Doctoral Research Paper of Daw Tin Tin Khaing

Study of in vitro culture of Gymnostachyum species

Tin Tin Khaing, A.L.T Perera[1], V.A Sumanasinghe2, D.S.A Wijesundara3
Postgraduate Institute of Agriculture
University of Peradeniya
Peradeniya, Sri Lanka

ABSTRACT

The genus Gymnostachyum (Acanthaceae) consists of beautiful flowering shrubs and herbs, native to Sri Lanka. Gymnostachyum zeylanicum can be used as an ornamental plant and a ground cover in landscape designing. The present research was conducted to find the optimum in vitro conditions for rapid multiplication of novel Gymnostachyum plants developed through mutation. Modified MS liquid and solid media consisting of MS salts + 0.1g/l myoinositol without hormones and modified with BAP + NAA, supplemented by 20g/l sucrose media at pH -5.7 were used as treatments in the in-vitro cultures.
The MS medium containing 5mg/lit BAP + 1mg/lit NAA gave the best results for callus regeneration as well as plantlet formation. Explants of leaf tissue and immature seed cultures showed high potential for regeneration under in vitro conditions. Callus formation and regeneration of the plantlets began about six weeks after culturing of leaf explants. The plantlets formed by leaf explants produced without roots and the plantlets from immature embryos developed roots.

Key words: Gymnostachyum, MS medium, explants,

If you want to have full paper, please contact to this address; tintinkhaing@gmail.com

Doctorate Research Proposal of Daw Myint Thu Zar, University Putra Malaysia

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 (Cambridge England). In several cases, due to insufficient inflorescences or number of pollen grains, two flowers will be examined instead of three and about 100 pollen grains will be counted instead of 500, respectively. (Porch TG & Jahn M., 2001).

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 (Tokyo, Japan) will be used to view the samples at 3 and 5 kV. All of the SEM microscopy sample images will be prepared using Adobe Photoshop software. (Adobe System, Mountain View, CA,USA).

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 KNO_3, 0.88 m mol/L MgSO_4, 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, Newhaven, UK). Specimens will be mounted on aluminum stubs, Sputter-coated with gold in a polaron E 5100 sputter coater ( Quorum Technologies) and viewed in a LEO Stereoscan 360 SEM ( LEO Electron Microscopy, Thornwood. NY,USA) at an accelerating voltage of 15 KV. Images will be recorded on Polaroid Type 55 Film (Polaroid, Cambridge, Massachusetts, USA).

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, Lenzkirch, Germany) throughout the period of sequential pollination and fixation.

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 NK₃PO_4. 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., Cary, NC, USA). Percentage data will be subjected to arcsine root square transformation and analysis of variance will be performed. The differences between means will be analysed by the Fisher’s Least Significant difference (LSD) test at the 0.05 level of significance.

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. Normal and abnormal seedlings will be counted after 3d and 7d sowing. Normal and abnormal seedlings will be classified according to ISTA classification (ISTA1996).

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, Jenway, UK). Seeds will be soaked in 75ml deionized water for 24hours at room temperature. Results will be expressed per gram of seed to take into account variations in seed size between lots (ISTA,1993).

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)

ထိုင္းႏိုင္ငံတြင္ တကၠသိုလ္တက္ခ်င္လွ်င္

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၁. Thammasat University for Political Science, Economics and Laws, http://www.tu.ac.th/
၂. Kassetsart University for Agricultural and Forestry, http://www.ku.ac.th/
၃. Mahidol University for all Medical fields, Industrial Ecology and Environment, http://www.mahidol.ac.th/
၄. Chularlongkorn University for All Science and Engineering fields, http://www.cu.ac.th/
၅. Ramkhangheng University for Political Science and Education, http://www.ru.ac.th/
၆. Assumption University for Business Administration, http://www.au.ac.th/
၇. National Institute of Development Administration for Development Studies, http://www.nida.ac.th/
၈. Ubon Rajathanee University for Greater Mekhon Subregion Development, http://www.ubu.ac.th/

