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‫‪ISSN 0021-1907‬‬
‫‪INIS ISRRAC EG‬‬
‫‪ISOTOPE & RAD. RES., 40(4), 887-899 (2008).‬‬
‫‪VARIATION IN RHIZOBIUM GROWTH DUE TO SEED‬‬
‫‪AND ROOT EXUDATES RELEASED FROM GAMMA‬‬
‫∗ ‪IRRADIATED GLYCINE MAX SEEDS‬‬
‫)‪KAMEL, H.A.(1) and ASKER, M.M.S.(2‬‬
‫‪(1) Radioisotopes Department, Atomic Energy Authority, Dokki, Giza,‬‬
‫‪Egypt and (2) Microbial Biotechnology Department, National‬‬
‫‪Research Centre, Dokki, Giza, Egypt.‬‬
‫‪Key words: Rhizobium leguminosarum, Glycine max, Gamma irradiation,‬‬
‫‪Seed exudates, Root exudates.‬‬
‫اﻻﺧﺘﻼف ﻓﻰ ﻧﻤﻮ اﻟﺮﻳﺰوﺑﻴﻮم ﻧﺘﻴﺠﺔ إﻓﺮازات اﻟﺒﺬرة واﻟﺠﺬر اﻟﻤﺤﺮرة‬
‫ﻣﻦ ﺑﺬور ﻓﻮل اﻟﺼﻮﻳﺎ اﻟﻤﺸﻌﻌﺔ ﺑﺄﺷﻌﺔ ﺟﺎﻣﺎ‬
‫هﺪاﻳﺔ أﺣﻤﺪ آﺎﻣﻞ و ﻣﺤﺴﻦ ﻣﺤﻤﺪ ﺳﻠﻴﻢ ﻋﺴﻜﺮ‬
‫ﺧﻼﺻـﺔ‬
‫ﻓﻰ هﺬﻩ اﻟﺪراﺳﺔ ﺷﻌﻌﺖ ﺑ ﺬور ﻓ ﻮل اﻟﺼ ﻮﻳﺎ ﺻ ﻨﻒ ﺟﻴ ﺰة ‪ ١٢٢‬ﺑﺠﺮﻋ ﺎت ﻣﺨﺘﻠﻔ ﺔ ﻣ ﻦ أﺷ ﻌﺔ ﺟﺎﻣ ﺎ‬
‫)‪ ٢٠٠-١٠‬ﺟ ﺮاى( ﺛ ﻢ أﺟ ﺮى ﻟﻬ ﺎ وﻟﺒ ﺬور اﻟﻌﻴﻨ ﺔ اﻟﻀ ﺎﺑﻄﺔ ) ﻏﻴ ﺮ ﻣﺸ ﻌﻌﺔ( ﺗﻌﻘ ﻴﻢ ﺳ ﻄﺤﻰ و ﻧﻘﻌ ﺖ‬
‫ﺟﻤﻴﻌﻬﺎ ﻓﻰ ﻣﺤﻠﻮل آﺒﺮﻳﺘﺎت اﻟﻜﺎﻟﺴﻴﻮم )‪ ١‬ﻣﻠﻠﻰ ﻣﻮل ﺑﺪرﺟﺔ ﺣﻤﻮﺿﺔ ‪ .(٦.٥‬ﺗ ﻢ ﺗﻘ ﺪﻳﺮ ﻗ ﺪرة إﻓ ﺮازات‬
‫اﻟﺒ ﺬور واﻟﺠ ﺬور ﻋﻠ ﻰ ﺗﺤﻔﻴ ﺰ ﻧﻤ ﻮ ﺑﻜﺘﺮﻳ ﺎ اﻟﺮﻳﺰوﺑﻴ ﻮم ﻋ ﻼوة ﻋﻠ ﻰ اﻟﺘﺤﺎﻟﻴ ﻞ اﻟﺒﻴﻮآﻴﻤﻴﺎﺋﻴ ﺔ ﻟﻬ ﺬﻩ‬
‫اﻹﻓﺮازات‪.‬‬
‫أﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أن إﻓﺮازات اﻟﺒ ﺬور اﻟﻤﺸ ﻌﻌﺔ و اﻟﻀ ﺎﺑﻄﺔ آﺎﻧ ﺖ أﻓﻀ ﻞ إﻓ ﺮازات اﻟﺠ ﺬور ﺣﻴ ﺚ أدت‬
