INFLUENCES OF HIGH TEMPERATURE STRESS
ON POLLEN QUALITY OF SOYBEAN [(Glycine max L.) Meer]
RESPONSE TO SEED YIELD AND SEED QUALITY
INTRODUCTION
Global climate change has emerged as an important environment challenge due to potential impact on biological systems of planet Earth (Wather et.al.,2002). Since the beginning of the industrial revolution (about 1750), the concentrations of CO2, methane and nitrous oxide have increased by 31%, 150% and 16%,respectively (Houghton et.al., 2001). The present day CO2 concentration (370 umol mol-1) has not been exceeded during the past 420,000 years and likely not during the past 20 million years (Petii et.al., 1999; Houghton et.al., 2001).
Human activities such as deforestation and burning of fossil fuel are mainly responsible for the recent rapid increases in atmospheric concentrations of greenhouse gases including CO2 (Kaufmann & Stern, 1997; Houghton et.al., 2001; Stott et.al., 2001). At the present rate of emission, CO2 concentration is subject to be in the range of 540-970 umol mol by the end of this century, which will potentially increase global near surface temperature by 1.4-5.8.C (Houghton e.tal.,2001) with some degree of delay, global warming will occur concurrently with increase in CO2. Therefore, it is important to quantify the interactive effects of increasing temperature and CO2 on crop production.
High quality planting seed is a key component of all grain cropping system. High quality seed is need to ensure adequate plant populations, with reasonable seeding rate, in a range of field conditions. Seed quality at planting represents the integrated effects of the environment during seed production and the conditions the seed were exposed to during harvest, conditioning, and storage. (Egli, D.B. et al. , 2005)
Soybean (Glycine max L.) Merr., has become the major source of edible vegetable oils and high protein feed supplements for livestock in the world. About 90% of the world’s soybean production occurs in the tropical and semi-arid tropical region, which are characterized by high temperature and low or erratic rainfall. In the tropic, most of the crops are near their maximum temperature tolerance; therefore crop yield may decrease even with minimal increases in temperature.
Reproduction plays an important role in the survival and succession of seed crop plants. The onset of the reproductive phase, its duration, and the quality and quantity of reproductive products are regulated by abiotics factors. Of the various abiotic factors, atmospheric temperature and CO2 concentration are subject to change in the near future. The climate change factors being tested in this study modify reproductive organs and processes. Elevated [CO2] and high temperature increased flower production in soybean (Nakamoto et.al., 2001; Zheng et.al., 2002).
Soybean seed yield components are also influenced by temperature. Soybean seed yield increased as temperature increased between 18/12 (day/night) and 26/20.C, but yield decrease (when plants were grown at temperature) greater than 26/20.C (Huxley et al., 1976; Sionit et.al., 1987). Raising temperature from29/20 to 34/20.C during seed fill decreased soybean seed yield (Dornobos and Mullen, 1991).
The number of pods per plant generally increased as temperatures increased to near 26/20.C (Sionit et.al., 1987). Plants grown at temperature exceeding 26/20.C had decreased pod numbers (Huxley et.al., 1976; Thomas and Raper, 1977, 1978; Sionit et. al., 1987). During flowering and pod set, temperatures as high as 30/20.C favored greater pod set (Lawn and Hume, 1985), but temperatures above 40.C severely limited pod formation (Mann and Jaworski, 1970).
Seeds per plant increased as temperatures from early vegetative growth to maturity increased from 18/12 to 26/20.C (Sionit et.al.,1987) and 26/19 to 36/29.C (Baker et.al., 1989). Season- long night temperature increases from 10 to 24.C did not influence seed number (Seddigh and Jolliff, 1984b). In contrast to these findings, Huxley et.al. (1976) observed fewer seeds per plant when day temperature was raised from 27 to 33.C and night temperature was increased from 19 to 24.C. Increase in temperature from 29/20 to 34/20.C during seed fill resulted in fewer seeds per plant(Dornbos and Mullen,1991). Seeds per pod was the seed yield component least affected by temperature (Huxley et al., 1976; Sionit et al., 1987; Baker et al., 1989).
