Mutants of Citrus macrophylla rootstock obtained by gamma radiation improve salt resistance through toxic ion exclusion

Highlights

• Citrus Macrophylla mutants are less sensitive to salinity than Citrus Macrophylla.

• MM4B was almost not damaged by salinity.

• MM4B and MMN1 were more efficient in Na + exclusion than Citrus Macrophylla.

• MM1A, MM4A, MM3B were more efficient in Cl-exclusion than Citrus Macrophylla.

• The capacity of Cl-exclusion in linked with a higher absorption capacity of NO3- in salinity conditions.

Abstract

Salinity is one of the biggest challenges that need to be faced in crop production. Citrus is highly sensitive to salt stress and obtaining rootstocks with improved resistance to salinity is key for the citrus growing industry. In this study, five mutants of Citrus macrophylla rootstock, obtained through gamma radiation and in vitro pre-selected for their resistance to salinity, were irrigated with a solution containing 100 mM of NaCl. After 8 weeks of exposure, the mutants were evaluated for their performance (growth, visual leaf damage) and chlorophyll, proline, starch, soluble sugars and ion contents to determine their degree of resistance to this salinity level.

In the saline conditions assayed, all the mutants showed better performance and less leaf damage than Citrus macrophylla. Our data suggest that this improved resistance to salinity was based on their capacity to accumulate less Na (MM4B and MMN1) or Cl⁻ (MM1A, MM4A and MM3B). Besides having the lowest Cl⁻ content, the mutants MM1A, MM4A and MM3B, had the highest NO₃⁻ concentrations in salinity. Furthermore, mutants did not show chlorophyll degradation and showed less leaf damage and acceptable plant growth. Other parameters including proline and soluble sugars, did not prove decisive in the salinity resistance of these genotypes.

Introduction

Soil salinization is the most common land degradation process and one of the major threats to this resource worldwide (WAD, 2018). The increasing mean global temperature and the lower rainfall are forcing growers to use low quality water for irrigation, and this, along with poor drainage from the sub-soil, leads to salt accumulation in the root zone. This increase in the salt concentration has profoundly detrimental effects on major morphological, physiological and molecular processes in non-tolerant plants (Dajic, 2006), and results in an estimated annual global cost of US$ 27.3 billion related to lost crop production (WAD, 2018).

Salinity induces osmotic stress and ion-specific toxicity due to the presence of the ions which compose the salt. Most of the studies, related with soil salinity problems mention that the resulting stress is caused by sodium chloride (NaCl) accumulation (Colmenero-Flores et al., 2020). Plants exposed to salinity undergo two stages of stress: a first phase in which an increase in osmotic pressure makes it more difficult for roots to extract water which causes dehydration symptoms to appear in the plant; and a second phase, in which Na⁺ and Cl⁻ uptake greatly increase, and whose accumulation at toxic concentrations leads to leaf damage, delayed growth and deficient crop production (Munns and Tester, 2008). In an attempt to withstand such conditions, many species have evolved mechanisms to tolerate NaCl stress including the boosting of osmotolerance, Na⁺ and Cl⁻ exclusion, and/or greater tissue tolerance (Adem et al., 2014). However, most commercially grown plants do not have these features and are usually sensitive to given levels of salinity. This is the case of citrus, a genus highly sensitive to salt stress (Mass, 1993), which comprises the most important group of commercial crops and which is mainly grown in arid and semi-arid areas (Inskeep and Bloom, 1985; FAO, 2017).

Because of the importance of citrus crops in the market, many authors have studied the effects of salinity on different rootstocks and cultivars (Gomez-Cadenas et al., 2003; Navarro et al., 2014; Sá et al., 2018; Shahid et al., 2019). Thus, it is known that the salt ions enter the root system, which acts as a first filter for the whole plant. Therefore, the correct choice of rootstock can increase the salt-tolerance potential of the plant by enhancing antioxidant activities, increasing the concentrations of osmoprotectants, and limiting the levels of Na and Cl in leaves (Shahid et al., 2019). Consequently, grafting on salt-tolerant rootstocks has the potential to alleviate the deleterious effects of salt stress on the scion (Maas and Grattan, 1999), helping the whole plant to endure saline conditions. As a result, in an evaluation of the most popular citrus rootstocks for their ability to withstand salinity (García-Sánchez et al., 2000; Navarro et al., 2014), Citrus macrophylla (MA) was found to be moderately resilient to salt stress (Gomez-Cadenas et al., 2003). MA has excellent features for it to be used as citrus rootstock making precocious trees and good-quality fruit producers. Furthermore, MA is resistant to root rot as well as being tolerant to boron (Bowmana and Joubertb, 2020). Therefore, MA seems to have an overall genetic background that can be useful as a starting point in the search for new rootstocks that are salinity resistant.

Citrus rootstock breeding programs carried out by sexual hybridization are hampered by citrus reproductive biology (apomixis) and the high heterozygosity of the citrus genomes (Ruiz et al., 2018). This implies to a long and laborious process although the probability of having individuals that combine all the desired traits is usually very low. This, combined with the fact that salinity seems to be regulated by many mechanisms, make any enhancement of salinity tolerance in citrus rootstock a very difficult task, so that alternative ways need to be found. One such way is gamma radiation, which has already been used to improve salinity tolerance in other species (Coto et al., 2014; Nikam et al., 2014; Wang et al., 2018); although to our knowledge, no new citrus rootstock has ever been obtained through gamma radiation.

In the present study, five MA mutants obtained by gamma radiation in previous experiments (Tallón et al., 2015), were selected for their seeming tolerance to salinity. The mutants and MA were subjected to salinity and their performance (growth, visual leaf damage) and their chlorophyll, proline, starch, soluble sugars and ion contents were recorded in order to determine their degree of salinity tolerance at a given salinity level and the underlying mechanisms that might be operating to regulate this trait.