တို႕ျဖစ္ပါသည္။ တကၠသိုလ္၀င္ခြင့္ အေသးစိတ္မ်ား၊ ၀င္ခြင့္ေလွ်ာက္လႊာမ်ားကို သက္ဆိုင္ရာ၀က္ဘ္ဆိုက္မ်ားတြင္ ၾကည့္ရႈ ရယူႏိုင္ပါသည္။ အျခားတကၠသိုလ္မ်ားမွာ ျမန္မာျပည္တြင္ ေရပန္းစားလွေသာ္လည္း တခ်ိဳ႕ထိုင္းလူမ်ိဳးမ်ားကိုယ္တုိင္ပင္ မၾကားဖူး ၾကသည္ကိုလည္း ေတြ႕ရပါသည္။ Webster University, www.webster.ac.th သည္ အေမရိကန္ႏိုင္ငံသို႕ သြားေရာက္ေနထိုင္ ပညာသင္လိုုသူမ်ား တဆင့္ခုန္ရာတကၠသိုလ္ ျဖစ္ေသာေၾကာင့္ နာမည္ႀကီးၿပီး Asian Institute of Technology, www.ait.ac.th သည္ ပညာသင္ဆုအေပးမ်ားျခင္း၊ International Program ခ်ည့္ သက္သက္သာရွိျခင္းႏွင့္ အာရွႏိုင္ငံမ်ားအခ်င္းခ်င္း နည္းပညာဆိုင္ရာ သုေတသနႏွင့္ ယဥ္ေက်းမႈတို႕ဖလွယ္ရာအျဖစ္ နာမည္ႀကီးေလသည္။သို႕ရာတြင္ ထိုတကၠသိုလ္မ်ားတြင္ အျခားဘာသာရပ္မ်ား အတြက္လည္း International Program မ်ားရွိပါသည္။ ၀မ္းနည္းစရာေကာင္းေသာအခ်က္မွာ ၂၀၀၄ခုႏွစ္တြင္ထုတ္ျပန္ေသာ အစိုးရပိုင္တကၠသိုလ္မ်ားဆိုင္ရာ ဥပေဒအရ ျမန္မာႏိုင္ငံမွဆယ္တန္းေအာင္သူကို ထိုင္းႏိုင္ငံမွအလယ္တန္းေအာင္သူႏွင့္သာ အရည္အခ်င္း တူသည္ဟု သတ္မွတ္ေပးထားၿပီး ရိုးရိုးဘြဲ႕အတြက္ တကၠသိုလ္တက္လိုသူသည္ GCE O' Level ေအာင္ၿပီးသူျဖစ္မွသာ လက္ခံပါသည္။ ထိုင္းသည္ အေျခခံပညာအတြက္ ၁၂တန္းရွိၿပီး အထက္တန္းကို ၂ႏွစ္တက္ရေသာေၾကာင့္ျဖစ္သည္ဟု ဆုိေလသည္။ မဟာဘြဲ႕ တက္လိုသူသည္ အေျခခံပညာ ၁၂တန္းႏွင့္ ရိုးရိုးဘြဲ႕အတြက္ ၄ႏွစ္တက္ေရာက္သင္ၾကား ေအာင္ျမင္ထားသူျဖစ္ရမည္ ဆိုေသာေၾကာင့္ အနည္းဆံုးစာသင္ႏွစ္ ၁၆ႏွစ္ ရွိရမည္ျဖစ္ရာ ျမန္မာႏိုင္ငံမွာဆယ္တန္းေအာင္ၿပီး တကၠသိုလ္ ၃ႏွစ္တက္လွ်င္ဘြဲ႕ရေသာေၾကာင့္ ၁၄ႏွစ္သာေက်ာင္းတက္ထားရာ မဟာဘြဲ႕အတြက္ ၀င္ခြင့္မမီႏိုင္ေတာ့ေပ။ ထိုအခါ GRE and GMAT စေသာ ေအာင္လက္မွတ္မ်ား၊ ဆိုင္ရာတကၠသိုလ္မ်ားဧ။္ ၀င္ခြင့္စာေမးပြဲမ်ား၊ လူေတြ႕စစ္ေဆးပြဲမ်ားကို ထပ္မံေျဖဆိုရ ေလသည္။ သို႕ေသာ္ Private University မ်ားတြင္ သီးျခားစည္းမ်ဥ္းမ်ား ရွိေလသည္။ အေသးစိတ္ သိလုိ ပါက www.moe.go.th တြင္ ၾကည့္ရႈ ေလ့လာႏိုင္ပါသည္။

ျမန္မာမိဘအမ်ားစု စိုးရိမ္ပူပန္ေနသကဲ့သို႕ ထိုင္းႏိုင္ငံသည္လံုျခံဳမွဳ မရွိေသာႏိုင္ငံ ဟုတ္မဟုတ္ကို ႏွစ္စဥ္ သန္းခ်ီေနေသာကမၻာလွည့္ခရီးသည္မ်ားက သက္ေသခံေနပါသည္။ ေက်ာင္းေဆာင္တိုင္းႏွင့္ တိုက္ခန္းမ်ားတြင္ အေဆာက္အဦတိုင္းတြင္ လံုျခံဳေရးအေစာင့္မ်ား ထူထပ္စြာခ်ထားၿပီး ရဲႏွင့္ လူအခ်ိဳးမွာ လူ၂၀၀လွ်င္ ရဲ တစ္ေယာက္ႏံႈး ျဖစ္ေလသည္။ မိန္းကေလးမ်ားကို လိုက္လံ ေႏွာင့္ယွက္ေသာ၊ ဇြတ္အတင္း ရည္းစားစာ လိုက္ေပး၊ ရည္းစားစကား လိုက္ေျပာေသာ ေယာက္်ားေလးမ်ား လံုး၀ မရွိပါေခ်။ စိတ္ပူစရာ တစ္ခုေကာင္း သည္မွာ ႏြဲ႕ေနေသာ ေယာက္်ားေလးမ်ား ထိုင္းတြင္ေက်ာင္းတက္ပါက အေျခာက္ ျဖစ္သြားႏိုင္ပါသည္။ ၃၀% ေသာ ထိုင္းေယာက္်ား ေလးမ်ားမွာ ႏြဲ႕ေနေသာ Girlish boy မ်ားျဖစ္ၾကၿပီး က်န္ ၃၀%မွာGay မ်ားျဖစ္ၾက ကာ ၂၀%ေသာ ထိုင္းေယာက်္ား စစ္စစ္မ်ားသည္ ညစဥ္ မေကာင္းေသာ ေနရာမ်ား၊ ကလပ္မ်ားသို႕ ပံုမွန္ သြားသည္ဟု social research တက္ေနသူ အခန္းေဖာ္ ထုိင္းမ ကိုယ္တိုင္ ေျပာျပေလသည္။ ထို႕ေၾကာင့္ ထိုင္းမမ်ားသည္ မိန္းကေလးမ်ားကို ဂရုစိုက္ ခ်စ္တတ္ ေသာ ျမန္မာေယာက်္ားကို ေတာ္ေတာ္ သေဘာက် ၾကေလသည္။ ထိုင္းႏိုင္ငံတြင္ တရား၀င္ေရာ၊ တရားမ၀င္ပါ ေရာက္ရွိေနထိုင္ေသာ ျမန္မာဦးေရ ငါးသန္းခန္႕ရွိသည္ဟုခန္႔မွန္းထားၾကပါသည္။