Weight per seed in soybean was increases in season-long temperatures from 18/12 to 26/20.C (Sionit et al., 1987), but as temperature increased above 26/20, weight per seed decreased (Hesketh et al., 1973; Huxley et al., 1976; Baker et al., 1989). Temperatures above 30/25.C during flowering and pod development reduced weight per seed, regardless of the temperature during seed fill (Egli and Wardlaw, 1980). Temperatures above 29/20.C during seed fill decreased soybean weight per seed (Dornbos and Mullen, 1991).
Gan et al. (2004) found that the seed yield of canola decrease by 15% when high temperature stress was applied before flowering, whereas the yield reduction was 58% when the stress was delayed to the period of flowering, and further to 77% when the stress was delayed to the pod developmental stage.
Physiologically, the high temperature stress during reproductive development may have affected flower abortion, sequent sink site, and later pod abscission resulting a decreased number of seeds per plant (Duthion and Pigeaire, 1991). Also, high temperature stress during reproductive development may have negatively affected cell expansion, cotyledon cell number and thus seed filling rate, resulting in the lowered weight per seed (Munier- Jolain and Ney1998).
Most studies on the effect of temperature on soybean seed yield have concentrated on increases in day temperature or concomitant increases in day/night temperature. There were no beneficial interaction between [CO2], Temperature and UV-B radiation on reproductive development processes such as pollen production, germination and tube length of soybean. Prasad et al. (2000b) investigated the effect of daytime soil and air temperature of 28 and 38.C, from start of flowering to maturity, and reported 50% reduction in pod yield at high temperatures.
Yield decrease due to high temperature and [CO2] could be due to the effect of on reproduction at both organ and process levels. High temperature inhibit pollen germination and pollen tube growth and genotypes differ in their sensitivity (Huan et.al., 2002; Kakani et.al., 2002). Decreased fruit- set at higher temperature was mainly due to poor pollen viability, reduce pollen production and poor pollen tube growth, all of which lead to poor fertilization of flowers (Prasad et al., 2003). Flower abortion also has been attributable to the decreased seeds per plant and seed yield in other crops such as Brassica napus (Angadi et al., 2000) B rapa ( Morrison and Stewart, 2002) and B. Juncea (Gan el at., 2004)
Pollen development, fertilization, and asynchrony of stamen and gynoceium development are sensitive to temperatures during flowering (Prasad et al.,1999; Croser et al.,2003; Boote et al.,2005). The lost of pollen or stigma viability under high temperatures stress might be the primary reason for the lowered number of seeds produce in the legume (Srinivasan et al., 1998; Davies et al., 1999; Hall, 2004).
Interactive effects of temperature and [CO2] on other legume (Ahmed et.al., 1993; Prasad et. al., 2002, 2003) showed that that the positive interactions observed between [CO2] and temperature on vegetative growth can not be translated to reproductive process. The physiology effects of high- temperature stress on reproductive development under typical field conditions are more pronounced the effects on vegetative development in many crop species (Hall,1992).
Significant negative correlation between pollen production and temperature were found in groundnut (Prasad et. al., 1999).Lower seed yield at high temperature under both ambient and elevated [CO2] conditions was shown to be due to decreased pollen viability in groundnut and bean (Prasad et. al., 2002, 2003). Pollen sterility and pollen production at high temperatures may also be associated with early degeneration of the tapetal layer of pollen (Porch and Jahn, 2001).The exact physiological reasons of pollen viability loss are not clearly known and need further investigation (Sailaja et.al., 2005).
A better understanding of the influence of high temperature during reproductive growth on soybean seed yield and quality is needed. Although pollen may successfully fertilize an ovule, enhanced high temperature may stay reduce seed production by inducing abortion. The clear effect of high temperature on pollen production and pollen grain germination will have major implications on the fertilization process and fruit set in sensitive crop under future climates. This paucity of information along with these variable responses makes it difficult to predict the consequences of enhanced high temperature on pollen production and plant reproductive success. In reality, plants in nature are exposed to multiple environmental conditions concomitantly, and their performance can be assessed only when plants are grown in multiple stress conditions.
