Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
4 International
th
( 95 )
Conference on Radiation
Sciences and Applications,
13-17/10/2014, Taba, Egypt
4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014) PP. 95 : 109
Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana)
Plant Under Abiotic Stresses
Abd El. Hamid Ali1, A. A. Abo Shosha1, M. K. M. Kassem2 and E.E.M. El-Dabaawy1
E.mail:[email protected]
ABSTRACT
Somaclonal variation is considered to be a useful source of variation
and has been demonstrated to be feasible in crop species like tomato, potato, wheat, rice, sorghum, maize and garlic (Evans et al., 1986; Lee and
Phillips, 1988; Adkins et al.,1990; Van den Bulk et al., 1990; Ali, 1998;
Badria and Ali, 1999 and Manjula et al., 2000) and stevia (Kuntal et al.,
2005; Moktaduzzaman and Rahman, 2009).
This work aimed to induced artificially new genetic variation in Stevia rebaudiana Brtoni by using somaclonal variants and gamma rays,
which might be useful for increasing the bioactive components and tolerant to abiotic stresses such as salinity. The micropropagated plants (six
weeks old) were treated with 0,750,1500 and 2250 rad doses of gamma
rays according to Mathius et al. (1995) to obtain the suitable explants.
Then explants were cultured to callus induction media, after that calli
were transferred to regeneration media supplemented with 0, 2000, 4000
and 6000 ppm 0f NaCl. Some plants were relatively tolerant to salt stress.
KEYWORDS
Stevia Rebaudiana,
Somaclonal Variation,
Gamma Rays, Salinity.
The selection for salinity tolerance treatments were done by using
Shoot-tip and node explants taken from micropropagated plants which
treated with 0.0, 750.0, 1500.0 and 2250.0 rad gamma ray doses were
transferred to callus induction medium MS4 and incubated at 25±2 oC
and the responded explants for callus induction data were recorded after
two weeks from culturing. Then calli were transferred to regeneration
medium 1 containing different concentration of NaCl ( 0.0, 2.0, 4.0, 6.0)
gm/L and incubated in the culture room at 25±2 oC under 16:8 light/
dark photoperiods with light intensity of about 2500 lux.After twenty
days of incubation, cultures were evaluated in the basis of the following
parameters:
Regeneration rate calculated as percentage of explants produced
shoot and the mean number of regenerated shoots per explant callus
source (ECS).
1.
2.
Genetic dept., Fac. Agric., Kafr el-sheikh univ., Egypt.
Plant Dept., Nuclear Res. Center, Atomic Energy Authority, Egypt
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4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
S
INTRODUCTION
tevia rebaudiana Bertoni, is a perennial
herb belongs to the Asteraceae family,
is one of the most valuable tropical medicinal plant . It is a natural sweetener
plant known as sweet leaf, sweet herbs
and honey leaf, which is estimated to be 300 times
sweeter than cane sugar (Liu and Li, 1995; Chalapathi and Thimmegowda, 1997). S rebaudiana is
originally a south American wild plant (Katayma et
al., 1976 ) , but it could be found growing in semiarid habitat ranging from grassland to scrub forest to
mountain terrain. The plant has gained wide access
to pacific rim countries, where is recent decades it
is being cultivated domestically, used in its raw leaf
form and is now commercially processed into sweetener . Stevia rebaudiana Bert is one of 154 members
of the genus stevia, which produces sweet steviol
glycosides (Soejarto et al., 1982).
Stevia plants were introduced to Egypt recentlly,
and there are many trails to adapte this plant and
growing it under Egyptian new reclamation areas.
The leaves of Stevia are the source of diterpene
glycosides, viz., stevioside and rebaudioside (Yoshida, 1986). Pure extract of stevioside is non-caloric
(Bhosle, 2004). Stevioside is regenerated as a valuable natural sweetening agent because of its relatively good taste and chemical stability (Yamazaki
et al., 1991; Toyoda and Matsui, 1997). Other attributes of this natural, high intensity sweetener include non-fermentable, non-discoloring, maintain
heat-stability at 100 oC and features a lengthy shelf
life. Now it is being cultivated in Japan, Taiwan,
Philippines, Hawaii, Malaysia and South America
for food and pharmaceutical products. Products can
be added to tea and coffee, cooked or baked goods,
processed foods and beverages, fruit juices, tobacco
products, pastries, chewing gum and sherbets.
Karyotypes of six species of the genus Stevia
from Southern Brazil were studied, utilizing root tip
metaphases. All species were diploid with 2n = 22
chromosomes. It was possible to identify each spe-
Abd El. Hamid Ali et al.
cies by chromosome morphology. The basic chromosome number for Brazilian species of Stevia is X
= 11. This number is also found in almost all South
American species (Frederico et al 1996).
Mutations can be induced by chemicals, or various types of ionizing radiation (X- rays, gamma rays,
neutrons, ultraviolet light, etc.). for induction of mutations in vegetatively propagated plants.
