Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.)

Abstract

In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO₄, AgNO₃ and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO₄, AgNO₃ and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO₄ or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO₄, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO₄. In another study, the basal induction and regeneration medium were fortified with AgNO₃ or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO₃ or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO₃ or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.Abbreviations: 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), IAA (Indole-3-acetic acid), AgNPs (silver nanoparticles), CuNPs (copper nanoparticles).

Keywords: Nano particles; auxins; callus; cytokinins; explant; mature embryo; nanotechnology; tissue culture.

Figure 1.

From synthesis to characterization of CuNPs. (a) Stock solution of prepared 100ppm CuNPs. (b) SEM image of CuNPs, (c) Zeta particle analysis of prepared CuNPs.

Figure 2.

From synthesis to characterization of AgNPs. (a) Stock solution of prepared 100ppm AgNPs. (b) SEM image of AgNPs, (c) Zeta particle analysis of prepared AgNPs.

Figure 3.

Callogenesis response of wheat genotypes to various induction medium. Bars sharing similar letters do not differ significantly (p < .05).

Figure 4.

Embryogenic callusing frequency of wheat genotypes in response to various concentrations of 2,4-D. Bars sharing similar letters do not differ significantly (p < .05).

Figure 5.

Regeneration response of wheat genotypes to various regeneration protocols. Bars sharing similar letters do not differ significantly (p < .05).

Figure 6.

Callus induction and plant regeneration from two wheat cultivars (AS-2002 and Wafaq-2001) and its response to various concentrations of Cu and Ag in salt and nanoparticles form supplemented with growth regulators and hormones. (a) Initial Callus formation (b) callus browning with higher doses of 2,4-D (c) Higher concentration of AgNPs causes callus browning (d) Excess amount of CuNPs promoting callus browning (e) embryogenic callus formation with optimized medium showing green spots (f) Morphology of developing roots (g) Regeneration in response to combined application of 0.015 mg/l CuNPs and 4 mg/l AgNPs (h) Fully developed shoots and branches (i) Toxic effects of Cu and AgNPs causes browning of callus and death of shoot initiation. (j) Fully developed mature plant with multiple shoots and roots system. (k) Regenerated well developed plants in ½ MS medium to improve root system. (l) Normal fertile plants grown in clay pots.