‫إﻟﻰ زﻳﺎدة ﻧﻤﻮ اﻟﺒﻜﺘﺮﻳﺎ وآﺬاﻟﻚ اﻟﺴﻜﺮﻳﺎت اﻟﻌﺪﻳﺪة اﻟﻤﻨﺘﺠﺔ ﻣﻨﻬﺎ واﻟﺠﺮﻋﺔ ‪ ٢٥‬ﺟﺮاى أدت اﻟ ﻰ أﻓﻀ ﻞ ﻧﻤ ﻮ‬
‫ﻟﻠﺮﻳﺰوﺑﻴ ﻮم وإﻧﺘ ﺎج اﻟﺴ ﻜﺮﻳﺎت اﻟﻌﺪﻳ ﺪة‪ ،‬ﺑﻴﻨﻤ ﺎ اﻟﺠﺮﻋ ﺔ ‪ ٢٠٠‬ﺟ ﺮاى أدت اﻟ ﻰ ﺗﺜﺒ ﻴﻂ ﻓ ﻰ ﻧﻤ ﻮ اﻟﺮﻳﺰوﺑﻴ ﻮم‬
‫واﻟﺴ ﻜﺮﻳﺎت اﻟﻌﺪﻳ ﺪة اﻟﻤﻨﺘﺠ ﺔ ﻣﻨﻬ ﺎ إذا ﻗﻮرﻧ ﺖ ﺑﺎﻟﻌﻴﻨ ﺔ اﻟﻀ ﺎﺑﻄﺔ‪ .‬أﻇﻬ ﺮ ﺗﺤﻠﻴ ﻞ اﻟﺴ ﻜﺮﻳﺎت اﻟﺬاﺋﺒ ﺔ ﻓ ﻰ‬
‫إﻓﺮازات اﻟﺒﺬور واﻟﺠﺬور ﺑﺈﺳﺘﺨﺪام ﺟﻬﺎز اﻟﻔﺼﻞ اﻟﻜﺮوﻣﺎﺗﻮﺟﺮاﻓﻰ اﻟﺴﺎﺋﻞ ﻋ ﺎﻟﻰ اﻟﻜﻔ ﺎءة وﺟ ﻮد ﺳ ﻜﺮﻳﺎت‬
‫اﻟﺠﻠﻮآﻮز‪ ،‬اﻟﺮﻣﻨﻮز و اﻟﻔﺮآﺘ ﻮز‪ .‬اﻧﺨﻔ ﺾ ﻣﺤﺘ ﻮى اﻟﺒ ﺮوﺗﻴﻦ ﻓ ﻰ إﻓ ﺮازات اﻟﺒ ﺬرة ﻋ ﻦ إﻓ ﺮازات اﻟﺠ ﺬور‬
‫وآﺎﻧ ﺖ أﻋﻠ ﻰ ﻗ ﻴﻢ ﻟﻬ ﺎ ﻧﺘﻴﺠ ﺔ اﻟﺠﺮﻋ ﺔ ‪ ١٠‬و ‪ ٢٥‬ﺟ ﺮاى ﻓ ﻰ إﻓ ﺮازات اﻟﺒ ﺬور واﻟﺠ ﺬور ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻰ‪.‬‬
‫أﻇﻬ ﺮت اﻟﻨﺘ ﺎﺋﺞ أﻳﻀ ًﺎ زﻳ ﺎدة اﻷﺣﻤ ﺎض اﻷﻣﻴﻨﻴ ﺔ اﻟﺤ ﺮة ﻓ ﻰ إﻓ ﺮازات اﻟﺒ ﺬور ﺑﺰﻳ ﺎدة اﻟﺘﺸ ﻌﻴﻊ ﺣﺘ ﻰ ‪٢٥‬‬
‫ﺟﺮاى ﺑﻴﻨﻤﺎ ﻓﻰ إﻓﺮازات اﻟﺠﺬور‪ ،‬ازدادت ﺑﺰﻳﺎدة اﻟﺘﺸﻌﻴﻊ ﺣﺘﻰ ‪ ٢٠٠‬ﺟﺮاى‪.‬‬
‫‪ABSTRACT‬‬
‫‪In this study, seeds of Glycine max Giza 122 were irradiated with gamma‬‬
‫‪rays from 60Co source at various doses (10 to 200 Gy), sterilized and soaked into‬‬
‫‪an aerated solution of CaSO4 (1 mmol and pH 6.5). The capacities of the‬‬
‫‪released seed exudates (SEs) and root exudates (REs) to promote Rhizobium‬‬
‫‪Accepted June 2008.‬‬
888
KAMEL, H.A. and ASKER, M.M.S.
