PHYSIOLOGICAL AND BIOCHEMICAL CHANGES DURING SEED DEVELOPMENT AND MATURATION IN MADHUCA LATIFOLIA ROXB
Seed development comprises a series of events involving cell division/histo differentiation, reserves deposition and desiccation. Development of Madhuca (Madhuca latifolia Roxb. seed has been divided into eight consecutive stages; 10, 18, 26, 34, 42, 50, 58, 66 days after fertilization (DAF) Seed colour was finally found to turn dark brown from initial creamish white. Length, circumference, fresh and dry mass increased gradually throughout. In contrast, water content (WC) and electrical conductance were declined (1.4and 94folds, respectively) suggesting deposition of reserves and improvement in membrane integrity in parallel to development. During seed development, sugar was declined (3 folds) while starch and protein contents were increased (4and 13 folds, respectively) suggesting their supportive role in germination and early seedling growth. Activities of antioxidants exhibited rising trend (1.6 21 folds) between initial and last stages of development advocating their defensive function. Data suggested that ideal time for Madhuca drupes/seeds harvesting was 66 DAF. Introduction Seed development in the life cycle of any higher plant is a crucial process providing the link between two different sporophytic generations (Baud et al. 2002) Generally, seed development involves an orchestrate programme of pattern, which includes formation, reserves deposition and desiccation. Formation of a seed is initiated after double fertilization that takes place in an embryo sac, and then repeated cell division occurs in order to establish the seed structures (Baud et al. 2002) Then, seed filling phase starts during which the water content (WC) declines and storage reserves accumulate, resulting into cell expansion. Carbohydrates, lipids and storage proteins are three chief storage reserves generally accumulated in most of the seeds. Seed filling is highly complex process, as many genes and a number of enzymes are involved to regulate storage of each component (Weber et al. 2005) For the process of seed filling, the monomers of various macromolecules viz. amino acids, fatty acids and monosaccharides are translocated through sieve elements of phloem to synthesize their long chain polymers (Weber et al. 2005) Next, the seed enters into maturation drying phase where changes in the seed size and huge fall in WC occurs (Berjak and Pammenter 2008) Maturation drying phase is absolutely absent in recalcitrants therefore, these seeds possess high amount of water (30 70% at the time of shedding from mother plant (Berjak and Pammenter 2008) Process of seed development is genetically programmed, and developing seed converts the precursors of carbon and nitrogen, into stable reserves required later for various processes (Weber et al. 2005) During development, as a by product of metabolic reactions reactive oxygen species (ROS) are generated, which are shown to play dual functions either cytotoxic (when exists in high amount) or playing a role in development, dormancy breakage and defence against biotic and abiotic stresses (Berjak and Pammenter 2008) These ROS are popularly known to react with all *Author for correspondence: <skeshavkant@gmail.com> 336 CHANDRA AND KESHAVKANT sorts of macromolecules, pigments and other cellular components, leading their degradation (Parkhey et al. 2014) Plant cells are having tremendous potency to prevent themselves from such oxidative injury by keeping the ROS under control via an efficient and highly redundant network of antioxidants (Chandra et al. 2015) Superoxide dismutase (SOD) catalase (CAT) guaiacol peroxidase (POX) and ascorbate peroxidase (APX) constitute the major enzymatic systems by which cells catabolize free radicals, thus minimizes the severity of oxidative damage (Chandra et al. 2015) SOD plays key role in conversion of superoxide into oxygen and hydrogen peroxide. While CAT, POX and APX catalyzes the conversion of hydrogen peroxide into water and molecular oxygen and therefore prevent further generation of free radicals (Chandra et al. 2015) Very limited reports are available concerning to the function of antioxidants during seed development however; no one has yet attempted to explore them in developing Madhuca seeds. The physiological and biochemical investigations conducted by He and Gao (2008) and Kok et al. (2013) on developing seeds of Chimonanthus praecox and Elaeis guineensis, respectively, provided comprehensive knowledge regarding seed metabolism. Certainly, information regarding developmental biochemical changes is an indispensable base in understanding the regulatory setup coordinating seed development and metabolism. Additionally, in depth knowledge regarding pattern and mechanisms of reserves accumulation in seed is pre requisite in any plant breeding programme (Kok et al. 2013) The metabolic changes taking place during seed development have been studied for several species (Baud et al. 