ထိုင္းႏိုင္ငံတြင္ ပညာသင္စားရိတ္သည္ တကယ္ ပညာတတ္သည့္ အတိုင္းအတာ၊ သင္ၾကားေရးႏွင့္ သင္ယူေရး ဆိုင္ရာ အေထာက္အကူျပဳ ကိရိယာမ်ား၊ စာၾကည့္တိုက္မ်ားႏွင့္ လိုအပ္ေသာ စာအုပ္စာတမ္း မ်ား ရရွိႏိုင္မႈ အတိုင္းအတာမ်ားႏွင့္ ႏိႈင္းယွဥ္လွ်င္ ျမန္မာႏိုင္ငံထက္ သက္သာသည္ ဟုပင္ ဆိုႏိုုင္ေလသည္။ ေနထိုင္မႈ စားရိတ္သည္လည္း ျမန္မာႏိုင္ငံထက္ အပံုႀကီး သက္သာေလသည္။ အင္တာနက္ကို ေက်ာင္းစာ ၾကည့္ တိုက္တိုင္းတြင္ ေက်ာင္းသားတိုင္း လြတ္လပ္စြာ သံုးစြဲႏိုင္ရံု သာမက ကိုယ့္အခန္းတြင္ တပ္ဆင္လို လွ်င္လည္း လြတ္လပ္စြာ တပ္ဆင္ႏိုင္ၿပီး ေလွ်ာက္လႊာတင္ၿပီး ၄ရက္အတြင္း အဆင္သင့္ သံုးႏိုင္ေလသည္။ တပ္ဆင္ခ ဘတ္ ၁၀၇၀ႏွင့္ လစဥ္ေၾကး ဘတ္၅၉၀ က်သင့္ေလသည္။ ေနထိုင္မႈ စားရိတ္မွာ သာမန္ ေက်ာင္းသားတစ္ဦးအတြက္ (ဟိုတယ္တြင္ သူေဌးသံုး သံုးဖို႕ မဟုတ္ပါ)

၁. တစ္နပ္စာ ဘတ္၆၀ (ေက်ာင္းကင္တင္းမွာ စားလွ်င္ တနပ္စာ ၁၅ဘတ္မွ ၂၅ဘတ္သာ)
၂. ေက်ာင္း အေဆာင္လခ ၄ေယာက္ တစ္ခန္းဆိုလွ်င္ ၇၅၀ဘတ္ (်အေညိေမိ ိသမာငအသမပ ဆိုလွ်င္ဘတ္ ၂၅၀၀မွ ဘတ္၄၀၀၀ၾကား)
၃. ေရတစ္ယူနစ္ ၁၀ဘတ္ (သတ္မွတ္ရက္တြင္ ႀကိဳတင္ ေၾကညာၿပီးမွ ေရေလွာင္ကန္ သန္႕ရွင္းရန္ ေရပိုက္လိုင္း ခဏျဖတ္ ေတာက္ျခင္းမွ အပ မည္သည့္အခါမွ ေရမပ်က္ပါ)
၄. လွ်ပ္စစ္မီး တစ္ယူနစ္ ၃ဘတ္ (ထရန္စေဖာ္မာ ေပါက္ျခင္း၊ မိုးႀကိဳးပစ္ ခံရျခင္းတို႕ေၾကာင့္ နာရီ၀က္မွ ၃ နာရီၾကာ ခဏ ပ်က္ျခင္းမွွအပ မည္သည့္အခါမွ မီးမပ်က္ပါ)သာကုန္က်ေလသည္။

ဘန္ေကာက္ၿမိဳ႕အတြင္း သြားလာေရးအတြက္ ကားလမ္း၊ ရထားလမ္းႏွင့္ တူးေျမာင္းမ်ား အတြင္းႏွင့္ ေက်ာက္ဖယား ျမစ္တြင္း ေျပးဆြဲေသာ ျမစ္ေၾကာင္း လမ္းသြား ေမာ္ေတာ္ဘုတ္မ်ား သံုးမ်ိဳးရွိေလသည္။ ကားလမ္းအတြက္ Taxi အငွား ကားမ်ား၊ ရိုးရိုးဘတ္စ္ကား၊ အဲယားကြန္း ဘတ္စ္ကား၊ van ဟုေခၚေလ့ ရွိေသာ Hi-Aceကားမ်ား၊ ခရီးတိုေျပးေသာ Pick-upကားေလးမ်ားရွိၿပီး ေစ်းအသက္သာဆံုးမွာ ရိုးရိုး ဘတ္စ္ကားျဖစ္ကာ ခရီးစဥ္ တေၾကာကို ၈ဘတ္ျဖစ္ပါသည္။ အဲယားကြန္း ဘတ္စ္ကားမ်ားမွာ Hybrid bus and ordinary bus ႏွစ္မ်ိဳးျပန္ကြဲၿပီး ခရီးစဥ္ အလိုက္ အနည္းဆံုး Hybrid ကားအတြက္ ၁၂ဘတ္၊ ရိုးရိုးကား အတြက္ ၁၁ဘတ္ျဖစ္ကာ ခရီးစဥ္ အစအဆံုး အတြက္မူ ၂၂ဘတ္ေပးရေလသည္။ van ဟု ေခၚေလ့ရွိေသာ Hi-Ace ကားမ်ားမွာမူ ခရီးစဥ္ နီးေ၀းအလိုက္ (ဆူးေလ-အင္းစိန္ ခရီးေလာက္ကို) ၂၅ဘတ္မွ ၉၀ဘတ္ (ဆူးေလ -တိုက္ႀကီး ခရီးေလာက္ကို) ေပးရၿပီး ေခ်ာင္ေခ်ာင္ ေအးေဆး သက္သာစြာ စီးႏိုင္ေလသည္။ ခရီးတို ေျပးေသာ Pick-up ကားေလးမ်ားမွာ (သမိုင္း- ဆူးေလ ေလာက္ ခရီးကို) ၇ဘတ္သာ ေပးရေလသည္။Taxi မ်ားမွာ စစခ်င္း ၃၅ဘတ္ တက္ေနၿပီး တစ္ကီလိုမီတာ ခရီးတိုင္း ၂ဘတ္ ေပးရေလသည္။ ရထားလမ္း အတြက္ Sky Train or BTS ေခၚ မိုးပ်ံရထား၊ Subway or MRTေခၚ ေျမေအာက္ရထား၊ ရိုးရိုးၿမိဳ႕ပတ္ ရထား ဆိုၿပီး သံုးမ်ိဳးရွိပါသည္။ BTS သည္ တစ္ဘူတာ ၁၀ဘတ္ႏံႈး ေပးရၿပီး ခရီးစဥ္ စဆံုးစီးပါမူ ၄ဘူတာစာ အလကား စီးရေလသည္။ MRT ေခၚ ေျမေအာက္ ရထားသည္ တဘူတာလွ်င္ ၁၂ဘတ္ ေပးရေသာ္လည္း ခရီးစဥ္ ရွည္ေလ၊ ေစ်းသက္သာေလ ျဖစ္ကာ ၁၈ဘူတာလံုး ခရီးစဥ္ စဆံုး စီးနင္းလွ်င္ ၃၆ဘတ္ သာက်သည္။ http://www.reed.eduwww.reed.edu/ ေက်ာက္ဖယား ျမစ္တြင္း ေျပးဆြဲေသာ ေမာ္ေတာ္ဘုတ္မ်ားမွာ ေမာ္ေတာ္ အမ်ိဳးအစား အလိုက္ ေစ်းႏံႈး အသီးသီး ရွိေလသည္။ Pulic Motor Boat မ်ားမွာ ခရီးစဥ္အလိုက္ ေစ်းအနည္းဆံုး ၈ဘတ္မွ ေစ်းအမ်ားဆံုး ၁၈ဘတ္ သာ ေပးရေလသည္။ http://www.in-bangkok.com/ ရထားႏွင့္ ျမစ္ေၾကာင္း ခရီးမွာ ကမၷာ့ အဆိုးရြားဆံုးဟု နာမည္ႀကီးေသာ ဘန္ေကာက္ၿမိဳ႕ဧ။္ ယာဥ္ေၾကာ ပိတ္ဆို႕မႈကို ေရွာင္ရန္ အေကာင္းဆံုးေသာ ခရီးစဥ္မ်ား ျဖစ္ေလသည္။