leguminosarum growth were investigated as well as biochemical analysis of the
exudates was carried out. SE of both control and gamma irradiated seeds
resulted in a higher Rhizobium population and polysaccharide production than
RE. Relative to control, the highly effective doses in Rhizobium growth and
polysaccharide production were 25 and 200 Gy; the former was a promoter
while the later was an inhibitor. HPLC analysis of soluble carbohydrates
revealed the presence of glucose (Glu), rhamnose (Rha) and fructose (Fru) in the
SE and RE. Protein content in SE was lower than that in RE; the highest values
were due to 10 Gy and 25 Gy in SE and RE, respectively. Free amino acids
content in SE was increased up to 25 Gy then decreased while RE was increased
by increasing gamma doses from 10 to 200 Gy.
INTRODUCTION
The intensity of root nodulation is considered to be affected by the Rhizobium
population in soil (Weaver and Frederick, 1974 a, b; Herridge et al., 1987; Kato
et al., 1997). Since bacteria on root surface generally proliferating using substances
released from roots, Rhizobium population on leguminous roots may be altered by
the quantity and quality of root exudates (Roszak and Colwell, 1987; Kolter et al.,
1993). It had been reported that some components of legume root exudates had
positive effects on Rhizobium growth (Van Egeraat, 1975; Parke and Ornston,
1984; D’Arey-Lameta and Jay, 1987 and Streit et al., 1996).
On the other hand, gamma radiation has been widely used to enhance the
storability of grains and legumes, improve seed germination, increase yield and
quality of product, and to induce genetic variability in crop plants (Anjum et al.,
1990; Ripa and Audrina 1993; Kamel, 1998; Viccini and Carvalho, 2001). High
doses of gamma radiation produce deleterious effects such as poor growth and
genetic damage. Relatively low doses usually alter growth characteristics whereas
lower doses have shown to stimulate plant growth (Watanabe et al., 2000).
In the present study, Glycine max seeds were gamma irradiated at various doses
(low, medium and high) and the capacities of seed and root exudates to promote
Rhizobium leguminosarum growth as well as biochemical analysis of the exudates
were investigated.
VARIATION IN RHIZOBIUM GROWTH DUE TO SEED …
889
MATERIALS AND METHODS
Seeds:
Seeds of soybean (Glycine max Giza 122) were purchased from Crop Institute,
Agriculture Research Centre, Giza, Egypt.
Irradiation of seeds:
Uniform soybean seeds were transferred to plastic bags and exposed to gamma
radiation using 60Co source at the rate of 1 Gy/58 sec for a time corresponding to
10, 25, 50, 100 and 200 Gy.
Rhizobium:
Rhizobium leguminosarum was kindly obtained from Microbiology
Department, Soil and Water Institute, Agriculture Research Centre, Giza, Egypt.
Preparation of soil extract:
Soil extract was prepared according to Iizuka et al. (2002). Clay soil and
distilled water were mixed by 1 : 1 (w/v) and shaken for 2 h. The suspension was
centrifuged at 3000 rpm for 10 min. The supernatant was filtrated through a
combination of glass fiber filter and cellulose nitrate membrane with 0.45 μm pore
size. The filtrate was evaporated to the volume equivalent to 60% of maximum
water holding capacity of the soil and the pH was adjusted to 6.5.
