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Open Access
Biochemical and molecular studies on post-harvest physiological deterioration of cassava (Manihot esculenta crantz)
(Department of plant physiology, College of agriculture,Vellayani, 2015) Saravanan, R; KAU; Roy Stephen
The project entitled “Biochemical and molecular studies on post-harvest physiological deterioration of cassava (Manihot esculenta Crantz)” was conducted at the Dept of Plant Physiology, College of Agriculture, Vellayani and at ICAR-CTCRI, Thiruvananthapuram during 2013 to 2015. The main objective of the work was to analyse the physiological biochemical and molecular mechanisms associated with post harvest physiological deterioration (PPD) and develop methods to delay the PPD in cassava. The initial screening of the cassava genotypes for their performance of PPD was done with 61 different genotypes including some released varieties. Significant differences were observed for PPD development and shelf-life of harvested roots. Cassava genotypes such as IMS2-8, 9S-172, 11S-53, IRS 2-10 and 9S-286 started showing the visible discolouration in the parenchyma tissue earlier (less than 3 days of storage). Genotypes such as 9S-7, 9S-98, 11S-31, 11S-86, 11S-14, CE63-3, CI43-2, CR43-2, CR54-A5, CR59-8R, Sree Athulya and Kalpaka showed low PPD scores and better shelf-life. Tissue imprinting for peroxidase enzyme showed that there was a remarkable increase in peroxidase activity in the root tissues with increasing PPD symptoms. There was no correlation between the root morphological traits and PPD severity. Carotene and starch content of root did not influence the PPD in the genotypes studied. Chemotypic profile of roots with PPD symptoms was used to classify the genotypes based on PPD. To develop an objective screening tool, Near Infrared Spectroscopy (NIRS) was utilized to analyse root samples for PPD. Principal component analysis (PCA) and chemometric tools clearly grouped the different PPD category in root tissues. Various stains such as saffranin, aniline blue, erythrocin, fast green and phloroglucinol stained the tissue specifically at vascular tissues and other cell components and were not suitable for detecting PPD. Storage techniques such as storing the harvested roots in de-aerated bags, wax coating and burying the roots under the soil were employed with selected cassava varieties like Sree Athulya, Sree Jaya, Vellayani Hrashwa, Kalpaka and Sree Padmanabha to delay PPD. Wax coating was suitable to reduce PPD for few weeks. Effect of different storage temperature on PPD was studied for five cassava varieties. Root respiratory flux was higher in roots stored at ambient conditions compared to high (40o C) or low temperature (4o C) storage. There were significant positive correlation between root respiratory flux at 3 and 9 days of storage to the CAT and POX activities studied in different cassava varieties. Roots were treated with various food preservatives at two different concentrations (0.5 and 1%). There was a weak, but significant reduction in symptom development in butylated hydroxy touline – (BHT, at 0.5 and 1% level) treated roots compared to other treatments. Nearly three folds increase in total phenol content was noticed in BHT and butylated hydroxy anisole (BHA) treated roots. The plant hormones related to wound response such as Salicylic acid and jasmonic acid were used to study the PPD response in roots under storage. The roots did not show marked influence to hormone application. Significant genetic variation was observed for PPD. The low PPD type genotypes such as 9S-7, 9S-98, 11S-31, 11S-86, 11S-14, CE63-3, CI43-2, CR43-2, CR54-A5, Sree Athulya and Kalpaka can be utilized for breeding programmes. High temperature storage of cassava at 40 oC resulted in reduced respiratory rate and increased antioxidant scavenging enzyme activity and also reduced the PPD. Differentiation of cassava roots at the metabolites level corresponding to visual symptoms and chemotypic profile of PPD and NIR spectroscopy offer a rapid screening tools. Among the different storage treatments, wax coating with antiseptic pre-treatment is most suitable and economical for increasing shelf-life of roots. Food preservatives like BHT and BHA have a significant, albeit marginal influence on PPD symptom development in cassava.