မည္သည့္ခရီးစဥ္ကို လိုက္ပါသည္ျဖစ္ေစ အလြန္အမင္း တိုးေ၀ွ႕ကာ ၾကပ္သိပ္ ညပ္ပိတ္ေနေအာင္ စီးရသည့္အခါ မည္သည့္ အခါမွ မရွိပါေခ်။ မတ္တပ္ရပ္စီးနင္းရေသာသူ အတန္ငယ္ လူမ်ားက ဘယ္သူမွ သည္လို ကားေပၚ ထပ္မတက္ ၾကေတာ့့သလို စပယ္ယာကလည္း အတင္းမေခၚ၊ ယာဥ္ေမာင္းကလည္း ေစာင့္မေခၚသည္မွာ အလြန္စိတ္ခ်မ္းသာစရာေကာင္းေသာ အက်င့္ပါေပ။ စပယ္ယာက အတင္းယာဥ္စီးခ ေတာင္းရန္ မလိုပါ။ အခ်ိဳ႕ဘတ္စ္ကားမ်ားတြင္ စပယ္ယာ မပါဘဲ ခရီးသည္မ်ားက ယာဥ္ေမာင္းထံကို စည္းကမ္းရွိစြာ အလိုအေလ်ာက္ သြားေရာက္ကာ ယာဥ္စီးခ ေပးၾကေလသည္။ ဘတ္စ္ကားလိုင္း အခ်ိဳ႕မွာ ည (၇)နာရီတြင္ သိမ္းၿပီး အခ်ိဳ႔မွာ ည ၁၁နာရီ ထိရွိကာ မိုးခ်ဳပ္ေသာေၾကာင့္ ၾကားကားေစ်းေတာင္းျခင္း မရွိရံုသာမက တရက္စာ စာရင္းပိတ္ၿပီးခ်ိန္တြင္ အလကား ေပးစီးပါေသးသည္။ ထိုင္းဥပေဒအရ ႏိုင္ငံျခားသားေက်ာင္းသားမ်ား အလုပ္လုပ္ခြင့္ မရွိေသာ္လည္း ႏိုင္ငံျခားသားအလုပ္သမားမ်ား ေက်ာင္းတက္ခြင့္ရွိေသာေၾကာင့္ အလုပ္ရၿပီးမွ ေက်ာင္းတက္မွသာ အလုပ္တဖက္ ေက်ာင္းတဖက္ ေနႏိုင္ပါမည္။ သို႕ေသာ္ မဟာဘြဲ႕အတြက္ ထိုသို႕ ေက်ာင္းတက္ပါက စာေမးပြဲက်ဖို႕ မ်ားေလသည္။ သည္မွ်ဆိုလွ်င္ ထိုင္းႏိုင္ငံတြင္ ေက်ာင္းေနရန္ႏွင့္ ေက်ာင္းတက္ေနစဥ္ ကာလအတြင္း ေနထိုင္ စားေသာက္ သြားလာႏိုင္ရန္ သတင္းအခ်က္အလက္ အေတာ္အသင့္ ျပည့္စံုၿပီဟု ယူဆမိပါသည္။
: Higher Education, Thai Education, ခြန္ျမလႈိင္
Copy from "Education for Myanmar Youths Blog"

Thesis Proposal of U Ko Ko, Kasetsart University

Insecticidal Activities of Melaleuca leucadendron Leaf Essential Oil Against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) and Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae)

Introduction
Insect pests cause heavy losses of stored grain quantitatively and qualitatively throughout the world (Madrid et al., 1990). Insect damage in stored grains may amount to 10% - 40% in countries where modern storage technologies have not been introduced (Shaaya et al., 1997). Insect pests cause the loss of stored food grain in tropical and semitropical environments (Tripathi et al., 2001). Chemical control of insects in storage has been used for a long time, but has serious drawbacks (Sharaby, 1988). Careless and indiscriminate use of synthetic pesticides has led to a number of wellknown problems. Some of these are contamination of food, soil, ground water, rivers, lakes, oceans, air etc. with toxic residues, side effects on non-target insects and other organisms, increase of the number of pest species resistant to pesticides, and pest resurgence. In addition, many non-lethal as well as lethal accidents occurred due to mishandling of highly toxic synthetic products.

