkonin et al. 2000), Wheat (He et al. 1989) and Cassava (Utsumi et al. 2017). MS3 (Table 1) medium containing 2.0 mg/L 2,4-D in combination with 0.5 mg/L NAA found to be the best for maximum callus induction (92%) (Table 2). Earlier reports also revealed that MS medium supplemented with 2,4-D is the optimum condition for the callus induction in ginger (Ibrahim et al. 2015; El-Nabarawy et al. 2015). The effect of 2,4-D for inducing callus might be due to their role in DNA replication and mitosis (Sen et al. 2014).

Effect of growth regulators combination (2,4-D, BA and NAA) on the callus formation of ginger

Plant growth regulators (mg/L)Callus induction frequency (%) in different nitrogen level

2,4-DBANAAStandard MS medium MS1 MS2 MS3 MS4
0.0--0.0h0.0e0.0h0.0h0.0h
1.0--15.66±1.45g8.66 ±1.76d17.66±4.40g21.0 ±2.51g14.66±2.60g
1.5--18.33±1.20fg9.66 ±0.66d23.0 ±1.0g32.33±2.33f25.0 ±2.30f
2.0--25.0 ±1.73de18.0 ±1.52c35.33±2.33ef42.33±2.96e42.66±3.17d
2.5--21.0 ±2.64efg15.66±1.76c32.33±1.76f34.33±2.33f33.66±1.85e
2.00.3-27.33±2.18de17.66±1.85c42.0 ±3.78de51.66±1.76d41.0 ±4.35de
2.00.5-29.66±0.66d20.0 ±0.57c49.66±1.20cd81.66±2.02b51.66±2.18c
2.01.0-24.33±2.02def17.66±1.45c48.0 ±2.30d66.66±4.63c57.33±3.17c
2.00.50.342.0 ±1.52c26.33±2.18b56.66±1.20bc81.66±2.3b66.33±2.02b
2.00.50.557.33±4.09a31.33±1.20a69.0 ±1.52a92.0 ±2.08a76.0 ±2.88a
2.00.51.050.66±2.02b29.66±2.02ab63.33±4.25ab75.0 ±2.30b70.0 ±2.64ab

Values are expressed as the mean ± SE, taking ten explants in each experiment with three replications. Within each group, values with different letters indicate significant difference at P ≥ 0.05 using Duncan’s multiple range test (DMRT)


Effect of BA on shoot induction

Well-developed white compact calli obtained from leaf sheath explants were transferred to auxin-free MS medium containing BA alone. The presence of BA is necessary for indirect organogenesis in ginger and the maximum number of shoots (7.33±0.33) induced on MS medium supplemented with 0.5 mg/L BA. High frequency (92.33%) of shoot induction was recorded in this concentration (Table 3). Similar results were also reported in Alpinia calcarata (Bhowmik et al. 2016).

Effect of BA on shoot regeneration from in vitro grown callus of ginger

BA (mg/L)Shoot induction (%)Mean number of shoots/callusMean plantlet length (mm)
0.016.0 ±2.88d2.0 ±0.57c51.66±1.66c
0.2567.0 ±3.05c5.33±0.88b68.33±4.40b
0.592.33±2.02a7.33±0.33a88.33±4.40a
0.7578.66±3.17b5.66±0.33ab78.33±3.33ab
1.072.33±1.15bc5.0 ±0.57b75.0 ±2.88b

Values are expressed as the mean ± SE, taking ten explants in each experiment with three replications. Within each group, values with different letters indicate significant difference at P ≥ 0.05 using Duncan’s multiple range test (DMRT)


Effect of auxins on in vitro rooting

In the present study, the addition of IAA and IBA to the culture medium enhanced the root induction and elongation. Among the auxins supplemented for in vitro rooting, the best result noted on 1.0 mg/L IBA with 97.66% rooting response (Table 4). These observations are in accordance with the result obtained in Coleus blumei (Rani et al. 2006), Solanum nigrum (Sridhar and Naidu 2011) and Curcuma amada (Raju et al. 2015).

Effect of auxins (IAA and IBA) on root induction from in vitro regenerated shoots of ginger

Plant growth regulators (mg/L)Days for root inductionRooting (%)Number of roots per explant

IAAIBA
0.00.015-2022.0 ±2.08g4.33±0.66e
0.5-16-1844.0 ±1.52f8.33±0.33d
1.0-15-1760.0 ±3.21d11.33±0.88c
1.5-16-1853.33±1.85e12.66±0.88c
-0.510-1269.66±2.90c16.33±0.88b
-1.010-1297.66±0.33a22.66±0.88a
-1.513-1583.0 ±2.08b18.33±0.57b

Values are expressed as the mean ± SE, taking ten explants in each experiment with three replications. Within each group, values with different letters indicate significant difference at P ≥ 0.05 using Duncan’s multiple range test (DMRT)


Acclimatization

The rooted plantlets were transferred to paper cups containing a mixture of vermiculite, soil and sand (1:2:1) (Fig. 1G). After 3 ∼ 4 weeks of primary hardening, 83% of plants survived and transferred for secondary hardening in earthen pots (Fig. 1H). It was noted that the survival rate was decreased to 71% under the shade house. The regenerates did not show any morphological variations, but genetic variations is yet to be investigated.

Fig. 1.

Callus induction and plantlet regeneration in ginger. (A) Sterilized leaf base explant. (B) White compact calli on the surface of leaf base explant. (C) Friable calli on MS3 medium. (D) Indirect organogenesis on shoot induction medium. (E) Root development in rooting medium. (F) Plantlets excised for hardening. (G) One month old primary hardened plant. (H) 2 month old secondary hardened plant. Bars: (A-C) 0.5 cm; (D-F) 1 cm; (G&H) 3 cm


Conclusion

We have developed an indirect plant regeneration protocol through leaf base explant for ginger, an important spice crop using worldwide. This method is simple, highly reproducible and yields a large number of acclimatized plants. This study indicates that leaf base explants are efficient in terms of indirect organogenesis and shoot multiplication. Production of friable calli by 2,4-D treatment and reduced nitrogen level are the highlights of this study. This protocol will provide a basis for rapid large scale production of ginger in a considerably short period and also useful for the future applications in ginger transformation studies.

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