Open Access
Physiological approaches for manipulating male sterility in thermosensitive genic male sterile system for hybrid rice seed production
(Department of Plant Physiology, College of Agriculture,Vellayani, 2019) Gayathri Rajasekharan, KAU; Roy Stephen
Thermosensitive Genic Male Sterile (TGMS) plants which are male sterile above critical sterility temperature (CST) and male fertile below CST can be utilized as an efficient system for developing hybrid rice suitable to the state of Kerala. In this context, a study on ‘Physiological approaches for manipulating male sterility in thermosensitive genic male sterile system for hybrid rice seed production’ was conducted at the Department of Plant Physiology, College of Agriculture, Vellayani during 2015 to 2018 to evaluate the environmental conditions required for complete male sterility of TGMS plants and to manipulate the male sterility by using plant growth regulators and also to understand the molecular mechanism associated with TGMS system. The male sterile trait of stable TGMS line EC720903 from IRRI, Philippines, was transferred to red rice variety of Kerala, Jyothi. The CST of EC720903 was determined as 26.9⁰C and the critical thermosensitive phase was identified as 15-22 days before flowering. The seeds of BC1F2 plants were used for the experiment. The seeds were sown in pots in the open field and Rain Out Shelter (ROS) at monthly interval from June, 2017 to May, 2018 to evaluate the environmental conditions required for complete male sterility. The newly developed TGMS line exhibited complete pollen and spikelet sterility throughout the study period at both the experimental conditions since the average temperature prevailed during the critical thermosensitive period of TGMS red rice line was above the CST of 26.9⁰C. Anatomical studies of anther also showed that the TGMS lines were pollen free which is a preferred character of an ideal TGMS plant. The TGMS plants inside ROS had higher plant height, number of productive tillers and they flowered early compared to open field. Hence this low cost structure which is covered with a UV stabilized transparent sheet can be used for commercial hybrid seed production using TGMS lines throughout the year. 259 To maintain the sterility expression during the critical stages, the potential of plant growth regulators (PGR), were evaluated at RARS, Ambalavayal. Three PGRs, ethrel (400 mg L-1, 800 mg L-1 and 1200 mg L-1), salicylic acid (400 mg L- 1, 600 mg L-1 and 800 mg L-1) and maleic hydrazide (MH: 600 mg L-1, 800 mg L-1 and 1000 mg L-1) were applied as foliar spray at two stages viz., panicle initiation and two weeks after panicle initiation. The PGRs were capable of enhancing pollen sterility in all the treated TGMS plants. MH induced significantly higher percentage of pollen sterility (83.49%) compared control plants (19.92%). The external application of salicylic acid at three different concentrations induced 62- 82% of pollen sterility in rice and ethrel caused more than 75% pollen sterility. It was also observed that the PGRs applied were not inhibitory to the primary metabolism of TGMS lines. Hence spraying of MH (1000 mg L-1) two times at the time of panicle initiation and fifteen days after panicle initiation can be recommended to maintain male sterility. The TGMS line was hybridized with two rice varieties as pollen parents, Aiswarya (red) and Swetha (white) using proximal hybridization to evaluate the potential of using TGMS line as a female parent in Kerala condition. Seed setting was significantly high for the cross involving Swetha (40.07%). The F1 progenies obtained from two crosses along with parents and female parent EC720903 were sown in pots during April-May to September-October. The F1 progenies attained early flowering and physiological maturity. The protein content at panicle initiation stage was also high. Plant height, photosynthetic rate, transpiration rate and stomatal conductance at panicle initiation stage of F1 progenies of Swetha were significantly higher. The F1 plants did not show sterility and the seed setting percentage was in the acceptable range. Complete spikelet sterility was exhibited by the TGMS line since temperature was higher than CST. Molecular characterization of TGMS plants maintained at sterility inducing and fertility inducing conditions was done using samples collected from leaf and young panicle at ten days after panicle initiation. Protein profiling with SDS-Poly Acrylamide Gel Electrophoresis showed an enhanced expression of proteins at 25- 260 35 kDa, 35-48 kDa and 245 kDa under sterility inducing condition. In the panicle, increased expression at 17 kDa and presence of protein band at 245 kDa was observed at fertility and it was between 48-63 kDa under sterility inducing condition. Microarray gene expression analysis of TGMS leaf and panicle revealed that the genes encoding proteins similar to programmed cell death protein 2, Nuclear ribonuclease Z, hormone degradation and conjugation were up regulated and genes encoding proteins similar to male sterility protein 2, pollenless3, pollen-specific protein SF21 and no pollen were down regulated in the TGMS leaf under sterility inducing condition. The genes involved in hormone degradation and conjugation and programmed cell death protein 2 and genes encoding ABC transporter proteins required for exine and sporopollenin synthesis, lipid transfer protein and wax synthase, sucrose transporter, male sterility protein 2, β-1,3- glucanase and tapetal programmed cell death were down-regulated in the panicle. Pathways involved in the IAA, GA, brassinosteroid and jasmonic acid biosynthesis and carbohydrate synthesis and transport was down-regulated during sterility inducing condition in the TGMS line leaf and panicle. The TGMS line can be recommended to the state of Kerala as a female parent for the development of suitable red rice hybrids because of its stable sterility. If the temperature of a particular location goes above 27⁰C in a particular season that period can be used for hybrid seed production. Application of MH (1000 mg L-1) at the time of panicle initiation and fifteen days after panicle initiation is more effective in sterility manipulation when there is drop in temperature. Occurrence of pollen free anthers in the TGMS line during sterility inducing condition is mainly due to the down-regulation of genes encoding ABC transporter proteins, lipid transfer protein and strictosidine synthase, glucosemethanol- choline oxidoreductase, male sterility protein 2, wax synthase and β- 1,3-glucanase.
