III. MATERIAL AND METHODS

Transcription

1 aterial and ethods

2 III. ATERIAL AND ETHODS Both field and laboratory experiments were conducted to study the influence of seasons on flowering behaviour, seed setting, seed yield and quality in parental lines of sunflower hybrids; occurrence of seed dormancy, natural dissipation and safe removal of seed dormancy in sunflower and efficacy of seed treatment chemicals and packing materials on storability of sunflower hybrids at the Department of Seed Science and Technology, Gandhi Krishi Vignana Kendra, University of Agricultural Sciences, Bangalore, during The details of the materials used and techniques adopted during the course of investigation are described in this chapter. 3.1 General description Experimental site The field experiments were conducted at the All India Coordinated Research Project on Sunflower, Zonal Agricultural Research Station, Gandhi Krishi Vignana Kendra, University of Agricultural Sciences, Bangalore, during Kharif and Rabi, The experimental site is located at latitude of North, a longitude of East and at an altitude of 930 above sea level. urther, the seed quality valuation of the produce obtained from various experiments including storage and dormancy studies were carried out in the Department of Seed Science and Technology, University of Agricultural Sciences, Bangalore Soil The field experiments were conducted on red sandy clay loam soils in Plot No. E- 18 (Kharif-2010), E-16 (Rabi-2010), E-19 (Kharif-2011) and E-16 (Rabi-2011) at ZARS, GKVK, Bangalore. The composite soil samples from the experimental site was drawn before the commencement of the experiment and analyzed for the different chemical properties. The results along with the methods followed for soil analyses are presented in Appendix-I.

3 3.1.3 Climatic conditions The meteorological data during the crop growth and storage periods recorded at the meteorological observatory at GKVK, Bangalore are depicted in Appendix II Isolation To avoid cross pollination and to produce genetically pure seeds, minimum isolation distance of 600 m was provided between the experimental plot and other cultivars of sunflower crop. 3.2 EXPERIENTAL DETAILS Experiment-1: Influence of seasons on flowering behaviour, seed yield and quality of parental lines of sunflower hybrids Source of seeds reshly harvested seeds of parental lines of newly released sunflower hybrids (Table 3.1 and Plates 3.1 to 3.2) were obtained from the AICRP on Sunflower, University of Agricultural Sciences, GKVK, Bangalore. The seed material were dried to safe level of moisture (<9%), graded to uniform size by using suitable sieves and then used for the study to avoid size variations, if any Treatment details Table 3.1. List of sunflower hybrids and their parental lines used for the study Hybrids Seed parent (A line) aintainer line (B line) Pollen parent (R line) KBSH-1 CS 234A CS 234B RHA 6D-1 KBSH-41 CS 234A CS 234B RHA 95C-1 KBSH-42 CS 851A CS 851B RHA 95C-1 KBSH-44 CS 17A CS 17B RHA 95C-1 KBSH-53 CS 335A CS 335B RHA 95C-1 Seasons (S): S 1 : Kharif; S 2 : Rabi

4 CS LINES CS-335 A RESTORER LINES Plate 3.1. Sunflower seeds of CS lines and restorer lines used for the study

5 Plate 3.2. Sunflower seeds of different 1 hybrids used for the study

6 3.2.4 Plan and layout of the experiment The experiment was laid out in a Randomized Complete Block Design (RCBD) with three replications during kharif and rabi in 2010 and The plan and layout of experiment adopted is illustrated in ig 3.1 and general view of the experimental site is depicted in Plate Plot size Gross plot size: Net plot size : 6.0 m x 3.5 m 5.4 m x 3.0 m 3.3 Cultural and after care operations Land preparation The experimental site was ploughed twice by tractor drawn B plough followed by passing the cultivator and harrow. The land was smoothened with wooden plank to bring in to fine tilth. The experimental site was laid out into sub-plots as per the plan and layout given in ig 3.1, a week before the sowing ertilizer application The experimental plots were applied with nitrogen, phosphorus and 60:90:60 kg. ha -1 in the form of Urea, Single Super Phosphate (SSP) and uriate of Potash (OP), respectively. The furrows were opened at 60 cm apart using marker and fertilizer placed in the furrows and mixed thoroughly with the soil. 50 per cent of nitrogen and entire quantities of phosphorous and potassium were applied as basal dose and the remaining 50 per cent of nitrogen was top dressed at 30 days after sowing at the time of earthing up Sowing of seeds and spacing In Sunflower, for effective synchronization of flowering, staggered sowing was followed. or KBSH-1 hybrid, male parent (RHA 6D-1) was sown eight days earlier than female parent (CS-234A), for KBSH-41 and KBSH-42, male parent (RHA 95C-1) was