Preparation of seed and root exudates:
Seed and root exudates were prepared according to the methods of Ayo Odunfa,
(1979) and Iizuka et al. (2002). Irradiated soybean seeds (600 g for each dose of
gamma radiation were divided into three replicates; 200 g each) were surface
sterilized by shaking in 20% Milton sterilizing fluid (1% sodium hypochlorite and
16.5% sodium chloride) for 5 min. The seeds were washed several times with
sterile distilled water and soaked into aerated 1 mmol CaSO4 (pH 6.5). Soaked
seeds were incubated in dark at 25oC. After 24 h, the medium was collected and
designed as seed exudates (SE, 0-24 h after soaking) and the imbibing seeds were
wrapped in a urethane foam rubber in such a way that the rubber held the seeds
while the roots remained free. The rubber was placed on mesh attached to a plastic
container filled with CaSO4 solution, so that, the primary roots immediately contact
with the medium just after germination. The containers were placed in racks
designed to keep the roots and the rooting medium in darkness. The containers
were placed in a growth chamber (25°C, 14/10 h light/dark, 150 μE m-2 s-1, 60%
humidity), the medium was collected within 24-96 h and termed root exudates
(RE). CaSO4 was precipitated from the collected media and removed by
centrifugation.
Inoculation preparation:
Rhizobium leguminosarum was preliminarily cultured for 48h with shaking (120
rpm) at 25ºC in yeast extract mannitol (YM) broth. The medium contained in g l-1;
10 mannitol, 1 yeast extract, 1 KH2PO4, 1 K2HPO4, 0.2 NaCl, 0.5 NH4Cl,
890
KAMEL, H.A. and ASKER, M.M.S.
0.13 CaCl2.2H2O and 0.18 MgSO4.7H2O, pH at 6.8 (Somasegaran and Hoben,
1994). The inoculum had been transferred into fresh (YM) broth for another day.
By this procedure, the Rhizobium culture reached the middle or late logarithmic
phase and cell density in the culture was estimated by measuring optical density at
540 nm.
Assays of SEs and REs effects on Rhizobium growth and polysaccharides
production:
Rhizobium growth rate was assayed with a medium composed of 4 ml soil
extract and 1 ml of either seed or root exudates. One ml of saturated CaSO4
solution (pH 6.5) was used as a control for the exudates. The Rhizobium suspension
of 25 µl (0.726 × 105 colony forming unit (CFU)/ml) was added to the medium.
The cultures were incubated at 25ºC and shaked at 120 rpm. The CFU and
polysaccharides produced were determined at 120 h by the standard plate method
(Yoon et al., 2006) and gravimetric method (Linker and Jones, 1966), respectively.
Quantitative analysis of SEs and REs:
a- Sugar analysis:
Soluble carbohydrates in the exudates were measured by colorimetric methods
using a phenol sulphuric acid reagent (Dubois et al., 1956).
Monosugars and oligosaccharides were determined by using HPLC (10A
Shimadzu). The apparatus was equipped by Shim-pack SCR-101N column
(Shimadzu 7.9 mm × 30 cm) and maintained at 40ºC. Separation was achieved by
pumping water through the column at 0.5 ml/min for 25 min. Monitoring was
performed by measuring changes in the refractive index (RI). Identification and
quantitative determination of mono and oligosaccharides were done using external
standard (El Sayed et al., 2005).
b- Free amino acid analysis:
Ten ml of the exudates was used for free amino acids determination and acetone
was added to the extract to precipitate protein (Green and Hughes, 1955).
Supernatant was dried under vacuum and amino acids were dissolved in 1 ml of
buffer solution containing sodium acetate (8.2 g/l), methanol (7.5 %), formic acid
(0.3 %), acetic acid (1.5 %) and octanoic acid (0.001%) then it had been filtered.
Total free amino acids in the exudates were measured by ninhydrine reagent
(Yemm and Cocking, 1955). Amino acid analysis was carried out using LC3000
Amino Acid Analyser (Eppendorf, Biotronic, Maintal, Germany) equipped with a
75 × 6.0 mm BTC guard column and a 145 × 3.2 mm BTC 2140 main column with
the following conditions:
Flow rate: 0.2 ml/min.
Pressure of buffer from 0 to 50 bar.
Pressure of reagent from 0 to 150 bar.
Reaction temperature 123°C.
