Measurement of Lipid Peroxidation and Phenol Contents Under Heat Stress Condition in Sugarcane Genotypes

The experiment conducted in the laboratory of Plant Breeding and Molecular Genetics, Faculty of Agriculture, Rawalakot, Pakistan. Leaves of thirteen genotypes of sugarcane used for this experiment and were treated in an oven for heat stress. The treated leaves were then used for the estimation of phenol contents and the measurement of lipid peroxidation against heat stress. Minimum moisture loosed by CPF-237 and maximum moisture loss showed by CO-1148. Maximum phenol contents were observed in S-2003-US-778 showed high results (58.78 mg GAE/100ml) and minimum were Rb-72 and CO-1148 showed the lowest values (36.17 and 37.5 mg GAE/100ml). The higher heat tolerance of genotype, CPF-237 related to higher membrane stability, and maintenance of high fv/fm ratio under heat stress and lower lipid peroxidation of membranes. Hence, the relative tolerance of a genotype to heat stress as reflected by its lower lipid peroxidation, and higher membrane stability and pigment concentration, related to the levels of activity of its antioxidant enzymes. By using morphological parameters, S-2003-US-778 was significantly different from Rb-72. Based on biochemical data, the genotypes S-2003-US-778, S-2003-US-694, CPF-237 and Co-1148 are heat tolerant genotypes and thus can be successfully planted in warmer areas to alleviate the socioeconomic status of farmers.


Introduction:
Sugarcane (Saccharum officinarum L.) is an important cash crop of Pakistan, plays energetic role to improve economic perspective of the farmers and survival of ever-expanding sugar industry in Punjab.Pakistan is significant cane producing country and categorized fifth in world cane acreage and 15 th in sugar production (Insert Reference).Sugarcane developed on over a million hectares and delivered the raw material for Pakistan's 84 sugar mills, which contain the country's second largest agro-industry after textiles (Rehman, 2009).Thus evolution of new high cane and sugar yielding varieties and upgraded production technology i.e., Better Management Practices (BMPs) are existing need for improving livelihoods of sugarcane growers and other crops and ultimately advancement of mill owners also (Nasir, 2006;Iftikhar et al., 2010).Addition of organic matter in soil recovers the physicochemical and biological properties.Assessment of adaptation, performance of various cane selections in different ecologies, and evaluation of agronomic characters of exotic cane varieties is necessary before a variety introduced for commercial cultivation.Cane yield and sugarcane evaluation of varieties were commonly done on stalk, stalk height, and stalk girth, stalk weight and fiber contents (Akhtar et al., 2000).The major commercial sugar lands in Pakistan are all located in its Punjab and Sindh regions: an ecological zone that is often subject to fluctuating periods of drought due to insufficient and unpredictable distribution of rainfall.This poses a serious problem for sugar cane, which has a 10 to 12-month crop cycle.When drought occurs during the formative growth phase (0-120 days after plantation) the crop experiences slow growth, which then results in low cane yield (Barnes, 1974).In order to recover sugar yields, it is necessary to develop cultivars that remain dynamic under low soil moisture availability.Screening for drought tolerance can be achieved by simulating moisture stress at the critical growth stage of the crop, either under controlled atmosphere conditions or under field conditions.
Little success has achieved in screening plants for drought tolerance by selection for morphological features due to genotype× environment interactions (Bendelow et al., 1955).Blum (1988) theorized that the simplest approach for drought tolerance would be to utilize the natural stress of the field environment to screen genotypes.Despite the relatively drought tolerant nature of the sugar-cane plant, varieties differ obviously in their tolerance to drought (Moore, 1987).Sugarcane varieties susceptible to drought will be wilt, with indication of reduced cane production early during drought, while tolerant varieties remain turgid, and maintain near-optimum growth for longer production (Moore, 1987).Tolerant varieties have the capability to reduce transpiration losses, while at the same time upholding adequate absorption of water from the soil.
The mechanism of the toxic consequence of organophosphate compounds involves the inhibition of acetyl-cholinesterase and other non-specific esterases through phosphorylation at the esterase centre's of the enzyme.This mechanism is the same for all insecticides of the group, irrespective of differences in their chemical structure (Lotti, 2001).The inhibition of the activity of cholinesterase enzymes reasons an increase in the level of endogenous acetylcholine in the organism and results in its binding to muscarinic and nicotinic receptors in both of the peripheral and central nervous systems.This rise in the CNS (Explain 1 th ) disturbs the balance between neurotransmitters and causes the onset of acute intoxication symptoms (Lotti, 2001).The symptoms of acute intoxication with organophosphates have well described, while the effects of chronic exposure to these compounds are not completely clear.Many authors postulate that they may have an effect on redox procedures in a number of organs, thus leading to disturbances in these procedures and causing enhancement of lipid peroxidation, both in acute and chronic intoxication by these compounds (Abdollahi, 2004;Sharma, 2005).As increased generation of reactive oxygen species and lipid peroxidation induced by these species underlies many diseases.It is extremely important to regulate the effect of organophosphate insecticides on lipid peroxidation processes (Mates, 1999;Yagi, 1987).
The objective of this study was to eliminate drought tolerant genotypes of sugarcane through heat stress.Such screening will aid in the cataloging of genotypes in the selection of commercial sugarcane cultivars.In the documentation of parents to improved genetically drought tolerance sugarcane cultivars developed for Pakistan.

Plant Materials
Thirteen Sugarcane genotypes have been collected from different sugarcane institutions of Pakistan and have been cultivated in the glasshouse of Faculty of Agriculture, Rawalakot.

Heat Stress in Laboratory:
Three leaves were selected randomly from thirteen sugar-cane genotypes for laboratory screening.The leaves were cut early in the morning from mature cane stalks between the 5th and 6th leaf from top visible dewlap.Leaf area calculated based on 3/4 of the product of length and breadth (Barnes, 1974).The leaves weighed and kept in an oven at 35°C for three hours.Percentage (%) moisture loss from the excised leaves was determined using the formula of Blum (1988): ((Fresh weight -Dry weight) / Dry weight) × 100.

Phenol Contents:
The total phenol content was measured by the method of Singleton et al. (1999).The total phenol content was determined by adding 0.5 ml of the aqueous extract to 2.5 ml, 10% Folin-Ciocalteau's reagent (v/v) and 2.0 ml of 7.5% sodium carbonate.The reaction mixture was incubated at 45°C for 40 min, and the absorbance measured at 765 nm in the spectrophotometer.Gallic acid used as a standard phenol.The mean of three readings used and the total phenol content expressed as milligram of gallic acid equivalents/ g extract.