HYPOTHESIS
The perturbation of anther and pollen development due to extended treatment at high temperature stress will correlate with seed yield reduction and that one mechanism of reproductive heat tolerance may involve the reduction of the effects of heat on seed quality.
GENERAL OBJECTIVES
To identify specific changes in anther and pollen morphology under high-temperature stress that may account for at least some of the differences observed between seed quality with yield of heat- tolerant and heat-sensitive soybean genotypes under high temperatures in the field.
The specific objectives of this research are as follow:
(1) To determine the interactive effects of high temperature on flower and pollen morphology, pollen production, pollen germination, and pollen tube length of soybean.
(2) To identify whether injury to the pistil or stamen during development is responsible for reduced fruit set under higher temperature stress.
(3) To evaluate the effects of day time high temperature on soybean seed yield components and seed quality.
MATERIALS AND METHODS
The experiments will be conducted in the field for three planting seasons (January 2008 to January 2009) at University Putra Malaysia (UPM) Experimental Field with three genotypes namely Dieng, Willis, and AGR 190 ; in split plot randomized block desing with three replication, genotypes as main plots and high temperature treatments, randomized within the main plots, as subplots.
The plots will be irrigated as required to reduce moisture stress during the crop development stage. Seeds will be sown 4 cm deep (two seeds per hole) with a planting distance of 40 x 20 cm. Row spacing and length will be 0.45 m and 3 m, respectively and seedling rate will be about 30 seeds per meter length of row.
Normal crop production practices will be followed and fertilizer at the rate of 50 kg Urea + 75 kg KCL + 75 kg TSP per hectare will be applied at planting by surface application (Adisarwanto and Wudianto, 1999). Weed control will be accomplished with mechanical means using tractor will be operated motivator for pre-planting, hand weeding and post emergence inter-row cultivation for post-planting.
To be create variation in plant higher temperature, plastic sheet covered will be used. The two level of growth stages treatment during R1 to R2 (the first flowering period) and R1 to R5 (during flowering and pod set) will be imposed of 29 to 30 C higher temperature and the rest for control treatment (Dornbos and Mullen, 1991). The first reproductive stage R1 and R2 are the beginning one flower at any node immediately below the uppermost node with a completely unrolled leaf (Fehr and Caviness, 1977). The first treatment will be arrived after R2 stage the plastic cover sheet will be take out and the plant be received normal temperature the same with control treatment. Then the second treatment will be the R1 to R5. Growth stage R5 is the beans beginning to develop and beyond R5 stage the plastic sheet will be take out through the normal temperature growth (Descriptions of soybean reproductive growth stages by Fehr and Caviness, 1977 are shown in Appendix Table 1.
To determine the interactive effects of high temperature on flower and pollen morphology, pollen production, pollen germination, and pollen tube length
of soybean.
Measurement of Floral Morphology
Soybean, belong to the family Fabaceae and subfamily Papilionoideae, has a flower with a tubular calyx of five unequal sepal lobes and a five-parted corolla consisting of a posterior standard petal, two lateral wing petals, and two anterior keel petals in contact with each other but not fused (Carlson and Larsten,1987).Lengths of flower, standard petal, and staminal column will be measured on 20 fresh flowers randomly picked from five plants of each genotype, 60 Day After Sowing (DAS).Flower length will be measured from the tip of the standard petal to the base of the calyx. The standard petals will be stretched out before measuring the length, and the length will be measured from the point of insertion to the distal end. The stamina column will be separated from the flower and the length will be measured.