7 KBSH-1 KBSH-42 KBSH KBSH-41 KBSH-44 KBSH KBSH-42 KBSH-53 KBSH KBSH-44 KBSH-41 KBSH KBSH-53 KBSH-1 KBSH-42 R-I R-II R-III ig 3.1 PLAN AND LAYOUT O IELD EXPERIENT Studies on influence of seasons on flowering behaviour, seed yield and quality of parental lines of sunflower hybrids Hybrids: KBSH-1(CS 234Ax6D-1); KBSH-41(CS 234Ax95C-1); KBSH-42 (CS851Ax95C-1); KBSH-44 (CS 17Ax95C-1); KBSH-53 (CS335Ax95C-1) 5.4 m 3.0m E W N S

8 Plate 3.3a. General view of the experimental plot during kharif, 2010 Plate 3.3b. General view of the experimental plot during rabi, 2010

9 sown eight days earlier than female parent (CS-234A and CS-851A, respectively) and for KBSH-44, male parent (RHA 95C-1) was sown four days earlier than female parent (CS-17A). While for KBSH-53 hybrid, female parent (CS-335A) was sown four days earlier than male parent (RHA 95C-1). In Kharif, sowing was done on July 30 th and 21 st during 2010 and 2011, respectively, while in rabi on 26 th November and 26 th December in 2010 and 2011, respectively with a seed rate of 1.25 kg per ha and 3.75 kg per ha seeds of male and female respectively. Sowing of female and male parents was done in marked rows with spacing of 60 cm between rows and 30 cm between plants in a row in 3:1 ratio. Before sowing, the seeds were treated with 5 g per kg of seeds to prevent occurrence of bud necrosis. Two seeds per hill were dibbled at about 4 cm deep into the soil surface and sufficient numbers of male parental lines were sown in separate block in the ratio 75:25 to facilitate adequate supply for pollen for pollination. After sowing, the experimental plots were irrigated so as to ensure maximum germination and to maintain optimum plant population Thinning Thinning was done on 15 th day after sowing for maintaining the optimum plant population with good seedlings.only one vigorous seedling was retained per hill Weeding Two hand weedings were done at 15 th and 30 th day after sowing (DAS) manually. Blade harrow was passed to remove the weeds in between the rows during the early period of crop growth in order to keep seed plot free of weeds Earthing up Sunflower plants tend to lodge because of large and heavy heads. Therefore, earthing up was done at 30 th day after sowing along with second hand weeding and top dressed with urea.

10 3.3.7 Plant protection Two sprays of Propiconozol at 25 th and 40 th day after 1.0ml per liter of water were given to control the leaf spot disease and Endosulfan was sprayed at 55 and ml per liter of water to control the leaf eating caterpillars Irrigation Irrigation was provided to the crop depending upon the soil moisture content and prevailing weather conditions during the period of experiment. ive irrigations were provided during the entire crop growth period to avoid moisture stress especially at the critical stages. The crop grown in rabi was irrigated at an interval of seven to eight days by adopting furrow method Roguing The pollen shedder and off-types which did not confirm to the specific characters of the parental lines were removed before flowering and pollination in order to maintain genetic purity. Besides, diseased and undesirable plants were also removed to prevent contamination Direction of rows The rows were facing North- South direction for efficient roguing from westward since the sunflower heads always face east (Heliotropism) Bagging of seed parent and pollen parental lines Capitulum of both seed parent and pollen parents were covered with cloth bags before the anthesis and bags were retained up to completion of flowering Artificial pollination During the flowering period, pollen from the male parental lines were collected by opening the cloth bags and replaced in the same position after collecting the pollen. Collected pollen were immediately pollinated on stigmatic surface of the female flowers by opening the cloth bags and covered again with the same bag. The pollination was