2002, Silveira et al. 2004, Saldivar et al. 2011, Pavithra et al. 2014) however there is an absolute lack of knowledge for Madhuca. Madhuca (Madhuca latifolia Roxb. family Sapotaceae) seeds are quite sensitive towards desiccation and low temperature hence got damaged even at 15degC (DFSC/IPGRI 2000) No information is available regarding biochemical changes taking place during developmental stages of Madhuca seeds. In this context, experiments were designed to profile the morphological (colour, length, circumference) physiological (Fresh mass (FM) Dry mass (DM) WC and electrolyte leakage) and biochemical (contents of sugar, starch and protein, and activities of SOD, POX and APX) changes that occur during the development of Madhuca seeds. Materials and Methods Madhuca (Madhuca latifolia Roxb. drupe bunches were obtained from 10 15 identified trees growing in the school campus of Attari village, located 8 km away from the Pt. Ravishankar Shukla University, Raipur, India. The flowers appear in March, which takes about 32 35 days for complete development. During April, the inflorescences were constantly and keenly monitored based on the appropriate physiological state at anthesis. Flowers having only pistil without stamen and corolla, indicating completion of fertilization, were tagged. The drupe bunches were harvested manually at defined developmental stages i.e. 10, 18, 26, 34, 42, 50, 58, 66 days after fertilization (DAF) Five bunches of drupes from each developmental stage were plucked from different Madhuca trees. Each bunch represents a single replicate for each analysis. Due to the small amount of endosperm available at stages 10 34 DAF, two bunches of Madhuca drupes were collected from each tree and pooled as a single replicate. The drupes were plucked from the bunch. The kernels in the form of liquid/gel like (up to 18 DAF) were collected from the drupes using an aspirator, whereas for the solid kernels, the fruits were immersed into liquid nitrogen (LN2) and then the seed was cracked to free the kernel. Thereafter, samples were grinded with LN2 and were stored at 80degC (U410, Eppendorf, Germany) for further use. Seeds of different developmental stages were scored for their size and colour. Length and circumference data of seeds were determined using scale and vernire callipers, respectively and expressed in terms of cm. PHYSIOLOGICAL AND BIOCHEMICAL CHANGES DURING SEED DEVELOPMENT 337 To monitor change in FM and DM data, five independent sets of 5 seeds each, were weighed using a four digit electronic balance (Sartorius, Sweden) before and after oven drying at 103 oC for 72 hrs (Parkhey et al. 2014) and expressed in g. Seed WC was determined gravimetrically (Parkhey et al. 2014) and expressed as g H2O/g FM. Ten seeds in five replications were soaked in 30 ml MilliQ water (MW) (Millipore, Gradient A 10, USA) and allowed to stand at room temperature (26 28C) for 24 hrs. The electrical conductivity was measured using a digital conductivity meter (CM 183, Elico, India) and expressed as mS/g FM. Amounts of total sugar and starch were determined following the method of Siloto et al. (2006) and Hodge and Hofreiter (1962) respectively and were expressed in mg/g FM. Protein was extracted following the method of Wang et al. (2006) and was assayed by Bradford (1976) Data was expressed as g protein/g FM, and BSA was used as standard. Weighed (0.2 g) amount of LN2 crushed seed powder was homogenized with 2 ml of borate buffer (0.2 M, pH 7.4) consisting 20% (w/v) polyvinyl polypyrolidone. The slury was centrifuged at 14,000 rpm for 15 mins at 4degC. The supernatant thus obtained was used as a source of antioxidant enzymes. Activity of SOD (EC 1.15.1.1) was estimated following Marklund and Marklund (1974) by estimating per cent inhibition of pyrogallol auto oxidation at 490 nm. One unit of SOD was defined as the quantity of enzyme required to inhibit the nitro blue tetrazolium photoreduction by 50% Activity was expressed as SOD units /min /g FM. POX (EC 1.11.1.7) was measured following Chance and Maehly (1955) The tetraguaiacol formed was measured at 470 nm, and its activity was expressed as mM/min/g FM. Activity of APX (EC 1.11.1.1) was assayed following Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm. Its activity was expressed as mM/min/g FM. All the experiments were performed in five individual biological replicates. The data obtained were subjected to one way ANOVA, and the mean differences were compared by the DMRT at p 0.05 level, using the SPSS software (Ver. 16.0) The correlation analyses between studied parameters were also performed. Results and Discussion Madhuca trees are potentially important due to various applications of its different parts. Madhuca trees started flowering during March and from April onwards drupe setting was initiated which ripens up to July. In practice, a seedling of it is propagated from small embryo enclosed within the hardy seed. Theoretically, the strength of any developing plant depends on the