Measurement of Pollen Number
Mature anthers will be collected from different flowers from five plants a day before anthesis to determine the number of pollen grains will produce per anther.60 DAS. Pollen number will be counted by placing a single anther in a water drop on a glass slide and will squash with a needle, and the pollen grains will disperse in the drop of water will be counted (Bennett, 1999). The number of pollen grains from five anther per genotype under each treatment will be counted.
Measurement of Pollen Viability
Pollen viability will be determined using acetocarmine strain prepared by boiling a solution of 40% acetic acid saturated with carmine. Viability will be determined by counting red-staining pollen (viable) out of a total of 500 pollen grains using Cambridge Instruments microscope (
Sample preparation
On the day of anthesis, flower will be collected from each treatment genotype combination between 08:00 and 09:00 h. Anthers and pistal will be dissected from the flowers and fixed in 0.1 M sodium cacodylate ( Ph 6.8) containing 2.5% glutaraldehyde and immediately refrigerated at 4 ∙C. Prior to viewing the floral structures, the sample will be washed in 0.1 M sodium cacodylate buffer, dehydrate in a graded ethanol series (10-70%) at room temperature, stained with 2% uranyl acetate at 70% ethanol, dehydrated in a grade ethanol series (70-100%) and critical-point dried using a transition fluid.
Microscopy
For SEM analysis, the critical-point-dried anthers and pistils will be mounted on aluminum stubs, the edges of the stubs will be warped with sliver tape and the stubs will be sputter-coated with gold / palladium using a Bal-Tec SCD 050 sputter coater (Blazers). If the anthers had not dehisced as a result of the heat treatment, the anther will be dissected along the stomium to observe the pollen. A Hitachi S- 4500 SEM microscope (
Pollen viability will be analysed with a split-plot analysis of variance with field and temperature as the main plot and soybean genotype as the subplot. Statistical analysis of percentage pollen viability will performed after arcsin transformation.
Measurement of Pollen Germination and Pollen Tube Length
Pollen germination will be determined by the handing-drop method (Sophie M.et al., 1996) using the following sucrose and salt solution:m1.2 mol/L sucrose solution and a mineral-salt solution consisting of 2.54 m mol/L Ca (NO3)2, 1.62 m mol / L H3BO3, 1.00 m mol/L KNO3, 0.88 m mol/L MgSO4, and 3.5 m mol/L NH3 with the Ph of this solution adjusted with 1 mol/l NaOH to 8.8. Equal parts of the sucrose and the salt solution will be mixed and one drop of surfactant will be added to prevent clumping of grains. Pollen will be scarped from all Petri dishes in random order using a clean teasing needle and will place in 0.01ml of the sucrose-salt solution on a microscope slide.
The slide will be inverted (to prevent anoxia) over a depression grid that will contain three drops of distilled H2O. After 20 min (trial runs indicated that pollen germinated with 15min), the pollen grain will be observed with a light microscope at X200; pollen grains will be considered to have germinated when tube length will be equal to or greater than their diameter. Three such 0.01 m L sub samples will be taken from each Petri dish and two field of view will be examined per sub samples.
Pollen Morphology
For the pollen morphology, fresh flowers will be collected between 19:00 h and 21:00 one day before anthesis and will store in FAA (Formaldehyde: glacial acetic acid : ethyl alcohol). Solution will be fixed in 3% glutaral dehydrate in 0.1 M phosphate buffer, PH 7.2 over night at 4C for SEM. After fixation, specimens will be rinsed in buffer, post fixedin2%Osmium tetroxide in 0.1 M phosphate buffer for 2 h, will rinse in distilled water, dehydrated in an ethanol series, and critical point will dry in a Polaron E 3000 Critical Point Dryer (Quorum Technologies,
To identify whether injury to the pistil or stamen during development is responsible for reduced fruit set under higher temperature stress.
