Biogenic iron oxide nanoparticles enhance callogenesis and regeneration pattern of recalcitrant Cicer arietinum L

Abstract

This study is the first report on the biosynthesized iron oxide nanoparticles (IONPs) which mediate in-vitro callus induction and shoot regeneration in economically important recalcitrant chickpea crop (Cicer arietinum L.). Here, we used leaf extract of Cymbopogon jwarancusa for the synthesis of IONPs in order to achieve a better biocompatibility. The bioactive compounds in C. jwarancusa leaf extract served as both reducing and capping agents in the fabrication process of IONPs. Field emission scanning electron microscopy (FE-SEM) revealed rods like surface morphology of IONPs with an average diameter of 50±0.2 nm. Energy-dispersive X-ray spectroscopy (EDS) depicted formation of pure IONPs with 69.84% Fe and 30.16% O2. X-ray diffractometry (XRD) and attenuated total reflectance-fourier transform infrared (ATR-FTIR) validate the crystalline structure, chemical analysis detect the presence of various biomolecular fingerprints in the as synthesized IONPs. UV-visible absorption spectroscopy depicts activity of IONPs under visible light. Thermo-gravimetric analysis (TGA) displayed thermal loss of organic capping around 500°C and confirmed their stabilization. The biosynthesized IONPs revealed promising results in callus induction, shoot regeneration and root induction of chickpea plants. Both chickpea varieties Punjab-Noor 09 and Bittle-98 explants, Embryo axes (EA) and Embryo axes plus adjacent part of cotyledon (EXC) demonstrated dose-dependent response. Among all explants, EXC of Punjab-Noor variety showed the highest callogenesis (96%) and shoot regeneration frequency (88%), while root induction frequency was also increased to 83%. Iron content was quantified in regenerated chickpea varieties through inductively coupled plasma-optical emission spectrometry. The quantity of iron is significantly increased in Punjab-Noor regenerated plants (4.88 mg/g) as compare to control treated plants (2.42 mg/g). We found that IONPs enhance chickpea growth pattern and keep regenerated plantlets infection free by providing an optimum environment for rapid growth and development. Thus, IONPs synthesized through green process can be utilized in tissue culture studies in other important recalcitrant legumes crops.

Fig 1. Stepwise schematic protocol for green synthesized IONPs using leaves extract of C. jwarancusa.

Fig 2. Field emission scanning electron microscope (FE-SEM) micrographs of IONPs showing nanorods at a resolution of (a) 10 KX (b) 25 KX (c) 50 KX (d) 100 KX (e) Particle size distribution of IONPs.

Fig 3. (a) X-ray powder diffraction (XRD) patterns of biosynthesized α-Fe₂O₃ demonstrating Braggs diffraction peak associated to Fe crystallites planes, (b) ATR-FTIR spectrum of green synthesized IONPs showing bond vibrations coming from surface attached bioactive compounds and Fe-O, (c) UV-Vis spectrum of IONPs, and (d) Thermogravimetric analysis (TGA) of the prepared IONPs.

Fig 4. Effect of IONPs (1, 5, 10, 15, 20 mg/L) on callogenesis of chickpea varieties; EA, embryo axes with removed root apex, EXC embryo axes plus attached part of the cotyledon.

Fig 5. Effect of IONPs (1, 5, 10, and 15 mg/L) on organogenesis of chickpea varieties; EA, embryo axes EXC.

Embryo axes plus attached part of cotyledon.

Fig 6. The hypothesized mechanism of action of hematite IONPs as nano-fertilizer to enhance regeneration capability of recalcitrant crops.

Fig 7. (a) Effect of different concentrations of IONPs on shoot length of proliferating shoots from different explants; EXC and EA, and (b) Iron content per dry weight of regenerated chickpea plantlets.