Open Access
Studies on the physiological and biochemical changes in relation to reproductive efficiency in Chickpea (Cicer arietinum L.)
(Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi, 1981) Seshadrinath, S; KAU; Sabra Abbas
The average yield of chickpea in India is as low as 700 kg/ha. One of the major causes for this low yield can be attributed to the low reproductive efficiency. The cause for low reproductive efficiency is mainly due to heavy shedding of reproductive structures. The present investigations were, therefore, initiated with the following objectives: 1) To study the genetic variability and diversity with reference to the flowering behaviour and shedding of flowers during the reproductive stage in order to evaluate the peak periods of flowering, shedding percentage and reproductive efficiency.
Open Access
Evaluation of CO 2 enrichment effects on resource utilization in cowpea and amaranathus
(Department of Plant Physiology, College of Agriculture, Vellayani, 2019) Srikanth, G A; KAU; Manju, R V
The current experiment entitled "Evaluation of CO2 enrichment effects on resource utilization in cowpea (Vigna unguiculata L.) and amaranthus (Amaranthus tricolor L.)" was undertaken with the objective to study the impact of CO2 enrichment on cowpea and amaranthus imder varying moisture, temperature and nutrient regimes. Four sets of pot culture experiments were conducted during 2015 to 2018 with two varieties of cowpea, Lola and Vellayani Jyothika and Arun variety of amaranthus. The technology used for CO2 enrichment was Open Top Chamber (OTC) system established under Department of Plant Physiology, College of Agriculture, Vellayani. Carbon dioxide was released from CO2 cylinders to one of the two OTC's bringing the CO2 level to 600 ppm and the second OTC worked as control at ambient CO2 for chamber effect. The experiments were laid out in CRD factorial. First experiment was conducted to study the varietal variation in cowpea in response to CO2 enrichment through OTC. Experimental plants were maintained in OTC from sowing to harvest. Observations were taken at biweekly intervals till 3 months. In this study highest values were recorded in variety Lola for number of leaves per plant (79.5), specific leaf area (208.78 cm^g"'), root weight (14.91 g), shoot weight (65.05 g), root shoot ratio (0.229), total dry matter production (79.98g), single pod weight (12.88 g), number of seeds per pod (12.34) and early flowering was also observed under elevated CO2 condition. In Vellayani Jyothika, highest values were recorded for number of pods per plant (11.75), total yield (102.59 g/plant) and total chlorophyll content (0.897 mg g"^), stomatal frequency (2203.84 cm'\ starch (14.26 mg g"') and reducing sugar (15.97 mg g"'), and fibre content (1.34 mg g"') under elevated CO2 condition. Parameters like total soluble protein recorded lower values (8.75) under CO2 enrichment. Quality parameters were modified with a reduction in total protein content (15.76, 14.75 mg g"') and increase in fibre content (1.34, 1.23 mg g '). Among the two varieties of cowpea, Vellayani Jyothika was found to be the best responding variety to elevated CO2 in terms of yield parameters and so was selected for further experiments. In the second experiment evaluation of plant response to elevated CO2 under different soil moisture regimes were evaluated. Two weeks old potted plants were shifted to OTCs. All the three sets of plants were maintained at field capacity (FC) initially. Soil moisture levels were brought down to 80% and 70%, in the second and third sets 30 days after planting and were maintained for a period of 30 days at these soil moisture regimes in OTCs. The result indicated an improvement in growth performances of cowpea and amaranthus under mild and severe moisture stress conditions (80% and 70% FC) in terms of increased number of leaves (46.82, 43.22), specific leaf area (360.43, 261.58 cm^ g-') root weight (9.02, 8.51 g), shoot weight (30.56, 22.16 g), root shoot ratio (0.327, 0.216) and dry matter production (38.98, 29.67g) respectively. The same trend was found in the case of amaranthus for number of leaves per plant (41.00, 37.66), specific leaf area (171.25, 157.59 cm^ g'), plant height (79.66, 72.32 cm), root weight (0.840, 0.416 g), shoot weight (4.740, 3.031 g), root shoot ratio (0.197, 0.130) and dry matter production (4.82, 4.71 g). In the case of cowpea CO2 enrichment induced early flowering in all the three soil moisture conditions. Significant increase in yield was also obtained under stress condition (78.51 and 77.08 g/plant) due to increase in number of pods per pod (8.67, 7.32), single pod weight (11.63, 10.36 g), number of seeds per pod (9.33, 8.75) both under 80% and 70% PCs. In cowpea, total chlorophyll content (1.671, 1.238 mg g"'), RWC (85.24, 77.97 %), stomatal frequency (2144.00, 1964.53 no cm"^), starch (6.12, 5.69 mg g"'), reducing sugar (12.48, 12.09 mg/g), phenol content (0.943, 0.801 mg g') free amino acid content (5.960, 4.823 mg/g), SOD activity (3.466, 4.230 g-Wnute-'), ascorbic acid content (6.87, 5.84 mg/lOOg). Reduction of transpiration rate (0.547, 0.335 mmol water m-^ s"') total soluble protein (6.02, 5.13 mg g'^), membrane integrity (% leakage) (37.80, 34.61%) under CO2 enrichment after stress. The same trend was found in the case of amaranthus total chlorophyll content (1.245, 1.206 mg g"'), RWC (84.98, 79.37%), stomatal frequency (691.16, 573.78 no cm'^), reducing sugar (17.61, 13.56 mg g"'), starch (2.66, 2.53 mg g"'), phenol content (6.20, 3.53 mg g"') free amino acid content (1.071, 1.036 mg g'), SOD activity (1.842, 1.526 g-'minute-'), ascorbic acid content (36.93, 28.40 mg/lOOg), reduction of transpiration rate (2.093, 1.410 mmol water m- s ) total soluble protein (15.42, 15.06 mg g '), membrane integrity (3.480, 3.017%) under elevated CO2. Evaluation of plant responses to elevated CO2 under different soil nutrient regimes was earned out in the third experiment. Potted plants of cowpea and amaranthus (Variety Arun) were used for conducting the experiment. Plants were maintained at FC at four nutrient levels throughout the crop period. The best performance given by plants receiving nutrients as per POP recommendation along with 25% extra nitrogen In cowpea, the values were recorded as follows, number of leaves (76.00), specific leaf area (468.95 cm^ g"^), dry root weight (42.0%), dry shoot weight (0.5%), root shoot ratio (4.9%). total dry matter production (117.58 g), number of pods per plant (16.66), single pod weight (15.83 g), number of seeds per pod (13.33) and total yield (169.53 g/plant). In the case of physiological and biological parameters also this level of nutrient application recorded maximum values for total chlorophyll content (1.528 mg stomatal frequency (2782.01 no cm"^), total soluble protein (20.25 mg g'), starch (13.88 mg g"'), reducing sugar (14.65 mg/g), total protein (15.25 mg g ) and fibre content (1.18 mg g"') and The highest number of root nodules per plant (45.26) and highest nutrient use efficiencies for N, P and K (1.013, 2.675, 0.293 g) were recorded under this treatment. In amaranthus, higher values were recorded in for number of leaves per plant (52.11), specific leaf area (316.20 cm^ g"'), dry root weight (2.13 g), dry shoot weight (4.34 g), root shoot ratio (0.64 g) and total dry matter production (10.47 g). Total chlorophyll content (1.542 mg g"'), stomatal frequency (705.64 no cm"^), total soluble protein (20.25 mg g"'), starch (3.29 mg g"'), reducing sugar (23.14 mg g"'). Calcium content (23.69 mg g'), Fe content (6.71 mg g"') and ascorbic acid content (43.51 mg/lOOg), under elevated CO2 condition. Though a C4 plant like amaranthus also responded to CO2 enrichment, extend of increase in growth and dry matter production was less compared to cowpea, which can be due to the mnate CO2 enrichment mechanism present in C4 systems. The fourth experiment was conducted to study the temperature and humidity interaction with CO2 enrichment. Potted plants of cowpea and amaranthus (variety Arun) were used for conducting the experiment. Plants were maintained at EC throughout the crop period as per POP recommendations of KAU. One set of plants were exposed to mist and the second set was maintained without exposure to mist. In cowpea, highest values were recorded by plants exposed to mist for number of leaves per plant (76.14), specific leaf area (471.07 cm^ g"'), dry root weight (21.74 g), dry shoot weight (72.46 g), root shoot ratio (0.302), dry matter production (104.2 g), single pod weight (15.93 g), number of pods per plant (18.75), number of seeds per pod (14.00), total yield (175.36 g/plant), RWC (96.48%), total chlorophyll content (1.651 mg g'^), stomatal frequency (2724.83 no cm"^), starch (13.29 mg g'^), reducing sugar (15.71 mg g"'), phenol (1.128 mg g"'). Free amino acid (6.398 mg g"'), SOD activity (3.56 g-Wnute-') and ascorbic acid content (9.36 mg/lOOg), Early flowering was induced in this set of plants. Parameters like transpiration rate (1.394 mmol water m-^ s"') total soluble protein (8.82 mg g"'), membrane integrity (43.92% leakage) recorded lower values under CO2 enriched treatments. In amaranthus, higher values were recorded under mist condition for number of leaves (48.51), specific leaf area (327.68 cm^ g"') dry root weight (2.160 g), dry shoot weight (6.74 g), root shoot ratio (0.517), dry matter production (8.90 g). RWC (95.38 %), Total chlorophyll content (1.382 mg g"'), stomatal frequency (694.02 no cm"^), starch (4.19 mg g'), reducing sugar (23.02 mg g"'), phenol (7.92 mg g"'), Free amino acid (1.536 mg g"'), SOD activity (2.44 g-'minute-') and ascorbic acid content (42.75mg/100g). Parameters like transpiration rate (12.36mmol water m-^ s ), total soluble protein (19.05 mg g') (25.40 %) and membrane integrity (% leakage) (6.47 % leakage) recorded lower values. Significant improvement in plant performance and increase in yield are seen under CO2 enrichment with mist exposure in the cases of cowpea and amaranthus The present study shows the existence of varietal variation in the crop responses under CO2 enrichment gives option for selection of varieties with better yield and quality under the changing climatic condition. Elevated CO2 concentration is found to be improve stress tolerance through better photosynthetic rate and activation of defence mechanism. Improved production technologies can be developed especially with mist facility with minimizing irrigation requirement. This can also be utilized for enhancement of antioxidant production which are economically valuable secondary metabolites. Exploitation of soils lacking sufficient nutrient and water can be done through the introduction of low cost CO2 enrichment methods. Since soil moisture and temperature are the primary determinants of nutrient availability and acquisition plant nutrition can strongly be influenced by global climate change. This necessitates site specific CO2 enrichment studies. The present study showed the existence of varietal variation in the crop responses under CO2 enrichment which gives option for selection of varieties with better yield and quality under the changing climatic scenario. Elevated CO2 concentration is found to improve the performance of plants grown under low soil moisture levels by improving the performance of photosynthetic machinery and by activating defence mechanisms. Based on this, improved high-tech agriculture production technologies with mist facility can be developed with minimum irrigation requirement which will ensure maximum water use efficiency. This also gives a possibility of utilizing the interactive effects of different cultivars, CO2 enrichment, and other abiotic factors for enhancing the production of antioxidants, many of which are economically valuable secondary metabolites. The present programme also opens up possibilities of quality improvement of agricultural products based on the interaction of elevated CO2 with factors like cultivars, growth stages, light, nutrient and abiotic stress factors. Intensification of cultivation and quality improvement are equally important to address the new challenges of global health because many of the economically important crops, when grown under field conditions at the elevated atmospheric C02were reported to have deleterious impacts on quality. The present study indicated the improved performance of cowpea under CO2 enrichment with additional nitrogen input. This points towards the changing nutrient requirement of crop plants under the current scenario of increasing CO2 concentration and suggests for bringing out modifications in the nutrient recommendations with additional nutrients, especially nitrogen. Exploitation of soils lacking sufficient nutrient and water also can be done through the introduction of low cost CO2 enrichment methods. Since soil moisture and temperature are the primary determinants of nutrient availability and acquisition, plant nutrition can strongly be influenced by global climate change. So this study also points to the requirement of site specific CO2 enrichment studies.