Plant Material
On the day of anthesis of flower population study, one of the two will be chosen plant (high-temperature stress treatment) will be covered with 0.178-mm-thick polyethylene film with a metallic structure and the other (control) will be left uncovered. Such a system has been shown to be a valuable method to increase temperature in the field with out negatively affecting other parameters (Rodrigi& Herrero, 2002). Temperature inside and outside the plastic cage will be monitored every 5 min with a data logger (Testostor 175-3; Testo,
Pollination Procedure
Pollen will be obtained from flowers of soybean genotype will collect just 1d before anthesis (balloon stage); anther will be removed and left to dry on piece of paper for 24-48 h at room temperature. The pollen will be sieved through a 0.26 µm mesh and frozen at 20.C until required. The duration of stigmatic receptivity will be evaluated through the capacity of the stigma to support pollen germination. For this purpose, flowers will be emasculated 1d before anthesis and hand- pollinated on the day of anthesis and thereafter every subsequent day. The pollination will be carried out until style abscission will occur.
Microscopic Observation
In all treatments the pistils will be fixed 24h after pollination in formalin:acetic acid: 70% ethanol (1:1:18; FAA). Pollen performance will be monitored in squah preparations after washing out the fixative with distilled water three times, 1h each, softening the pistil in 5% sodium sulphite in the autoclave for 10min at 1 kg cm-2 and staining with 0.1% aniline blue in 0.1 NK3PO4. Preparations will be examined under an ortholux II microscope (Leitz, Wetzlar, Germany) will be equipped with UV epifluorescence with a hand pass 355-425 exciter filter and LP 460 barrier filter (Hedhly A. ET AL., 2003)
Statistical analyses will be performed using the SAS GLM (V. 15; SAS Institute, Inc.,
Seed-Set
To determine the effects of temperature on seed – set, individual flowers were tagged and will be followed through final harvest at maturity. A total of 40 flowers will be randomly tagged in each treatment on 9 to 16 plants (about four flowers per plant) between 3 and 5 days after first flowering. Seed – set expressed as percentage will be defined as the proportion of 40 tagged flowers that produce seed.
Seed Growth
Once pod will be started, 40 pods at an identical stage of development will be tagged on 10 plants ( four per plant) and their development and growth will be monitored by destructive harvests of four randomly will be selected pods from three to four different plants at 5,10,17,27,41,55 and 70 days after tagging and at maturity. Plants grown at 29 to 30 C during R1 to R2 growth stages and R1 to R5 growth stages on 45 DAS, and those at normal temperature on 43DAS. So that the reproductive structures will be of a same age (7 days) after start of pod.(when 59% of plants will start pod.
Time series data will be obtained from these harvests will be used to determine the individual seed growth rate and effective seed – filling duration was the length of time (days) from start of seed growth (Y=0) to the time when the average maximum seed size will be reached.
To evaluate the effects of day time high temperature on soybean seed yield components and seed quality.
Seed Yield and Yield Components
Each experiment, the plants will be grown in 4 row plots each, 3 m long with 0.45 m spacing between rows. The middle two rows of each plot will be harvested at maturity (R8) when 95% of the pods will be reached their mature pod color (brown); will threshed by hand and seed moisture will be measured for seed moisture determination using oven dry on wet weight basis. 100 seeds of each plot will be kept in the oven dry for 24 hours at 105C. Yield of each plot will be adjusted to 14% seed moisture content.
Yield components such as pods per plant, seeds per pod from the average of five plant samples and 100 seeds weight will be recorded by 100 seeds sample from each plot at harvest maturity. These seeds will be stored in a controlled environment room (10C, 50% RH) until seed quality evaluations will be made.
Seed Quality Evaluation
All seed samples will be harvested at R8 during the three experiments will evaluate for viability (Standard Germination Test), vigor (Spped of Germination Days, evaluation after germination in hot water stress treatment).
Seed Viability
For seed viability, the standard germination test will be carried out will record 3d and 7d after emergence for each experiment and for each harvested plot. Standard germination tests will be based on ISTA recommendations (ISTA, 1996), But onlt one hundred seeds from each plot will be sown in plastic boxes contain will wash, oven dry (sterile) and moisten sand. Each boxes will be covered with another box as lid and will place for 7d in germination room at the temperature of 30±3.C.