As a logical consequence of the undesirable side effects of these products, there is a growing awareness in industrialized and also in developing countries, of the toxicological and environmental problems involved in the use of synthetic pesticides. This awareness has led to a steadily increasing movement towards a more environmental – oriented, sustainable agriculture with low or no input of toxic synthetic pesticides and other agricultural chemicals in an attempt to preserve and protect the environment as well as human health.

The plant kingdom is by far the most efficient “factory” of compounds. It synthesizes secondary metabolic products which can partly be considered as weapons to defend plants against pests and diseases which have competed with them since time immemorial.

Historically, chemicals derived from plants have been an important source of insecticides. The insecticidal properties of the alkaloid nicotine, for example, have been known since 1800s (Metcalf et al., 1962; Ware, 1986). Botanical materials such as pyrethrum, rotenone, and nicotine were among the first compounds used to control agricultural insect pests (Perkins, 1985; Grainge and Ahmed, 1988). With the advent of effective and economical synthetic insecticides, however, plant-based insecticides used in agriculture were displaced. As technological (resistance, resurgence and secondary outbreaks) and cultural (health and environmental) problems involved in the use of synthetic pesticides have increased, interest in naturally occurring plant compounds as alternative sources of pest control has been renewed (Perkins, 1985). Currently, novel, as well as established, secondary plant compounds is being evaluated as alternatives for crop protection in many countries.

Several commercial produced plant-derived compounds have exhibited good potential as insecticides. Azadirachtin, a triterpenoid from the neem tree, Azadirachta indica A. Juss (Meliaceae), has growth-regulating and antifeedent properties against numerous insect pests (Warthen, 1979; Rembold, 1988). Commercial preparations of many household insecticides are augmented with pyrethrins, a complex of esters extracted from flowers of Chrysanthemum cinerariefolium (Treviranus) (Compositae) (Bell et al., 1990). Alkaloids that have been used commercially include ryanodine, the active ingredient in ryania (Ryania spp., Flacourtiaeae) the principal components extracted from the seeds of sabadilla, Schoenocaulon spp., Liliaceae (Ware 1986; Bell et al., 1990). Although too expensive for commercial synthesis, rotenone, an isoflavonoid procured primarily from two genera of tropical legumes, several Derris and Lonchocarpus spp., (Fabaceae), has been moderately effective against insects with chewing mouthparts (Bell et al., 1990).

Plants are rich sources of natural substances that can be utilized in the development of environmentally safe methods for insect control. The deleterious effects of certain purified phytochemicals or crude plant extracts on insects are manifested in several ways including toxicity (Hiremath et al., 1997), growth retardation (Breuer and Schmidt, 1995), feeding inhibition (Klepzig and Schlyter, 1999; Wheeler and Isman, 2000), oviposition deterrence (Dimock and Renwick, 1991; Hermawan et al., 1994; Zhao et al., 1998), suppression of calling behavior (Khan and Saxena, 1986) and reduction of fecundity and fertility (El-Ibrashy,1974; Muthukrishnan and Pushpalatha, 2001). Although a wide variety of effects provide potential alternatives for the use of synthetic chemical insecticides, certain plant families, particularly Meliaceae, Asteraceae, Rutaceae, Labiaceae, Annonaceae and Canellaceae are viewed as exceptional promising sources of plant – based insecticides (Jacobson, 1989; Schmutterer, 1990). Entomo-toxic properties of extracts from plants belonging to several other families have also been frequently reported (Hermawan, et al., 1994; Sadek, 1997; Rodriguez – Saona and Trumble, 1999).

Moreover, it is well known that some insecticides from plants have been used for a long time; for instance, pyrethrum, obtained from the flower heads of C. cinerariifolium (=C. cinerariaefolium), was already known during the time of the Persian King Darius the Great (521-486 BC). Botanical insecticides are broad-spectrum in pest control, and many are safe to apply, unique in action, and can be easily processed and used. Botanical pesticides are biodegradable (Devlin and Zettel 1999). It also reduces environmental contamination and human hazards (Grainge and Ahmed, 1988).

Essential oils are believed to act as allelopathic agents or as irritants that protect plants from predation by insects and infestation by parasites (Simpson, 1995). Essential oils and their constituents have also been shown to be a potent source of botanical pesticides (Singh and Upadhyay, 1993). Jilani et al., (1988) reported that turmeric oil repelled various grain insects. Clove oil was shown to be toxic against Sitophilus oryzae (L.) and Rhyzopertha dominica (F.) (Sighamony et al., 1986). The essential oils of several spices like anise (Pimpinella anisum L.) and peppermint (Mentha piperita L.) have been found to have fumigant toxicity to four major stored product pests, R. domonica, Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L.) and S. oryzae (Shaaya et al., 1991). Ho et al., (1996) found that the essential oil of garlic is insecticidal to T. castaneum and S. oryzae. The essential oils of nutmeg seed (Myristica fragrans Houtt) (Huang et al., 1997) and cinnamon bark (Cinnamomum aromaticum Nees) (Huang and Ho, 1998) are also toxic to T. castaneum and S. oryzae.