11 performed every day between 8 A.. to 11 A.. for a period of 14 days after initiation of flowering Harvesting and threshing The seed crop was harvested when it attained physiological maturity i.e., when the back side of the capitulum turned lemon yellow colour. At this stage, seed moisture is expected around 24 per cent. The heads of male parent and boarder rows were harvested first and threshed separately. The heads of the female rows were then harvested from the net plot area in all the plots. The harvested heads were dried under sun for three days on threshing floor. Threshing was done by beating the heads with the sticks manually. The seed was cleaned and net plot seed yield was recorded after thorough drying and expressed in kilograms. 3.4 Collection of experimental data Sampling procedures or recording various biometric observations, five randomly selected plants were tagged at vegetative growth stage in each treatment and all observations were recorded on these selected plants Observations recorded on growth parameters Plant stand at maturity The number of survived normal and healthy plants before harvest were counted and recorded Plant height (cm) at harvest The plant height was measured from the base of the plant at ground level to the growing tip (base of the head) at maturity but before harvest. The average plant height was worked out and expressed in centimeters.

12 Days taken for opening of first ray floret The number of days taken to initiate first flower in each genotype from the date of sowing was recorded and expressed in days from the date of sowing (DAS) in each plot Days to 50 per cent flowering The number of days taken to 50 per cent flowering was recorded based on the fully flowered plants from the date of sowing in each plot and expressed in days Days to completion of flowering The number of days taken to complete flowering from the date of sowing was recorded and expressed in days from each plot Observations on seed yield parameters The observations on seed yield parameters were recorded from the net plot area and on the randomly selected plants Capitulum diameter (cm) The distance between two diagonally opposite edges of the capitulum was recorded and the mean is expressed in centimeters Capitulum weight (g) After recording the capitulum diameter, the individual wet capitulum weight was recorded and the mean weight is expressed in grams Number of filled seeds per capitulum Well filled seeds of the selected heads were counted and mean number of filled seeds per head was recorded Number of unfilled seeds per capitulum After separating the well filled seeds, the remaining unfilled seeds were counted and the mean number of unfilled seeds per head was recorded.

13 Total number of seeds per head The seeds obtained separately from each head of the five sampled plants were cleaned and separated into filled and unfilled seeds. The sum of these two types of seeds was taken as total number of seeds and the mean was worked out and expressed in number Per cent seed filling per capitulum formula. Percentage of seed filling per capitulum was calculated by using the following Number of filled achenes per capitulum Seed filling (%) = x 100 Total number of achenes per capitulum Bulk seed yield per plant (g) After threshing of randomly selected dried heads, the seeds were separated, dried thoroughly, cleaned and weighed. The mean raw seed weight was recorded and expressed in grams per plant Bulk seed yield (q ha -1 ) The dried heads from each net plot were threshed, cleaned and the weight of the seed was recorded. Seed yield was computed and expressed in quintals per hectare Seed recovery (%) The seed recovery percentage was calculated by using the formula. Weight of good seed retained on recommended screen Seed recovery (%) = x 100 Total weight of bulk seed Processed seed yield (q ha -1 ) Based on the data of seed yield per ha, after cleaning and grading the processed seed yield was computed and expressed in quintals per hectare using the formula. Processed seed yield (q ha -1 ) = Seed yield (q ha -1 ) x Seed recovery (%).

14 3.4.4 Observations on seed quality parameters Seed moisture content (%) The moisture content of the seed was determined by the low constant temperature oven method as per ISTA (2010). ive grams from each treatment in two replicates of seeds were taken in aluminium boxes and dried in oven at 103±1 C for 17±1 hours. After oven drying, the metal boxes along with the seeds were cooled in desiccators over silica gel for 30 min and the weight was recorded. The moisture content was expressed on wet weight basis using the following formula. Where, 2-3 oisture Content (%) = x weight of the empty aluminum box + lid (g) 2 - weight of the empty aluminum box + lid + seed material before drying (g) 3 - weight of the empty aluminum box + lid + seed material after drying (g) seed weight (g) rom the seeds taken out randomly, one hundred seeds were counted manually from each treatment in eight replications and weighed. The mean weight of the sample was recorded as hundred seed weight and expressed in gram Seed density (g/cc) Seed density was worked out by water displacement method by using toluene. Known amount of seeds were filled with known quantity of toluene in a measuring cylinder. The raise in volume was recorded and the seed density was determined by using the formula given below and expressed in g/cc. Weight of seeds (g) Seed density (g/cc) = Volume of toluene displaced

