Elucidating the morpho - physiological and molecular changes during seed priming in bitter gourd (Momordica charantia L.)

Abstract

A study to elucidate the morphological, biochemical and molecular changes leading to enhanced germination on priming in bitter gourd variety Preethi was conducted at College of Agriculture, Vellanikkara, Thrissur, during 2020-2023. Seeds of bitter gourd variety Preethi were dried to 6.84 per cent, and the quality parameters of the sourced seeds were assessed. They were divided into three equal sub-lots (5.0 kg each). Two sub-lots were packed in 700-gauge polyethylene bags and sealed airtight and one each stored for a period of six month under i). Ambient environment (Am.S), ii). Refrigerated storage (Rf.S) at ~18OC-20OC. The third sub-lot was packed in moisture-vapour proof aluminium foil bags seeds and stored for a period of six months at Medium term storage (MTS) facility at the ICAR-NBPGR (National Bureau of Plant Genetic Resources), Regional station, Vellanikkara. At the end of the storage period i.e., six months, the quality parameters of the stored seeds under the various storage environments were assessed following which they were further divided into 11 equal parts and subjected to 10 priming treatments (T1 to T10) following standard procedure, while unprimed seeds served as the control (T11). The priming treatments included T1: KNO3 0.3% for 2 h, T2: KNO3 150 ppm for 3 h, T3: KH2P04 10-2 M for 24 h, T4: PEG 6000 -1.5 MPa for 24 h, T5: GA3 100 ppm for 24 h, T6: Solid matrix priming with Perlite for 48 h, T7: Solid matrix priming with Cocopeat for 48 h, T8: Psuedomonas fluorescens 1x106 cfu.ml-1 for 24 h, T9: Hydration hot water at 50°C for 4 h and T10: Hydration cold water soaking for 24 h. Three separate studies as enumerated below were conducted to compare the effect of priming on germination of stored seeds under three different storage conditions viz. Ia) Effect of priming on seeds stored under ambient environment, Ib) Effect of priming on seeds stored in refrigerated storage (~18-20 OC) and Ic) Effect of priming on seeds stored in medium-term storage (~ 4-6 OC). Seed quality parameters viz., number of days to emergence, speed of germination, germination per cent, seedling root length (cm), seedling shoot length (cm), seedling dry weight (g), seed moisture content (%), germination capacity (GC), speed of germination, mean germination time (MGT), time taken for 50% germination (T50), energy of germination (GE), germination index (GI), coefficient velocity of germination (CVG) and seedling vigour indices were assessed after priming treatments in each experiment. In addition to the physiological observations, biochemical parameters viz., total soluble sugar (%), total protein (%), total oil content (%), H2O2 content (μmol g_1), lipid peroxidation (MDAμmol g_1), total dehydrogenase activity (OD), α–amylase activity (μmol g_1), catalase activity (μmol g_1), electrical conductivity of seed leachate (μScm-1), leakage of amino acid (μg leucine eqiv.ml -1) and leaching of sugar (μg glucose eqiv.ml -1) were recorded along with seed structural parameters viz., total seed coat thickness (μm), thickness of testa (μm), thickness of tegmen (μm), embryo length (μm), endosperm length (μm), endosperm breadth (μm), seed length (μm) and relative water absorption (RWA). Comparison of quality parameters before and after storage under the three environments pointed out that in addition to a reduction in germination, root length and shoot length the seedlings, the vigour indices I and II had also decreased in comparison to the estimates recorded prior to storage, while parameters like MGT and T50 of stored seeds were observed to have increased after six months of storage. The per cent reduction of speed of gemination, germination per cent, seedling root length, seedling shoot length, seedling dry weight, GC, GE, GI, CVG and vigour indices was however, the least in seeds stored in Am.S condition. Hence, it became evident that the per cent deterioration in seed stored at low temperatures is low, conversely, it reinforced the fact that storing bitter gourd seeds at low temperatures (MTs and Rf.S) would help maintain seed quality longer. The effect of seed priming treatments on germination (%) after six months of storage under i) S1: Ambient storage (Am.S), ii) S2: Refrigerated storage (Rf.S) and iii) S3: Medium-term storage (MTS) was anlaysed separately, each following a Completely randomized design with three replication and 11 priming treatments, in order to assess the best priming treatment under each storage environments studied. Germination of seeds stored under varying environmental conditions were found to be significantly influenced by the priming treatment they were subjected to. Germination of aged seeds from the three storage environments when subjected to Solid matrix priming with perlite (T6: Perlite for 48 h) cocopeat (T7: Cocopeat for 48 h) and hormonal priming with GA3 (T5: GA3 100 ppm for 24 h) were significantly superior over untreated seeds. Priming with PEG 6000 (T4: PEG 6000 -1.5 MPa for 24 h) was found to adversely affected germination irrespective of the storage environment the seeds were derived from. Although bio-priming seeds from Am.S with P. fluorescens (T8: P. f at 1x106 cfu.ml-1 for 24 h) and hydropriming (T9: Hydration hot water at 50°C for 4 h) was beneficial, it was not so in seeds derived from Rf.S and MTS. Similarly, hydration with hot water @ 50°C for 4 h was not found very beneficial in seeds stored under Rf.S and MTS as compared to Am.S. The impact of storage environment (S), priming treatment (T), and their interaction on physiological, biochemical and seed structural quality indices of seeds stored were analysed following a Completely randomized design with three replications and two factors viz., Factor I: Storage environments (S1, S2 and S3) and Factor II: Priming treatments (T1 to T11). Results indicated that, irrespective of the priming treatments, the quality indices of seed stored in MTS followed by Rf.S were found to be significantly superior to those stored under Am.S. It was evident that storing seeds at low temperatures (~ 18-20 0C in Rf.S, ~ 4-6 0C in MTS) was beneficial in bitter gourd. It resulted in higher germination as well as other physiological, biochemical and structural indices of the seed. Irrespective of the storage conditions, the quality indices of the aged seeds were found to be significantly influenced by the priming treatment they were subjected to. Solid matrix priming (SMP) with Perlite (T6: solid matrix priming with perlite for 48 h) invariably followed by solid matrix priming with cocopeat (T7: solid matrix priming with cocopeat for 48 h) and hormonal priming with GA3 (T5: 100 ppm for 24 h) were found to be significantly superior to other priming treatments as well as untreated seeds. Priming with PEG 6000 (T4: -1.5 MPa for 24 h) was found to be detrimental and registered the least seed indices. The interaction between seed priming treatment and storage conditions was found to significantly influence germination as well as other quality indices of the aged seeds. Results pointed out that germination and most seed indices studied were significantly superior in solid matrix primed seeds involving perlite (T6: solid matrix priming with perlite for 48 h) under both low temperature storage (MTS and Rf.S), as well as ambient storage (Am.S). Solid matrix priming with cocopeat (T7: solid matrix priming with cocopeat for 48 h) and hormonal priming with GA3 (T5: 100 ppm for 24 h), were found to be next best to SMP with perlite under low temperature storage environments viz., MTS and Rf.S. However, unlike in the low temperature storage environments, under ambient storage conditions, bio-priming with P. fluerescens (T8: P. f at 1x106 cfu.ml-1 for 24 h) was found to be next best to SMP with perlite and superior over SMP with cocopeat and hormonal priming with GA3. However, apart from untreated seeds, osmopriming seeds with PEG 6000 (T4: PEG 6000 -1.5 MPa for 24 h), hydration with cold water (T9: Hydration hot water at 50°C for 4 h) as well as with hot water (T10: Hydration cold water soaking for 24 h), were found to be detrimental under low temperature storage (MTS and Rf.S) and ambient storage as well. The results obtained thus point out that loss of germination, vigour and other quality parameters over storage is inevitable. However, the deterioration of seed indices in bitter gourd due to ageing can be slowed down to the maximum extent by storing them under Medium-Term storage (MTS). Storing seeds under refrigerated storage (Rf.S) is highly beneficial compared to storing them under ambient conditions (Am.S). Irrespective of the environment under which the seeds were stored, solid matrix priming of bitter gourd seeds with perlite (48 h) was the best priming treatment as it resulted in significantly superior germination, growth indices of seedlings, biochemical parameters and seed structural changes over untreated seeds. SMP with cocopeat (48 h) and hormonal priming with GA3 (100 ppm for 24 h) were next best to SMP with perlite in bitter gourd. Subjecting seeds of bitter gourd stored either in low temperature environment like medium term storage and refrigerated storage or under ambient environments to solid matrix priming with perlite would result in enhanced germination and seed quality indices. Solid matrix priming with cocopeat or hormonal priming of the aged seeds with GA3 (100 ppm for 24 h) were the next best priming options, if the seeds were stored at low temperature environments. However, in seed stored under ambient environments, bio-priming with P. fluorescens (1x106 cfu.ml-1 for 24 h) would be the best priming option, next to solid matrix priming with perlite.