Open Access
Nutrio-physiological and molecular analyses and carbon dioxide enrichment studies of coconut palms (Cocos nucifera L.) with foliar yellowing
(Department of Plant Physiology, College of Agriculture, Vellayani, 2017) Deepa, S; KAU; Manju, R V
A study entitled “Nutrio-physiological and molecular analyses and carbon dioxide enrichment studies of coconut palms (Cocos nucifera L.) with foliar yellowing” was carried out with the objective to analyze the nutrio - physiological changes occurring in the palms affected with foliar yellowing and to assess the impact of enhanced carbon dioxide on phytoplasmal response. In this study, palms showing mid whorl yellowing (MWY), root (wilt) affected palms (RW) and healthy palms were selected from two different locations viz the Instructional Farm, College of Agriculture, Vellayani (location 1) and Venganoor region (location 2) in the Thiruvananthapuram district. Coconut seedlings showing yellowing were selected from the Instructional Farm, College of Agriculture, Vellayani to study the effect of enhanced carbon dioxide on phytoplasmal response. Catharanthus and brinjal plants with ‘little leaf symptom’ maintained at the Department of Plant Pathology were utilised for grafting into healthy catharanthus and brinjal plants and these were also taken for studying the phytoplasmal response under elevated carbon dioxide condition. Physiological and biochemical analyses revealed significant variations in all the parameters studied. Palms with mid whorl yellowing (MWY) showed a significant reduction in the chlorophyll a, b and total chlorophyll contents, relative water content, membrane integrity and phenol content compared to the healthy palms. An increase in the protein (88%), carbohydrate (25.14%), reducing sugar (28%) and starch content (28.33%) was noted in palms with MWY. In the case of antioxidant enzymes there was build up of polyphenol oxidase (145.38%) and peroxidase activities in palms with MWY compared to the healthy palms. A similar trend was observed in almost all cases of RW affected palms. Nutrient analyses revealed significant alterations in the nutrient content of the selected palms. Significant reduction in the nitrogen (47.74%), magnesium (22.72%) and zinc content (24.19%) was observed in palms with yellowing compared to the healthy palms. Accumulation of elements like potassium (37.96%) calcium (40.79%), iron (54%) and copper (35.75%) was observed in palms with mid whorl yellowing compared to the healthy palms. These variations in the nutrient levels can have an influence on symptom development in coconut palms. The results on the soil nutrient analyses did not show any particular role in development of yellowing symptoms. Phytoplasma cells were detected in the phloem tissues of both mid whorl yellowing and root wilt affected palms under Scanning Electron Microscopy (SEM) study. Morphological and anatomical analyses of fresh roots indicated browning and necrosis of roots and vascular disintegration in MWY and RW affected palms. The presence of phytoplasma in the selected samples were checked by nested PCR analysis using phytoplasma specific universal primers-P1/P7- R16F2n/ R16R2, Phytoplasma 16S rDNA specific semi nested primers 1F7/7R3 - 1F7/7R2 and by real time PCR technique using real time primers QPF2/R2. Semi- nested PCR yielded an amplicon of 493 bp in all the MWY and RW palms in both the locations. Real time PCR yielded an amplicon of 140 bp in the RW palms, 3 MWY palms in location 1. Nested PCR with phytoplasma specific universal primer pairs P1/P7-R16F2n/ R16R2 yielded an amplicon of 1.2 kb in MWY palm and RW palm. Sequence analysis of the mid whorl yellowing phytoplasma revealed 89% similarity to the root wilt phytoplasma. In experiment II, nested PCR with universal primer pairs showed no amplification in the coconut seedlings and hence only catharanthus and brinjal grafts with phytoplasmal infection were kept in Open Top Chamber (OTC) with 500 ppm Carbon dioxide concentration for a period of one month. After the exposure period, the plants kept in OTC showed an increased rate of growth and development with phytoplasmal symptoms. Physiological and biochemical analyses showed a significant increase in the reducing sugar content (63%), protein content (147%) and PAL activity (32%) and significant reduction in the phenol content (37%), and polyphenol oxidase activity (62%) in the infected plants kept in OTC compared to the infected plants kept in open condition. Molecular analyses of the infected samples using real time PCR kept in OTC and open condition with the DNA samples isolated at the 15th and 30th day of exposure was done. No significant variation was observed in the phytoplasmal load after elevated carbon dioxide exposure. The role of phytoplasma in causing MWY was established by molecular and anatomical studies. Sequencing of the PCR product revealed that MWY phytoplasma can be a variant of root (wilt) phytoplasma. The present study indicated that the overall health status of the palms with MWY was highly deteriorated. Significant variation in the nutrient profile was noted which emphasise the need for proper nutrient management. Semi- nested PCR was found to be more accurate and specific in phytoplasmal detection which can be utilised for phytoplasmal indexing and mother palm and disease free seedling selection. Elevated carbon dioxide showed an improved growth and development and activated defense response to phytoplasma in the infected plants but the CO2 induced modifications in phytoplasmal load was not detected. The results indicated a better tolerance strategy to environmental conditions. phytoplasma under changing