15 Germination (%) One hundred seeds of four replicates were drawn at random from each treatment and the germination test was conducted using between paper (BP) method as per ISTA (2010). The rolled towels were incubated in a germination chamber maintained at 25±1 0 C and 90 per cent relative humidity. The germinated seedlings were evaluated on fourth and tenth day as first and final counts, respectively and percentage germination was expressed based on normal seedlings Seed oil content (%) and oil yield (kg ha -1 ) Estimation of seed oil content (%) was done with the help of NR at AICRP on Sunflower, Gandhi Krishi Vignana Kendra Campus, University of Agricultural Sciences, Bangalore. Oil yield per hectare was worked out by multiplying oil content and graded seed yield per hectare and expressed in kilogram per hectare Seed protein estimation (1951). The total seed protein was estimated as per the method prescribed by Lowry et al. a) Preparation of acetone powder The seed samples was homogenized with chilled acetone in a pestle and mortar, then the slurry was filtered immediately under suction using Whatman No.1 filter paper and washed with excess chilled acetone to remove the pigments. Then acetone powder was air-dried overnight and stored in sealed containers at 4 0 C until analysis. b) Preparation of reagents 1. Solution A: 20g of Anhydrous sodium carbonate + 4 g of Sodium hydroxide were dissolved in 1000ml. 2. Sodium potassium tartarate solution (1.35%): 1.35 g of Sodium potassium tartarate was dissolved in water and diluted to 100ml. 3. Copper sulphate solution (5.5%): 5.5g of Copper sulphate was dissolved in water and diluted to 100ml.

16 4. Solution B: 1.0ml of 1.35% Sodium potassium tartarate ml of 5.5% Copper solutions were mixed together. 5. Solution C: 50ml of solution A was mixed with 1ml of solution B just before use. 6. olin Ciocalteau Reagent (CR): Commercial grade reagent was diluted 1:1(V/V) before use. 7. Standard Bovine Serum Albumin (BSA) solution: 2mg of BSA was dissolved in water and diluted to 10 ml to get a working standard solution containing 200 mg/ml. c) Sample extraction One hundred mg of oven-dried sample was extracted in 10 ml of 0.1 Sodium phosphate buffer, ph 7.0 for one hour on a magnetic stirrer at room temperature. The extract was centrifuged at 10,000 rpm for 15 min. The supernatant was used for the estimation of total seed protein content. d) Estimation of protein The required quantity (0.1ml) of supernatant was diluted to 1 ml with distilled water and the 5 ml of solution C was added and mixed. After 10 minutes, 0.5 ml of CR was added and mixed immediately. After incubation for 30 minutes, the absorbance of the solution was recorded at 660 nm against a reagent blank. A standard graph was constructed with BSA as a standard with the concentration of 20 to 120 mg. The standard curve was used to estimate the protein content of the sample and expressed in percentage Electrical conductivity (EC) of seed leachate (ms/ppt) Twenty five seeds of two replicates were taken randomly from each treatment in a beaker. Then the seeds were soaked in 50ml of double distilled water for 24 h at 25±1 0 C. The steeped water from soaked seeds was collected and the electrical conductivity (EC) of seed the leachate was measured in a digital conductivity meter (odel: Systronic conductivity meter 306). After subtracting the EC of the distilled water from the value obtained from the seed leachate, the actual EC due to electrolytes was estimated and expressed in ms/ppt.