Salinity of the soil either natural or caused by
irrigation in arid environment and excessive use of
fertilizers is a great problem in agriculture. Breeding
for salinity tolerance is not easy task, as it represents
a complex problem, it can be preferably handled by
a multi-discipline approach.
The present investigation has the following objectives:
1- Establish a new protocol for Stevia rebaudiana
somaclonal variants production via tissue culture techniques to identify the suitable sources
of explants, media, growth regulators and physical factors.
2- Assessing the possibility of obtaining salt tolerance stevia plants via tissue culture (somaclonal
variants) and gamma irradiation.
3- Study the effect of gamma rays on the growth
traits of micropropagated plants.
MATERIALS AND METHODS
The present study was carried out at Plant Tissue Culture Laboratory (Biotechnology Unit), Plant
Research Department, Division of Isotopes Applications, Nuclear Research Centre, Atomic Energy Authority, Egypt. And Genetics Department, Faculty of
Agriculture, Kafrelsheikh University, Egypt. Plant
analysis was done in food technology research institute, Egypt.
Plant Materials.
Seedlings and seeds of Stevia rebaudiana Bertoni var. spanti used in the present study were provided by Sugar Crops Research Institute, Agric. Re-
Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
search Center, Ministry of Agric, Egypt.
Gamma irradiation treatments were done
in Atomic Energy Authority, Nuclear Research Center, Inshas, Egypt. Treated stevia seeds were surface
sterilized in 70% (v/v) ethanol for 1-2 min, followed
by dipping in 5% (v/v) sodium hypochlorite (NaOCl) plus 0.1% Tween 80 for 20 minutes, after that,
seeds were rinsed thoroughly three to five times in
sterile distilled water under sterile conditions. Then
the seeds were transplaced into water agar medium
for germination.
In vitro micropropagation.
1. Explants sterilization.
Shoot tip and nodal segments (1.5-2.0 cm) were
washed under running tap water and rinsed with
small amount of soap for 10 minutes, followed by
distilled water. After that, the explants were soaked
in 5% (v/v) Clorox for 10 minutes after has been
washed three times with sterile distilled water, then
immersed in 0. 15% mercuric chloride (HgCl2) solution for 1 minute. Finally, four washes with sterilized distilled water were used to remove all traces
of HgCl2. All steps had been done under sterilized
( 97 )
conditions. The purpose of this procedure was to disinfect the plant tissue from fungi, bacteria, and other
sources of contamination, without harming the regenerative capacity of the explant.
2. Type of explant.
Two explants (shoot tip and nodal segments)
were used from micropropagated plants in this experiment. Every type of explant were cultured in
jars containing 40 ml of MS ( Murasshige and
Skoog 1962) supplemented with 0.5mg/l benzyl
adenine (BA) and solidified with 8.0 gm/l agar, five
explants were cultured on each jar. Cultures were incubated in growth room at 25±2 ºC under 16 hr light
and 8 hr dark provided by florescent light intensity of
2500 lux. The following data were recorded: number
of shoots, number of leaves, shoot length and length
of inter node.
3. Somaclonal variants production.
3.1. Callus induction media.
For induction callus, MS (Murashige and
Skoog, 1962) medium with five different combination of growth regulators (MS1, MS2, MS3, MS4,
MS5) were used in this study (Table 2).
Table (1): The composition of different media used for callus induction of Stevia plants.
Medium code
Composition
According to
MS1
MS+1mg/L 2,4-D
Uddin et al. (2006)
MS2
MS+2mg/L 2,4-D
Uddin et al. (2006)
MS3
MS+3mg/L 2,4-D
MS4
MS+1.5mg/L NAA+1mg/L BA
MS5
MS+1.5mg/L 2,4-D +1mg/L
BA
Uddin et al. (2006)
Moktaduzzaman and Rahman
(2009)
Moktaduzzaman and Rahman
(2009)
2. Regeneration media.
To regenerate plants from callus, four types of
MS medium different in their growth regulator combinations (NAA and BA) were used as following:
1. The first regeneration medium contained MS basal salts supplemented with 1.8 mg/L BA (6.Ben-
zyle adenine) and 0.120 mg/L NAA (Naphthalene
acetic acid) Moktaduzzaman and Rahman
(2009).
2. The second regeneration medium contained MS
basal salts supplemented with 1mg/L BA and
0.5gm/L NAA.
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4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
3. The third regeneration medium contained MS
basal salts supplemented with 1.5mg/L BA and
0.1mg/L NAA (Moktaduzzaman and Rahman,
2009).
4. The forth regeneration medium contained MS
basal salts supplemented with 2mg/L BA and
0.134gm/L NAA (Moktaduzzaman and Rahman, 2009).
All used media were solidified with 0.8% agar
and the pH was adjusted to 5.8 before autoclaving.
