Use of compact and friable callus cultures to study adaptive morphological and biochemical responses of potato (Solanum tuberosum) to iron supply

Highlights

• Effect of Fe nutrition on callus morphology is dependent on callus type and culture duration.

• Fe nutrition influenced levels of photosynthetic pigments in potato calli.

• Friable and compact callus cultures exhibit differential tolerance to chlorosis.

• Fe deficiency enhanced ferric reduction capacity and phenolic production.

• Antioxidant enzyme activity is lowered in response to Fe deficiency.

Abstract

The efficient utilization, storage and buffering of iron (Fe) is necessary to prevent the development of iron deficiency or toxicity related stresses in plants. It is of practical interest to develop effective in vitro plant tissue culture schemes for generating chlorosis-tolerant and/or Fe-biofortified plants. The objectives of this study were to induce the formation of compact and friable calli from leaf explants of potato and to investigate adaptive responses to Fe nutrition using these in vitro cell culture-based systems. The severity of chlorosis of the callus cultures increased with the withdrawal of Fe supplementation from the growth medium and the culture duration. Ferric chelate reductase (FCR) activity was found to be higher in the compact compared to the friable potato callus cultures. Chlorophyll and carotenoids content in compact calli increased with an increase in Fe supply but in friable calli carotenoids content declined and the chlorophyll level was unaffected. Phenolic content in Fe-deficient compact calli was significantly higher than in friable ones but a reverse trend was observed as the culture period was extended. Peroxidase activity was reduced in both compact and friable calli cultured under Fe-deprived condition compared to those on Fe-sufficient medium. Friable and compact calli displayed differential morphological and biochemical responses to Fe deficiency. These calli also appeared to vary in their sensitivity to Fe-deficiency and the response to Fe nutrition seemed to be related to the culture duration.

Introduction

Mineral stresses (deficiencies or excesses) constitute a key constraint to crop production worldwide resulting in agricultural and economic losses. The functions of mineral nutrients are normally of a cellular nature and therefore are just as critical to cultured cells as to whole plants. Cellular mineral nutrition and mechanisms by which plants resist mineral stresses indicate that resistance mechanisms that will function in whole plants can be selected (Meredith, 1984). In vitro culture offers a remarkable tool for examining the morphological, biochemical and molecular adaptations and/or changes in plant in response to nutrition and environmental stresses.

In plants, Fe it plays a critical role in photosynthesis, respiration, hormone synthesis, nitrogen fixation, osmoprotection, pathogen defence, production and scavenging of reactive oxygen species, sulphate assimilation, DNA synthesis and repair (Le and Richardson, 2002, Hansch and Mendel, 2009, Zamboni et al., 2012, Hindt and Guerinot, 2012, Kerkeb and Connolly, 2006, Darbani et al., 2013). Excess Fe is toxic since unbound iron can react with oxygen to generate free radicals that destroy cellular components including proteins, DNA, lipids and sugars (Perron and Brumaghim, 2009). Iron however, is mostly unavailable to plants due to the formation of insoluble Fe complexes at neutral or basic pH (Guerinot and Yi, 1994), low soil moisture, high nitrate and carbonates content and low organic matter (Hansen et al., 2006). Fe deficiency in plants causes chlorosis which has been linked to a reduction in crop quality and yields as well as a decrease in nutritional value of edible plant parts (Abadia et al., 2011, Hindt and Guerinot, 2012, Bert et al., 2013, García-Mina et al., 2013). Iron content in potato is between 30 and 45 μg/gDW representing about 3–5% of the recommended daily allowance (Legay et al., 2012, Navarre et al., 2009). Potato is the major non-cereal food crop of great economic importance and increasingly serving as a significant source of food, income and employment worldwide (King and Slavin, 2013, Barrell et al., 2013). Low levels of iron in plant-based foods and insufficient intake of iron can result in anaemia which is the most prevalent and serious nutritional disorder affecting an estimated 30% of the world's population (Hirschi, 2009, WHO, 2011). Understanding how plants absorb and use iron is important for meeting increasing global nutritional demands.

Plants have evolved a conserved set of coordinated responses for the uptake and utilization of Fe in order to maintain balanced levels within cells and prevent toxicity caused by excess Fe (Darbani et al., 2013, Kim and Guerinot, 2007, Vert et al., 2003). Under conditions of low iron bioavailability, plants utilize specialized responses to enhance Fe uptake and mobilization for key cellular metabolic processes. Strategy I plants like potato employ a reduction-based strategy which involves the release of protons for rhizosphere acidification which enhances the solubility of Fe, reduction of Fe (III) to Fe (II) and the transport of Fe (II) from the roots to other parts of the plant (Legay et al., 2012, García-Mina et al., 2013, Thomine and Vert, 2013, Walker and Connolly, 2008). Responses to iron nutrition in plants include hormonal signalling, root morphological modifications, secretion of phenolics and other root exudates, alterations in ferric reduction activity and oxidative stress responses (Hindt and Guerinot, 2012, García-Mina et al., 2013, Rodríguez-Celma et al., 2013, Zamboni et al., 2012, Legay et al., 2012, Robinson et al., 1999).

Improvement in plant's Fe-use efficiency has the potential to boost crop yields and enable the growth of plants in environments limited by low Fe content and heavy Fe fertiliser (Goos and Johnson, 2000, Hansen et al., 2006, García-Mina et al., 2013). In vitro plant tissue culture can be used to screen for tolerance to Fe-deficiency or to enhance Fe nutritional content through biofortification, for example, in potatoes and complement field selection methods of acquiring valuable plants tolerant to Fe stress (shortage or excess) conditions. The objectives of this study were to induce the formation of compact and friable calli from leaf explants of Solanum tuberosum L. (cv, ‘Iwa’) and determine the effect of Fe nutrition on these calli. It was previously found that friable and compact calli induced from potato explants exhibited differential sensitivity to cadmium exposure (Ashrafadeh et al., 2015). It is not known if there is a differential adaptive response to Fe deficiency between these two types of potato calli. Moreover, studies on the mechanisms underlying iron uptake and of the responses of potato callus cultures to Fe nutrition is virtually non-existent. Therefore, another objective was to find out if there is a differential adaptive response to Fe deficiency between the two types of calli. Such studies are, however, prerequisites for the design of efficient in vitro selection schemes for the development of Fe-efficient and/or biofortified potato variants.