17 Total dehydrogenase activity (TDH) Ten sunflower seeds from each treatment were preconditioned by soaking in water for 24 hours. Then, seed coat was removed and they were soaked in 2ml of 0.5 per cent tetrazolium solution for four hours at 25 ± 1 C in dark. The stained seeds were washed thoroughly with distilled water. The red colour formazan developed was eluted from the stained embryos by soaking with 5ml of 2-methoxy ethanol in screw caped vials until complete elution. The extract was decanted and the colour intensity was measured with the help of UV-Vis Spectrophotometer (odel SL 171) at 480nm with suitable blank. The TDH was expressed in terms of Absorbance (Perl et al., 1983). 3.5 Experiment-II Studies on seed maturation, occurrence of seed dormancy, dissipation of seed dormancy and its safe removal in sunflower hybrids and its parental lines over seasons reshly harvested seeds of sunflower hybrids and their parental lines obtained from Experiment-I was used to study the occurrence of dormancy, natural dissipation and its safe removal Seed maturation, occurrence and natural dissipation of dormancy In general sunflower capitulum takes about 8-12 days for complete flowering and days to reach physiological maturity from the day of opening of flowers which depends on season. Seeds were harvested at different seed developmental stages viz., 10 DAA, 20 DAA, 30 DAA, 35 DAA and 40 DAA. The fresh seeds were dried to safe moisture level (<9%) and evaluated for various quality attributes Observations recorded Capitulum weight (g) After recording the capitulum diameter, the individual capitulum weight was recorded and the mean weight is expressed in grams.

18 Seed moisture content (%) The seed moisture content was estimated as described under section Seed weight (g) per capitulum The seeds harvested at different stages were dried to safe moisture level (<9.0%) and seeds were weighed and the mean weight was expressed in grams Seed dormancy Seed dormancy period was recorded from 0 to 70 days after harvest with an interval of 10 days. Hundred seeds from each hybrid were subjected to germination test in three replications. The number of fresh un-germinated seeds and the normal seedlings were counted on 10 th day (final count) and expressed in percentage Germination (%) The seed germination test was conducted as described under section ean seedling length (cm) Ten normal seedlings were taken at random in each treatment and replication at the time of final count of germination test. The shoot and root lengths were measured from collar region to point of attachment of cotyledons and from the collar region to the tip of primary root, respectively. Sum total of the shoot and root lengths constitute total length of seedling. The mean seedling length in each treatment and replication was calculated and expressed in centimeters ean seedling dry weight (mg) The seedlings used for measuring shoot and root lengths were also used for estimating dry weight after removing the cotyledons. These seedlings were dried in hot air oven at 80±1 C for 24 hours. After drying, the mean seedling dry weight was recorded and expressed in milligrams.

19 Seedling vigour index [SVI] The seedling vigour indices were calculated as per formula given by Abdul Baki and Anderson (1973). SVI-I = Germination (%) x ean seedling length (cm). and SVI-II = Germination (%) x ean seedling dry weight (mg) Electrical conductivity (EC) of seed leachate(ms/ppt) section Electrical conductivity (EC) of seed leachate was recorded as described under Total dehydrogenase activity Total dehydrogenase activity was recorded as described under section Safe removal of seed dormancy reshly harvested and dried seeds of sunflower hybrids and their parental lines collected from Experiment-I were subjected to various dormancy breaking treatments viz., plant hormones (GA 3, Ethrel), physical (water soaking, heat), varmi-wash etc. The treated seeds were tested for germination and other quality attributes Treatment details The seeds were soaked in plant hormones at predetermined concentration for 18 hours and dried under shade. Then they were tested for germination and other parameters Physical treatment The seeds were subjected to drying in hot air oven as per the treatment details. The treated seeds were tested for germination by between paper method as per ISTA (2010).

20 Treatments T 1: Control T 2 : Water soaking T 3 : Pre drying at 45 0 C for 8 hours T 4 : Pre chilling at 4 0 C for 72 hours T 5 : GA 3 (100 ppm) T 6 : Ethrel (25 ppm) T 7: KNO 3 (0.2%) T 8 : Thiourea (0.1%) T 9 : Vermiwash (1:1) Observations recorded Germination (%) The seed germination test was conducted as described under section ean seedling length (cm) ean seedling length was measured as described under section ean seedling dry weight (mg) ean seedling dry weight was estimated as described under section Seedling vigour index Seedling vigour index was computed as described under section Experiment-III Studies on the efficacy of seed treatment chemicals and packing materials on storability of sunflower hybrids General description The laboratory experiments were conducted at the Department of Seed Science and Technology, University of Agricultural Sciences, GKVK, Bangalore during