VARIATION IN RHIZOBIUM GROWTH DUE TO SEED …
891
Amino acids derived from column were quantified with ninhydrine at 440 nm
for primary amino acids and at 570 nm for proline.
c- Protein analysis:
After assaying the effects of SEs and REs on Rhizobium growth, (NH4)2SO4 was
added to give 90 % saturation (30.15 g) to 50 ml of the exudates and kept overnight
in the refrigerator to precipitate protein (Green and Hughes, 1955). Protein was
separated by centrifugation then dissolved in 0.5 ml distilled water. Dialysis was
carried out in dialysis membrane stock No. 250-7U (Sigma Chemical, USA)
against distilled water for 24 h (water changed each 8h). Total protein in the
exudates was measured by colorimetric method of Bradford (1976). The
electrophoretic detection of protein by sodium dodecyle sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) was carried out using the method of Laemmili
(1970).
Protein content in the samples was adjusted to 2 mg/ml. SDS was added to the
sample at a rate of 4 mg SDS/l mg protein then 50 μl 2-mercaptoethanol was
applied to each 950 μl of the sample and the mixture was heated at 100°C in water
bath for 3-5 min.
Electrophoresis was performed in a vertical slab mold (Hoefer Scintific
Instruments, San Francisco, CA, USA, Model LKB 2001, measuring 16×18×0.15 cm).
Electrophoresis was carried out at 30 mA at 10°C for 3 hours.
Protein was stained with silver nitrate using the method described by Sammons
et al. (1981). This method is sensitive to 2 μg of protein in a single band.
Analysis of data was carried out using gel documentation system (Software
AAB, Advanced American Biotechnology 1166E, Valencia Dr. Unit 6 C, Fullerton
Ca 92631).
The clusters are based on arithmetic mean (UPGMA) which is represented by a
dendogram (Phenogram).
Statistical analysis:
The obtained data were subjected to one way ANOVA and the differences
between means were at the 5% probability level using Duncan’s new multiple
range tests. The software SPSS, version 10 (SPSS, Richmond, USA) was used as
described by Dytham (1999).
RESULTS AND DISCUSSION
Rhizobium growth and polysaccharide production:
Prior to analyzing the components of soybean SE and RE, their ability to
promote Rhizobium proliferation was tested. The effect of SE and RE on
Rhizobium growth is shown in (table 1).
892
KAMEL, H.A. and ASKER, M.M.S.
The addition of SE of either control or gamma irradiated soybean seeds
enhanced Rhizobium CFU than RE. SE of gamma irradiated seeds up to 50 Gy
significantly increased Rhizobium CFU while 200 Gy caused non-significant
decrease in the CFU relative to control. On the other hand, RE released from
gamma irradiated seeds up to 100 Gy significantly increased CFU than control.
These results agree with the previous results of Kato et al. (1997) where the
seed exudates of common bean (Phaseolus vulgaris L.) had a higher potential for
Rhizobium proliferation than root exudates. Also, Iizuka et al. (2002) reported that
seed exudates of both cultivars Glycine max L. c.v. Enrei and Tachinagaha always
resulted in higher Rhizobium population than root exudates and attributed the
difference in the ability between SEs and REs to promote bacterial growth to the
difference in the organic matter content between them. Kato et al. (1997)
mentioned that plants releasing compounds from seeds and roots directly affect
Rhizobium infection and root nodulation.
Regarding the amount of polysaccharide production by Rhizobium grown in the
culture medium containing either SE or RE, data in table (1) show significant
increase in the amount produced due to SE of seeds exposed to gamma dose up to
100 Gy relative to control. Also, RE of seeds irradiated with 25, 50 and 100 Gy
doses caused significant increase in the polysaccharide production.
Bacterial polysaccharides are necessary for a functional Rhizobium legume
symbiosis. It plays essential roles in the formation of infection thread, nodule
development and is important for the adaptation and survival of Rhizobium under
different environmental conditions at both the free living and symbiotic stages
(Kannenberg and Brewin, 1994; Lloret et al., 1998). A modification of extracellular polysaccharides has been described frequently as a response to different
environmental and physiological conditions (Lloret et al., 1998). In the current
study, the variation in the amount of polysaccharide produced by Rhizobium grown
either on SE or RE of gamma irradiated seeds was mainly due to variation in
bacterial growth and not due to the direct effect of gamma radiation on the
Rhizobium (table 1).