The Melaleuca essential oils have a high repellent effect upon the ant, Wasmannia auropunctata (Ochetomyrmex auropunctatus) which is a pest of both forest and plantations and fruit crops (Menendez et al., 1992). Alonso et al., (1996) demonstrated that undiluted cajuput oil inflicted 100% mortality to Andrector ruficornis (Cerotoma ruficornis) in lablab (Lablab purpureus cv. Rongai) in Cuba and has distinct repellent effects. Alonso – Anelot et al., (1994) found that essential oil of Melaleuca quinquenervia leaf has a potential for insect repellency on T. castaneum whereas the tea tree oil from Melaleuca alternifolia in water carrier could eliminate flea on dogs, cats, hamsters and white rats (Fitzjarrell, 1995).

Cajuput oil is also known to prevent plant infection of Herpes Simplex Type 1 on plant (Nawawi et al., 1999) and inhibits the growth of fungi, bacteria and yeast (De Colemnares et al., 1998; Dhirendra et al., 1989; Dubey et al., 1983; Farag et al., 1998).

Insect damage in stored grains may amount to 10% - 40% in countries where modern storage technologies have not been introduced (Shaaya et al., 1997). Therefore, there is a need for diverse kinds of safe insecticide or repellents for use on food grain. Generally, synthetic insecticides and fumigation are the main methods in the stored products pest control. However, increased public concern over the residual toxicity of insecticides applied to stored grain, the occurrence of insecticide resistant insect strains and the necessary precautions to work with traditional chemical insecticides calls for new approaches to control stored product insect pests. (Yildirim et al., 2001). Thus, there is a considerable interest in developing natural products that are relatively less damaging to the mammalian health and environment than existing conventional pesticides, as alternatives to non-selective synthetic pesticides to control the pests of medical and economic importance. (Tunc and Sahinkaya, 1998 and Keita et al., 2000).

M. leucadendron is easily found and grown in Southeast Asia countries. There is no report on the activities of M. leucadendron leaf essential oil against stored pests. So the purpose of this study is to investigate the activities of M. leucadendron against S. zeamais, one of the major stored pests in maize and T. castaneum, one of the major stored pests in groundnut.

Objectives

The objectives of this study are:
1. To study biological activity of Melaleuca essential oils on two stored pests, Sitophilus zeamais and Tribolium castaneum.

2. To increase the efficacy of Melaleuca essential oils using synergistic and/or formulation.

Literature Review

Sitophilus zeamais (Motschulsky)

Sitophilus zeamais (Maize weevil) is a small insect and belongs to order Coleoptera and family Curculionidae. It is a serious pest of stored grain throughout the warmer parts of the world. Infestation often starts in the field, and is later carried into the grain stored. The main host for this insect pest is maize and alternative hosts are sorghum, rice and other cereals.

Distribution

This pest is virtually cosmopolitan throughout the warmer parts of the world, extending as far north as Japan and southern Europe. It has been recorded from Europe (Spain, Italy, Turkey); Africa (Angola, Arabia, South Africa, East Africa, Ethiopia, Ghana, Madagascar, Madeira, Mauritius, Morocco, Mozambique, Nigeria); Asia (India, Tibet, Malaysia, Molucca Isles, Borneo, Japan); Australasia (Australia-New South Wales, Queensland, West Australia; New Guinea, New Zealand, Pacific Islands); USA (Texas, Florida); Central America (Costa Rica, Mexico, West Indies); South America (Argentina, Brazil, Guyana, Honduras, Chile, Equador, Guatemala, Nicaragua, Venezuela).

Life History

The female lays white and oval eggs inside the grain by chewing a minute hole in which each egg is deposited, followed by the sealing of the hole with a secretion. These eggs hatch into tiny grubs which stay and feed inside the grain and are responsible for most of the damage. Mature larvae are plump, legless and white, about 4 mm long. Pupation takes place inside the grain.

The adult beetle emerges by biting a circular hole through outer layers of the grain. They are small brown weevils, virtually indistinguishable from each other, about 3.5–4.0 mm long with rostrum and thorax large and conspicuous. The elytra are uniformly dark brown. Each female is capable of laying 300–400 eggs, and the adults live for up to 5 months and are capable fliers.

The life cycle is about 5 weeks at 30 ˚C and 70% RH; optimum conditions for development are 27–31˚C and more than 60% RH; below 17˚C development ceases (Dennis, 1983).

Damage

A thin tunnel is bored by the larva from the surface of the grain kernel. Circular exit holes on the surface of the grain kernel are characteristic.

Control

Infested buildings should be thoroughly cleaned and sprayed with BHC (Benzene Hexa Chloride) or malathion, and any parts of the building which cannot be reached with sprays should be fumigated with the use of recommended fumigants. Infested grtain can be mixed with malathion w.p. which is generally successful in achieving control.
Fumigation of infested grain with methyl bromide, or an ethylene dichloride and carbon tetrachloride mixture is effective but should only be carried out by approved operators because of the toxicity hazards.

Tribolium castaneum (Herbst)

Tribolium castaneum (red flour beetle) belongs to order Coleoptera and family Tenebrionidae. This species is a serious secondary pest throughout the warmer parts of the world in food stores. Infestation is apparently by the appearance of adults on the surface of the grain: there is extensive damage to previously holed or broken grains, or grain damage to previously damaged by other pests. Damage is done by both larvae and adults.

Distribution

This pest is cosmopolitan in warmer countries.

Life History

The female lays small, cylindrical and white eggs scattered in the produce. The larvae are yellowish-white, about 6 mm long when fully grown. The head is pale brown, and the last segment of the abdomen has two upturned dark pointed structures. The larvae live and develop inside the grain till pupation. The damage to the stored grains is done by the larvae. The pupa is yellowish-white, later becoming brown, the dorsum hairy, and the tip of the abdomen having two spine-like processes. The adult is rather flat, oblong, reddish-brown in colour, and about 3-4 mm long. Each female is capable of laying 400-500 eggs and the adults are long-lived, under some circumstances living for a year or more. The life period from egg to adult is 35 days at 30 ˚C. Adults fly in large numbers in the late afternoon (Dennis, 1983).