Seed Vigor
For the seed vigor evaluations will be counted standard germination, hot water treatment and conductivity test for all the experiments.
Germination Test
The same procedure outline follow for the standard germination test, a first count of normal seedlings which will strong, and at least 3.75 cm long will be made at 3d after sowing as described in the AOSA seed vigor testing rules (2001).
Hot Water Stress Test
The hot water stress procedure will be used a modification of that outlined by Kueneman (1983). Freshly will harvest and un weather seeds will be wrapped with muslin cloth and dipped for 70 seconds in 75.C water and immediately rinse in 25.C water for 1 minute. The hot water treated seed will be sown in oven dried sterilized fine sand in a germination room (grid house) at a depth of 1.5 cm in rows 5 cm apart and approximately 5cm between seeds within rows. Loose sand will be placed over the seeds, and will water tightly every day to keep the sand moist. Emerges seedlings (with the cotyledons completely above the sand surface) will be sown in three replication units of 100 seeds per plot. Evaluation of seedling will be done by the procedures of ISTA (1996).
Conductivity Test
Electrical conductivity of seed leachates will be performed on 25 seeds will obtain from each harvested plot with conductivity meter (Model 4301,
Data Analysis
To test the significance of high temperature, genotype and developmental growth stage and their interactive effects on flower morphology, pollen number per anther, pollen germination percentage, and pollen tube length data will be statistically analysis of variance (ANOVA) by Genstat 6 for windows (Genstat 6 Committee, 1997).
Analysis of variance (ANOVA) will be used to determine if significant differences will be present among means using SAS software version 15. Significant season treatment interactions will be occurred for several variables studied, data will be analyzed and will presents separately for each season. Genotype effect will be tested for significance using replication x genotype interaction mean square as the error term (Ea). Temperature on different development stage x genotype interaction will be tested for significance using error mean square (Eb). Mean separation will be carried out with the least significant difference (LSD). Error mean square (Ea) will be used in calculating the LSD ( = 0.05, 0.01) for comparing genotype differences. Error mean square (Eb) will be used to calculate the LSD ( = 0.05, 0.01) for making comparisons between temperature treatments and between temperature treatments with genotype.
For seed quality evaluations all seed samples will be harvested at harvest maturity (R8) during the experimental seasons will be evaluated for viability ( standard germination test), vigor (speed of germination days evaluation after germination in hot water stress treatment).
Seed germination test will be carried out and will record 3 day and 7 day after emergence for each treatment and each harvested plot. Seed germination test will be based on ISTA recommendations (ISTA, 1996). Normal and abnormal seedling will be classified according to (ISTA, 1996) classification. In addition to standard germination hot water treatment and conductivity test will be conducted for seed vigour evaluations for all the treatments (AOSA Seed Vigour Testing Rules)
EXPECTED OUTCOME
The response of soybean seed yield and seed quality to high temperature stress on reproductive growth stages R1 to R2 and R1 to R5 will have to be elucidated, and may will be related to the number and location of seeds on the plant. A plant with a small reproductive load may be able to maintain seed quality to a greater extent when subsequently high temperature-stressed than plants with a large reproductive load.
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APPENDIX
Table 1 Development Stage Description for Soybean
| Stage NO. | Description |
| R1 | One flower at any node |
| R2 | Flower at node immediately below the uppermost node with a completely unrolled leaf |
| R3 | Pod 0.5 cm (1/4 inch) long at one of the Four uppermost nodes with a completely unrolled leaf |
| R4 | Pod 2 cm (3/4 inch) long at one of the Four uppermost nodes with a completely unrolled leaf |
| R5 | Beans beginning to develop (can be felt when the pod is squeezed) at one of the four uppermost nodes with a completely unrolled leaf |
| R6 | Pod containing full size green beans at one of the four uppermost nodes with a completely unrolled leaf |
| R7 | One pod on the main stem has matured pod color |
| R8 | 95% of pods brown |
Source: Fehr and Caviness (1977)
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