Biochemical analysis:
Duke et al. (1983) suggested that imbibing soybean seeds exuded macromolecules from ruptured embryo cells and low molecules by negative diffusion.
This assumption led us to attempt the determination of carbohydrates, protein and
free amino acids concentrations in the SE and RE.
Total soluble carbohydrates:
The amount of total soluble carbohydrates (table 2) in control and gamma
irradiated seeds were higher in SE than RE. Regarding the effect of seed
irradiation, 10 Gy increased soluble carbohydrates to the highest contents of 90.91
and 71.29 µg/g seed while 200 Gy decreased it to the lowest contents of 30.44 and
26.84 µg/g seed in both SE and RE, respectively.
VARIATION IN RHIZOBIUM GROWTH DUE TO SEED …
893
Table (1): Rhizobuim leguminosarum growth and polysaccharide production
after 120 h growth in mixture of soil extract and either seed or root
exudates.
0
Rhizobium growth
(CFU × 106 ml-1 exudates)
SE
RE
7.477 ± 0.456b 4.216 ± 0.424c
Polysaccharide
(mg ml-1 exudates)
SE
RE
8.600 ± 0.306c
4.021 ± 0.392c
10
10.567 ± 0.274a
6.075 ± 0.341ab
14.607 ± 1.014a
4.781 ± 0.288c
25
11.883 ± 0.563a
6.639 ± 0.452a
12.33 ± 0.441b
6.404 ± 0.581a
50
11.267 ± 0.361a
6.064 ± 0.169ab
12.833 ± 0.333b
5.977 ± 0.217ab
ab
b
11.833 ± 0.333
6.411 ± 0.286a
5.281 ± 0.433bc
7.833 ± 0.441c
5.107 ± 0.288bc
Gamma dose
(Gy)
b
100
8.283 ± 0.563
200
7.343 ± 0.627b
5.542 ± 0.263
Data are mean of three replicates ± standard error.
Means having the same letter in the same column are non-significantly different at 5%.
Table (2): Carbohydrates, protein and free amino acids contents (μg/g seed) in
either seed or root exudates.
0
Carbohydrates
Protein
Free amino acids
(μg/g seed)
(μg/g seed)
(μg/g seed)
SE
RE
SE
RE
SE
RE
71.4 ± 2.9b 16.9 ± 1.3d 25.9 ± 1.7e 109.9 ± 0.6b 143.6 ± 3.0a 164.4 ± 5.3d
10
90.9 ± 5.0a 71.3 ± 1.3a 75.5 ± 1.6a 115.2 ± 8.1b 154.9 ± 0.3a 173.2 ± 6.9d
25
88.1 ± 7.9a 64.7 ± 1.7a 69.4 ± 1.8ab 154.3 ± 4.1a 158.9 ± 1.0a 181.9 ± 5.3d
50
75.1 ± 8.4ab 39.4 ± 3.2b 53.0 ± 1.3c 104.9 ± 5.4b 118.6 ± 14b 236.9 ± 14.7c
100
34.1 ± 4.6c 23.3 ± 4.6cd 40.5 ± 3.1d 104.1 ± 3.6b 96.9 ± 7.7b 356.6 ± 5.6b
200
30.4 ± 0.7c 26.8 ± 0.9c 65.9 ± 2.8b 85.5 ± 0.7c 102.9 ± 2.3b 448.9 ± 9.5a
Gamma
dose (Gy)
Data are mean of three replicates ± standard error.
Means having the same letter in the same column are non-significantly different at 5%.
In some previous studies, the concentration of sugars in SE was higher than RE
(Iizuka et al., 2002; Kato et al., 1997), but in other studies, the opposite was true
(Vancura and Hanzlikova, 1972).
HPLC analysis:
HPLC analysis (table 3) shows the presence of three monosugars (glucose,
rhamnose and fructose) in both SE and RE. The total values of monosugars in SE
were higher than that in RE. Glucose represents the major sugar followed by
rhamnose and fructose consecutively. There were no oligosaccharides found in the
SE of control.