Damage

Infestation is apparent by the appearance of adults on the surface of the grain; there is extensive damage to previously holed or broken grains, or grain damaged by other pests. Damage is done by both larvae and adults.

Control
The shelled grain should be thoroughly admixed with BHC (Benzene Haxa Chloride) dust or powder, if locally permitted. Fumigation should only be carried out by approved operators.


Melaleuca leucadendron

The M. leucadendron is commonly known as cajuput tree, punk, weeping paperbark, milk wood, white wood, paper bark tree, swamp tea tree, tea tree, white tea tree and brown tea tree (Bailey, 1958). It is classified in the kingdom Plantae, class Magnoliopsida, order Myrtales and family Myrtaceae.

M. leucadendron is a large tree, native to Southeast Asia, the Pacific Islands and Australia. It can be found in many countries such as Indonesia, Malaysia, Vietnam, the Philippines, Thailand, Cuba, Venezuela, Turkey, United States and Hawaii (Alonso et al., 1996; De Colmenares et al., 1998; Kitanov et al., 1992). The tree can be used for many purposes such as essential oil, medicinal properties, fuel wood, timber, charcoal and shelter belts (Brinkman and Vo, 1991).

The oil of M. leucadendron leaf derived by steam distillation (known as cajuput oil or tea tree oil) is green in color with a camphor odor and is used medicinally such as antiseptic, stomachic, stimulant, analgesic, antirhematic, expectotant, and for treatment of intestinal worms (Usher, 1974; Kitanov et al., 1992). This essential oil contains 1, 8 – cineole, nerolidol, alloromadendrene, viridiflorol and α – terpineol as the major components (De Colmenares et al., 1998; Pino et al., 2002).

Botanical Insecticides

Several higher plants produce secondary metabolites that are considered as defensive phytochemicals inhibiting herbivory and infestation by microorganisms (Green and Hedin, 1986; Klocke, 1989; Rosenthal and Berenbaum, 1991). In addition, plant allelochemicals act as repellents, growth regulators and antifeedants (Jermy, 1990). Therefore, these plants may provide new sources of natural pesticides and antifeedants which can be used to replace heavy insecticides applications (Grainge and Ahmed, 1988; Ananthakrishnan, 1992). Antifeedant or feeding deterrent is often highly specific in action, and advantages in integrated pest management (IPM) applications (Whitehead and Bowers, 1983).

The indiscriminate use of chemical pesticides has given rise to many serious problems, including genetic resistance of pest species, toxic residues, increasing costs of application, environment pollution, hazards from handling etc. (Ahmed et al., 1981; Khanam et al., 1990). The development of cross- and multi-resistance strains in many important insect species, resulting from the continuous use of chemical insecticides, has been reported from all over the world (Dyte, 1970; Pasalu and Bhatia, 1983; Dyte and Halliday, 1985; Irshad and Gillani, 1990; Zettler and Cuperus, 1990; Zettler, 1991).

For several years, many plant species are being investigated for their natural products to be used as pest control. For example, some active ingredients of seeds and leaves of the tropical neem tree, Azadirachta indica, especially the tetranortriterpenoid azadirachtin, influence the feeding behavior, metamorphosis, fecundity and numerous insect species belonging to various orders (Jacobson, 1989). Neem oil, obtained by cold-pressing seed, can be effective against soft-bodied insects and mites but is also useful in the management of phytopathogens (Isman et al., 1996).

The use of neem leaves in protecting stored grain and other commodities is as age-old practice in India. Pruthi (1937) and Pruthi and Singh (1950) reported the efficacy of neem leaves in protecting stored grain from insect attack. Jotwani and Sircar (1965) showed that the neem kernel was effective as a grain protectant against stored grain insect pests.Yadav (1983) found that neem kernel protects leguminous seeds from attack by Callosobruchus maculatus (F.) and C. chinensis (L.)

Pandey et al., (1976) observed that 1 to 3 parts of neem oil per 100 parts of seed effectively protects Bengal gram (Cicer arietinum) seeds from damage by the bruchid C. chinensis for at least 135 days, without any adverse effects of treatment on germination. Ketkar (1976) reported the efficacy of neem kernel powder and neem oil in controlling the grain pests, under existing godong (warehouse) conditions.


Apart from those natural products in current use such as pyrethrins, rotenone, nicotine, ryania, sabadilla and neem oil, several substances of plant origin have been identified as having toxic, repellent, antifeedant, and/or growth- and development inhibiting-potential on arthropod pests (Coats, 1994). The potential of essential oils to act as fumigants has been demonstrated for stored-product pests (Klingauf et al., 1983; El-Nahal, et al., 1989; Singh et al., 1989;, Shaaya et al., 1991; Rice and Coats 1994; Saraç and Tunç 1995). These results indicate that essential oils may be efficacious and safe replacements for conventional synthetic fumigants.

In Togo, Adhikary (1981) compared neem leaf and seed powder with conventional insecticides for protecting corn stored in sacks or unpeeled corn cobs in bins against S. zeamais, Tribolium spp. R. dominica and Cathartus spp.

As spices are commonly used in cooking in various parts of the world, they are generally considered to be relatively harmless to humans compared with non-edible plants. In addition, spices possess various insect control properties. For example, acetone and hexane extracts of peppercorn are toxic to several species of stored product insects (Su, 1984). Chopped garlic and ethyl acetate extracts of garlic were found to repel T. castaneum and S. zeamais (Ho et al., 1987). The essential oil of garlic has also been demonstrated to be insecticidal to T. castaneum and S. zeamais (Ho et al., 1996). The essential oil of several spices, such as anise (P. anisum) and peppermint (M. piperita) have fumigant toxicity to four major stored product pests, R. dominica, T. castaneum, S. oryzae and O. surinamensis (Ho et al., 1994) and star anise (Illicium verum Hook F) (Ho et al., 1995) showed insecticidal to T. castaneum and S. zeamais and suppressed progeny production. The main constituent of star anise, anethole, was also insecticidal to these two stored product insects and suppressed progeny production (Ho et al., 1997).