Open Access
Biochemical and molecular studies on post-harvest physiological deterioration of cassava (Manihot esculenta crantz)
(College of Agriculture, Vellayani, 2015) Saravanan, R; KAU; Roy, Stephen
The project entitled “Biochemical and molecular studies on post-harvest physiological deterioration of cassava (Manihot esculenta Crantz)” was conducted at the Dept of Plant Physiology, College of Agriculture, Vellayani and at ICAR-CTCRI, Thiruvananthapuram during 2013 to 2015. The main objective of the work was to analyse the physiological biochemical and molecular mechanisms associated with post harvest physiological deterioration (PPD) and develop methods to delay the PPD in cassava. The initial screening of the cassava genotypes for their performance of PPD was done with 61 different genotypes including some released varieties. Significant differences were observed for PPD development and shelf-life of harvested roots. Cassava genotypes such as IMS2-8, 9S-172, 11S-53, IRS 2-10 and 9S-286 started showing the visible discolouration in the parenchyma tissue earlier (less than 3 days of storage). Genotypes such as 9S-7, 9S-98, 11S-31, 11S-86, 11S-14, CE63-3, CI43-2, CR43-2, CR54-A5, CR59-8R, Sree Athulya and Kalpaka showed low PPD scores and better shelf-life. Tissue imprinting for peroxidase enzyme showed that there was a remarkable increase in peroxidase activity in the root tissues with increasing PPD symptoms. There was no correlation between the root morphological traits and PPD severity. Carotene and starch content of root did not influence the PPD in the genotypes studied. Chemotypic profile of roots with PPD symptoms was used to classify the genotypes based on PPD. To develop an objective screening tool, Near Infrared Spectroscopy (NIRS) was utilized to analyse root samples for PPD. Principal component analysis (PCA) and chemometric tools clearly grouped the different PPD category in root tissues. Various stains such as saffranin, aniline blue, erythrocin, fast green and phloroglucinol stained the tissue specifically at vascular tissues and other cell components and were not suitable for detecting PPD. Storage techniques such as storing the harvested roots in de-aerated bags, wax coating and burying the roots under the soil were employed with selected cassava varieties like Sree Athulya, Sree Jaya, Vellayani Hrashwa, Kalpaka and Sree Padmanabha to delay PPD. Wax coating was suitable to reduce PPD for few weeks. Effect of different storage temperature on PPD was studied for five cassava varieties. Root respiratory flux was higher in roots stored at ambient conditions compared to high (40o C) or low temperature (4o C) storage. There were significant positive correlation between root respiratory flux at 3 and 9 days of storage to the CAT and POX activities studied in different cassava varieties. Roots were treated with various food preservatives at two different concentrations (0.5 and 1%). There was a weak, but significant reduction in symptom development in butylated hydroxy touline – (BHT, at 0.5 and 1% level) treated roots compared to other treatments. Nearly three folds increase in total phenol content was noticed in BHT and butylated hydroxy anisole (BHA) treated roots. The plant hormones related to wound response such as Salicylic acid and jasmonic acid were used to study the PPD response in roots under storage. The roots did not show marked influence to hormone application. Significant genetic variation was observed for PPD. The low PPD type genotypes such as 9S-7, 9S-98, 11S-31, 11S-86, 11S-14, CE63-3, CI43-2, CR43-2, CR54-A5, Sree Athulya and Kalpaka can be utilized for breeding programmes. High temperature storage of cassava at 40 oC resulted in reduced respiratory rate and increased antioxidant scavenging enzyme activity and also reduced the PPD. Differentiation of cassava roots at the metabolites level corresponding to visual symptoms and chemotypic profile of PPD and NIR spectroscopy offer a rapid screening tools. Among the different storage treatments, wax coating with antiseptic pre-treatment is most suitable and economical for increasing shelf-life of roots. Food preservatives like BHT and BHA have a significant, albeit marginal influence on PPD symptom development in cassava.