KAMEL, H.A. and ASKER, M.M.S.
894
Table (3): HPLC analysis of soluble carbohydrates as a molar ratio in either seed
or root exudates.
Seed exudates
Root exudates
Gamma
dose
Monosugars
Monosugars
Oligosac.
Oligosac.
(Gy) Glu. Fru. Rha. Total
Glu. Fru. Rha. Total
0
1.0
0.5
0.0
1.5
0.0
1.0
0.2
0.1
1.3
1.2
10
4.3
2.3
2.9
9.5
19.0
2.2
0.7
0.4
3.3
2.5
25
4.1
3.3
2.1
9.5
16.0
1.5
0.5
0.4
2.4
2.0
50
2.7
1.4
2.7
6.8
21.0
1.3
0.9
1.0
3.2
1.6
100
1.7
1.3
1.5
4.5
15.0
1.2
1.0
1.2
3.4
1.5
200
0.4
0.1
0.1
0.6
1.0
0.9
0.1
0.0
1.0
1.0
In gamma irradiated seeds, the amounts of monosugars and oligosaccharides
were higher in SE than RE. The highest values of both fractions were due to 10 and
25 Gy while the lowest one was due to 200 Gy in the SE and RE.
In addition to the three monosugars determined in this study, raffinose,
cellobiose, maltose, sacharose, galactose, arabinose, xylose and ribose sugars were
determined in the seeds and seedling exudates of barley, wheat, cucumber and
bean. Also, differences in the number and nature of the oligosaccharides in seed
and seedling exudates were also observed in other species (Vancura and
Hanzlikova, 1972). In the current study, the obtained results declared that, gamma
irradiation of Glycine max seeds induced a difference only in the amount of monosugars and oligosaccharides that were found in SE and RE.
Free amino acids:
Depending on the results of Rhizobium growth and polysaccharides production
(table 2), the SE and RE of control, 25 Gy (inductive dose) and 200 Gy (inhibitory
dose) were used for studying amino acid analysis and protein finger print.
Relative to control, a non-significant increase was detected in the total free
amino acids in SE due to 10 and 25 Gy while 50, 100 and 200 Gy caused
significant decrease in amino acids content (table 2).
In RE, 50, 100 and 200 Gy caused significant increase in the amino acids
content and the increase was proportional to the dose. Amino acids analysis (table
4) shows the presence of 16 amino acids in both SE and RE. In SE, the number of
amino acids was decreased by increasing gamma dose from 25 to 200 Gy. On the
contrast, their number was increased in RE by increasing dose from 25 to 200 Gy
relative to control.
Of the sixteen amino acids detected, isoleucine, leucine, tyrosine, phenyl
alanine, histidine and proline amino acids were present in all samples analysed. In
control, the amino acids glycine, valine and methionine were occurred in SE only
while lysine was occurred in RE.
VARIATION IN RHIZOBIUM GROWTH DUE TO SEED …
895
Table (4): Amino acids analysis in seed and root exudates.
Amino acids
Aspartic acid
Serine
Glutamic acid
Glycine
Alanine
Cystine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Proline
Total %
Control
SE
RE
2.946
3.872
1.392
2.179
1.866
0.846
0.481
1.054
0.785
1.387
1.594
21.724
1.741
7.349
3.614
53.019
3.060
1.040
6.617
82.417
98.99
99.99
%
25 Gy
SE
0.514
1.045
1.195
0.345
18.969
40.752
2.499
34.682
100
RE
0.363
2.005
5.500
6.494
5.361
0.919
4.066
1.292
3.090
6.097
8.611
3.320
3.629
48.252
98.99
200 Gy
SE
RE
0.961
0.976
5.077
0.471
2.317
1.183
0.371
8.817
1.186
1.381
0.449
48.168
1.101
1.146
0.890
0.020
1.071
41.671
84.775
102.37
99.665
Aspartic acid, serine, cystine and arginine were occurred in RE due to seed
irradiation (the first three occurred due to 25 Gy while the latter occurred due to
200 Gy). Glycine and methionine were disappeared from SE due to seed
irradiation.