21 3.6.2 Source of Seed reshly harvested seeds of sunflower hybrids obtained from the National Seed Project (Crops), University of Agricultural Sciences, GKVK, Bangalore were used for the study. The seeds were dried to safe level of moisture (<9%), graded manually to uniform size and then used for the storage study (Plate 3.4) Preparation of seeds for storage After recording the initial observations, seeds were treated with 2.5g/kg and Imidacloprid 5g/kg and then packed in three packing material viz., cloth bag, super grain bag and polypouches as per the treatment details (Plate 3.5 and 3.6). These packed seeds were stored under ambient conditions at the Department of Seed Science and Technology, University of Agricultural Sciences, GKVK, Bangalore for a period of twelve months (from August, 2010 to July, 2011) under ambient conditions EXPERIENTAL DETAILS Treatments The details of the treatments are given below: Crop: Sunflower Hybrids (H): Three H 1 - KBSH-41 H 2 - KBSH-44 H 3 - KBSH-53 Seed treatments (T): our T 1 - Control (untreated) T g/kg of seed T 3 - Imidacloprid, powder form 5g/kg of seed T 4 - Imidacloprid, liquid form 5ml/kg of seed

22 KBSH-41 KBSH-44 KBSH-53 Plate 3.4. Seeds of sunflower hybrids used for the storage study

23 Packing materials (P): Three P 1 - Cloth bag P 2 - Super grain bag [polypropylene < 300 gauge)] P 3 -Polypouches (500 gauge) Treatment combinations: 3 x 4 x 3 = 36 Number of replications: Three Experimental Design: actorial CRD Seed treatment methodology Soon after recording the initial observations on various seed quality attributes, the seeds were subjected to manual treatment with different chemicals viz., Imidaclopridpowder 5g/kg and Imidacloprid-liquid 5 ml/ kg of seed. urther, the seeds of all genotypes were treated with powder formulation of Thiram 75% 2.5g per kg of seeds (Plate 3.5 and 3.6). Treated seeds were shade dried and then packed in three packing materials viz., cloth bag, super grain bag and polypouches. The packed seeds were stored for a period of twelve months under ambient conditions of Bangalore where the temperature and the relative humidity was ranging from to 29.18ºC and to per cent, respectively (Appendix-II). Seed samples were drawn at bimonthly initially for a period of six months and at monthly intervals subsequently and evaluated for various seed quality attributes Observations recorded The observations like seed moisture (%), 100 seed weight (g), germination (%), shoot length (cm), root length (cm), mean seedling length (cm), mean seedling dry weight (mg), seedling vigour index based on mean seedling length (SVI-I), seedling vigour index based on mean seedling dry weight (SVI-II), electrical conductivity of seed leachate (ms/ppt), total dehydrogenase activity (A 480 ), seed infection (%) and seed oil content (%) were recorded. The procedure adopted for determining these quality attributes are detailed here under.

24 Plate 3.5. Packing materials and chemicals used for the storage study of sunflower

25 Plate 3.6. Seeds of sunflower hybrids treated with chemicals and stored in different packing materials under ambient condition

26 Seed moisture content (%) The seed moisture content was estimated as described under section Hundred seed weight (g) Hundred seed weight was computed as described under section Germination (%) The seed germination test was conducted as described under section ean seedling length (cm) ean seedling length was computed as described under section ean seedling dry weight (mg) ean seedling dry weight was computed as described under section Seedling vigour index Seedling vigour index was estimated as described under section Electrical conductivity (EC) of seed leachate (ms/ppt) section Electrical conductivity (EC) of seed leachate was recorded as described under Total dehydrogenase activity Total dehydrogenase activity was recorded as described under section Seed health test Detection and identification of seed mycoflora was done by blotter paper method (TP) as per ISTA (2010). Twenty five seeds of two replicates were placed equidistantly in sterile glass petridishes of 12.5 cm diameter containing three moist blotter papers (Whatman No.1). Then the petridishes were incubated at 20±1?C for ten days with 12 hours light and 12 hours dark cycles. After incubation, seeds were examined under stereo

27 binocular microscope for the presence of seed mycoflora and the different fungi found on the seeds were recorded as number of seeds infected by storage fungi and expressed in percentage Seed oil content (%) The seed oil content was conducted as described under section Statistical analysis The data obtained from various experiments were subjected to statistical analysis. The analysis of variance and interpretation of data was done as per procedure given by Gomez and Gomez (1984). The level of significance used in and students T tests was P=0.05. Critical difference values were calculated whenever test was significant. The germination percentages were converted into Arc sine transformation and then subjected to the statistical analysis.