Open Access
Marker assisted transfer of thermosensitive genic male sterility to high yielding red kernelled varieties of rice (Oryza sativa L.)
(College of Agriculture, Vellayani, 2015) Niya, Celine V J; KAU; Roy, Stephan
The project entitled “Marker assisted transfer of thermosensitive genic male sterility to high yielding red kernelled varieties of rice (Oryza sativa L.)” was conducted in the Department of Plant Physiology, College of Agriculture, Vellayani during 2011 to 2014. The main objectives were to develop molecular markers associated with TGMS gene and to transfer TGMS character to red rice background. For the present investigation, two TGMS lines were imported from International Rice Research Institute (IRRI) namely, TGMS1 and TGMS2which are from two different TGMS sources ID24 and IR32364 respectively. The TGMS lines along with two popular red rice varieties of Kerala, Uma and Jyothi were sown on monthly intervals for their initial phenological study. The critical sterility temperature and period of TGMS lines were characterised in the field using tracking method. The activity of antioxidant enzymes plays an important level in causing sterility in rice pollen grains, though the reason behind male sterility is unknown. Morphological, agronomic and floral traits were studied during the specific crop growth stage and were recorded as per the standard evaluation systems given by IRRI. Phenological, floral and morpho-agronomic characterisation of TGMS lines revealed that the line TGMS1 performed better with short stature, early maturity, more productive tillers, wider glume opening, higher panicle and stigma exertion, more filled grains and longer panicles. Stages of panicle development were determined by various methods viz., physical method, morphological index method and tracking method. The results have shown that TGMS1 is a better candidate for Kerala condition with critical sterility period of 15-22 days before heading and the sterility inducing average temperature of 27.25 o C. For hybridisation, TGMS1 plants were grown under sterility inducing condition and they were crossed with a popular red rice variety, Uma to produce F1. The F1 s were then selfed to get F2 population. A gene specific primer was designed for the red pericarp colour (Rc) which can be used as a background selection marker. Fifty F2 plants were used for marker analysis. Microsatellite analysis was done to find out the SSR markers polymorphic to the tms gene. DNA was isolated from the 50 F2 plants and performed PCR using 45 SSR markers. Capillary electrophoresis was done for the allele sizing of PCR products. Among 45 SSR markers used, three primers RM 3351, RM23 and RM31 could differentiate Uma, Jyothi andTGMS1, TGMS2. A set of twenty one primers were able to distinguish TGMS1 and Uma. The F2 plants sterile as per the TGMS markers were found to be sterile under sterility inducing condition. Under low temperature their fertility was transformed indicating the presence of tms gene. Linkage analysis using MAPMAKER version 3.0, seven linkage groups and a few unlinked primers were found. A segregation ratio of 2.57:1 was obtained between fertile and sterile lines. This ratio explains the monogenic nature of tms gene.Among 20 polymorphic markers, six (RM23, RM31, RM3351, RM 212, RM258 and RM244) were found significant. These on further analysis using χ 2-test revealed the possible association between the sterility phenotype and marker. From the present study, the sterility tms gene got successfully transferred into 14 sterile F2 plants and RM31, RM23, RM3351, RM212, RM244 and RM258 can be used for the evaluation of TGMS1 X Uma hybrids and their F2s.

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