Free amino acids were detected in the seed and root exudates of cowpea and
sorghum (Ayo Odunfa, 1979) and in the exudates of seeds and seedlings of barley,
wheat, cucumber and bean (Vancura and Hanzlikova, 1972). The results of the
total free amino acids presented in table (2) disagreed with the results of Vancura
and Hanzlikova (1972); Ayo Odunfa (1979); Kato et al. (1997) and Iizuka et al.
(2002). The proportional increase in the type and amount of free amino acids in RE
may be due to increase in the activity of protease enzyme.
Protein:
There were no available publications dealing with the effects of gamma
irradiation on the protein in SE and RE.
Total protein:
In control and treated seeds, the total protein content in SE was lower than that
in RE (table 2). In SE, all gamma doses caused significant increase in protein
relative to control; the highest level was due to 10 and 25 Gy, respectively. In RE,
only 25 Gy caused significant increase while 200 Gy caused significant decrease in
protein content relative to control.
896
KAMEL, H.A. and ASKER, M.M.S.
Protein finger print:
Results of protein finger print (fig. 1) can be summarized in the following
points:
(1) Peptides of 38, 49, 55 and 60 KDa were occurred only in SE while peptides of
26, 29, 32, 57, 61, 66, 86, 90 and 94 KDa were occurred in RE.
(2) Peptides of 20, 22, 23, 27, 28, 31, 33, 36, 37, 42, 45, 47, 50, 51, 53, 58, 59, 63,
67, 72, 80 and 91 KDa were occurred due to seed irradiation while peptides of
21, 26, 29, 32, 38, 49, 55, 57, 60, 61, 66, 76, 86, 90 and 94 KDa were
disappeared due to seed irradiation.
(3) Peptides of 27, 45, 67, 72 and 91 KDa were occurred due to 25 Gy while
peptides of 28, 33, 36, 42 and 58 KDa were occurred due to 200 Gy.
Fig. (1): SDS-PAGE patterns of total protein in SE (lanes 1 to 3) and RE
(lanes 4 to 6) released from gamma irradiated soybean seeds. M is
the marker, lanes 1 and 4 for the control, lanes 2 and 5 for the 25 Gy
while lanes 3 and 6 for 200 Gy.
VARIATION IN RHIZOBIUM GROWTH DUE TO SEED …
897
Cluster:
Dendogram of the cluster shows that the similarity index was affected by the
type of exudates and gamma dose while in SE, the similarity was decreased from
95.47 to 81.75 when gamma dose increased from 25 to 200 Gy but similarity in RE
was decreased to 88.24 due to 200 Gy (fig. 2).
Proteins and peptides were found in seed and seedling exudates of barley,
wheat, cucumber and bean (Vancura and Hanzlikova, 1972) and its amount was
decreased in the order of cucumber, barley, wheat, bean. Atak et al. (2004) referred
that to genetic alterations produced by ionizing radiation due to the ionization and
the excitations of the DNA molecule and mentioned that, there are two effects of
ionizing radiation on the heredity material; gene mutations and chromosome
breaks.
Fig. (2): Cluster figure of total protein in SE (lanes 1 to 3) and RE (lanes 4 to
6) released from gamma irradiated soybean seeds. Lanes 1 and 4 for
the control, lanes 2 and 5 for the 25 Gy while lanes 3 and 6 for
200Gy.
CONCLUSION
Irradiation of Glycine max seeds with different doses of gamma radiation
caused a variation either in the amount or type of the biochemical components
(soluble carbohydrates, free amino acids and protein) that were analyzed in the
seed and root exudates. This variation induced changes in Rhizobium growth and
polysaccharide produced. Results of Rhizobium growth and produced
polysaccharides had led to assume that the irradiation of Glycine max seeds with
gamma radiation up to 25 Gy may increase the number of nodule formation and
consequently increase the amount of atmospheric nitrogen fixation. In the future, it
will be essential to identify the effective compounds in seed and root exudates that
affect Rhizobium population as well as nodule formation in the field.
Acknowledgement:
The authors are grateful to Naja M. Hessein, M.Sc. student, Fellowship
Program, Egyptian Academy of Scientific Research and Technology for her
technical assistance.
898
KAMEL, H.A. and ASKER, M.M.S.
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