The addition of acetone extracts of black pepper and nutmeg seeds reduced the number of F1 progeny and prolonged the developmental period of S. zeamais (Haryadi and Rahayu, 2003). Himalayan cedarwood oil drilled from the wood chips of Cedrus deodara was found to possess its toxicity to the pulse beetle (Singh and Rao, 1985) and protection against rice weevil (Singh et al., 1989).

Materials and Methods

Insects

Two species of insects from Department of Agriculture, Ministry of Agriculture and Co-operative, Thailand will be used in this study: S. zeamais (maize weevil) and T. castaneum ( red flour beetles). S. zeamais will be reared on rice 12–13% moisture content while T. castaneum will be reared on rice bran. The cultures will be maintained in the laboratory at 29-32 ˚C and 70–80% RH.

Extraction of Essential Oils

Fresh leaves or fruits of selected plants will be collected from several places or bought from local market, where no pesticides are applied. The fresh leaves will be cut into small pieces and placed in 5 l extracting flask before steaming on the heating mantle for 6–8 h. Then, essential oil will be collected and kept in the refrigerator for further use.

Repellency Test

Petri dishes of 9 cm in diameter will be used to confine insects during experiment. Melaleuca essential oil will be diluted in ethanol to different concentrations (1%, 2%, 3% and 4%) and ethanol absolute will be used as control. Filter papers (9 cm diameter) will be cut in half. One ml of Melaleuca essential oil will be applied to one half of the filter paper as uniformly as possible with a micropipette. The other half (control) will be treated with 1 ml of ethanol absolute. Both treated half and control sides will then be air–dried to evaporate the solvent completely. Full disc will be carefully remade by attaching tested halves to control halves with sellotape. Precautions should be taken so that attachment will not prevent the free movement of insects from one half to another, but the distance between the filter-paper halves remained sufficient to prevent seepage of test samples from one half to another. Each remade filter paper will be placed in a petri dish with the seam oriented in one of four randomly selected different directions to avoid any insecticidal stimuli affecting the distribution of insects. Ten insects will be released at the center of each filter-paper disc and a cover will be placed on the petri dish. For each essential oil, five replicates will be used and the experiment will be repeated once. Counts of the insects present on each strip will be made after 1 h and at hourly interval up to the fifth hour.


Fumigant Toxicity

Filter papers (2 cm diameter) will be impregnated with aliquots of 25 µl of an appropriate concentration of Melaleuca essential oil. Ethanol absolute will be used as control. After evaporating the solvent or ethanol for 2 min in air, each filter paper will be placed on the underside of the screw cap of a glass vial (2.5 cm diameter, 5.5 cm height). The cap will be screwed tightly onto the vial containing ten adults of either species of insects. Each concentration and control will be replicated five times, and the experiment will be repeated once. After 24 h, the insects will be transferred to holding cages with culture medium in incubator for the determination of end-point LC50 and LC95 values.


Contact Toxicity

The filer paper impregnation method will be used to examine the contact toxicity. Filter papers (9 cm diameter) will be impregnated with aliquots of 0.5 ml of an appropriate concentration of different essential oils. Ethanol absolute will be used for the control in these experiments. The filter papers will be air dried for 1 h. A glass ring (5.4 cm diameter, 2.5 cm depth) will be placed on each filter paper before 20 insects will be confined to each glass ring. Sticky substance will be applied to the inner surface of each ring to prevent the insects from climbing onto the side of the ring. For adult S. zeamais and T. castaneum, five replicates will be set up for each essential oil concentration and control, and experiment will be repeated once and kept them till new generation.

Flour Disk Bioassay

Aliquots of 200 µl of a suspension of wheat flour in water (10 g in 50 ml) will be dropped onto a clean plastic placed in a tray. The disks will be left in the fume hood overnight to dry, after which they will be put in an oven at 60 ˚C for 24 h. Melaleuca essential oil will be diluted in ethanol to get different concentrations (2%, 4% and 6%) and ethanol absolute and no ethanol will be used as two controls. After evaporation of the solvent, the disks will be placed in glass vials (diameter 2.5 cm, height 5.5 cm). Ten group-weighed, unsexed adults will be added to each preweighed vial containing the disk. Five replicates will be prepared. After three days, the glass vials with flour disks and live insects will be weighed again, and mortality of insects, if any, will be recorded. In order to eliminate the decrease in weight of flour disks due to the evaporation of ethanol and Melaleuca essential oil, the weight of one and two flour disks treated with ethanol or various concentrations of Melaleuca essential oil will be recorded. After three days, the disks will be weighed again to determine the decrease in their weight. The weight of the flour disks used in the experiments will be corrected for any decrease in weight due to evaporation and drying. Nutritional indices will be calculated with some modifications: relative growth rate (RGR) = (A-B)/B x day-1, where A = weight of live insects on the third day (mg)/No. of live insects on the third day, B = original weight of insects (mg)/No. of insects; relative consumption rate (RCR) = D/B x day-1, where D = biomass ingested (mg)/No. of live insects on the third day; efficiency of conversion of ingested food (ECI)(%) = (RGR/RCR) x 100. For antifeedant action, the formula described by Isman et al. (1990) will be modified in calculating the feeding deterrence index (FDI)(%) = (C-T)/C x 100, where C = the consumption of control disks and T = the consumption of treated disks, as the control and treated disks will be placed in separate vials in no-choice tests.


Data Analysis

Data in all experiment will be analyzed by SAS program, and Least Significant Difference Test (LSD) will be analyzed to compare the treatment means (P <>Experimental Place and Duration

Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand (June - 2007 to Jan - 2009).

Study Supported

All of these studies are under financial support by the government of The Union of Myanmar under technical support by FAO for Myanmar Oil Crops Development Project.

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