Four replicates were used for each treatment, each
containing four explants. The jars were incubated
at 25±2 oC under 16:8 light/dark photoperiods with
light intensity of about 2500 lux.
3. Rooting formation.
For rooting formation, the regenerated shoots
were transferred to a combination of two MS medium strength ( Full MS and Half MS) supplemented
with 0.0 and 1.5 mg l-1 IBA (Indole Butyric Acid)
(Moktaduzzaman and Rahman, 2009; Mubarak,
2009).
4. Acclimatization stage.
Acclimatization can take place by allowing
the in vitro plants gradually get used to a lower relative humidity, which is the case in vivo. The soil
composition is very important during acclimatization stage and differs from composition during acclimatization stage on the growth and development
of Stevia rebaudiana. Rooted shoots were washed
from agar in running tap water to get rid of residues.
Then the roots were washed with sterilized distilled
water. Plantlets were transferred into plastic pots 8.0
cm diameter filled with 1:1 (v/v) peatmoss and sand
after autoclaving at 121oC for 20 min. Plantlets were
covered immediately with polyethylene bages for 15
days to maintain their humidity and placed in growth
chamber at 85% relative humidity with 16 h light
and 8 h darkness at 25 oC. After that, polyethylene
bages were opened gradually and slowly reduced
and irrigated contiuously. The healthy plants were
transferred to the field for more growth.
Abd El. Hamid Ali et al.
3. Gamma irradiation treatments.
Micropropagated plants (six weeks old) were treated with 0.0, 750.0, 1500.0 and 2250.0 rad doses of
gamma ray. The treated shoot tip and node explants
were transferred to MS medium with 0.5 mg benzyladenine/litre for bud multiplication.
4. In vitro selection for salinity tolerance using
gamma irradiated plants.
The selection for salinity tolerance treatments
were done by using Shoot-tip and node explants
taken from micropropagated plants which treated
with 0.0, 750.0, 1500.0 and 2250.0 rad gamma ray
doses were transferred to callus induction medium
MS4 and incubated at 25±2 oC and the responded explants for callus induction data were recorded after
two weeks from culturing. Then calli were transferred to regeneration medium 1 containing different
concentration of NaCl ( 0.0, 2.0, 4.0, 6.0) gm/L and
incubated in the culture room at 25±2 oC under 16:8
light/dark photoperiods with light intensity of about
2500 lux.After twenty days of incubation, cultures
were evaluated in the basis of the following parameters:
1. Regeneration rate calculated as percentage of
explants produced shoot.
2. The mean number of regenerated shoots per explant callus source (ECS).
RESULTS AND DISCUSSION
The results are presented in main parts to facilitate demonstrations and discussion, these sections
are:
1. in vitro micropropagation.
2. Somaclonal variants production.
3. In vitro selection for salinity tolerance using
gamma irradiated plants.
4. Effect of gamma irradiation on stevia plants via
tissue culture.
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Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
The data presented in table (2) showed the mean
number of shoots, number of leaves, length of shoot
and length of inter node for two explants (shoot tip
and node) produced under MS medium supplemented with 0.5 mg/l benzyl adenine (BA).
Table (2): Effect of different explants on Stevia rebaudiana for number of shoots, number of leaves, length of shoot
and length of inter node after two weeks.
Type of explant No. of shoots
Shoot tip
Node
LSD 0.05
0.01
2.4
2.3
0.497
0.697
No. of leaves/
shoot
28.3
20
104.145
146.014
Regarding number of shoots character, shoot tip
and nodal segments explants gave very close values
(2.4 and 2.3) which supported with figure 3.
Concerning number of leaves character, the results indicated that shoot tip explant gave high value
(28.3), while nodal segments explant gave low value
(20).
For length of shoot character, shoot tip explant
gave the highest value (3.5 cm) compared with nodal
segments (3 cm).
The data revealed that shoot tip and nodal segments explants gave very close values for length of
inter node character (1.4 and 1.2 cm).
These results are in agreement with those obtained by Patil et al. (1996) and Mubarak et al.
(2008) they mentioned that micropropagation of stevia by shoot tip explants, gave the highest number of
plantlets than nodal segments. The multiplied shoots
were cultured on modified MS medium supplemented with different concentrations of growth regulators.
1.2. Somaclonal variants production.
This part was done for the evaluation of tissue
Length of shoot
(cm)
3.5
3
1.103
1.546
Length of inter
node(cm)
1.4
1.2
0.162
0.228
Shoot tip
Node
Fig. (1) Shoot growth from the different explants {A(Shoot
tip) and B(Node)}.
culture response of Stevia rebaudiana Bertoni using
four explants (node segments, inter node, leaf and
shoot tip) and using MS medium supplemented with
five different concentration of growth regulators to
select the best explant which produced embryogenic
calli and then select the best medium which produced
the high number of shoots from the four regeneration
media.
1.3. Callus induction.
Four explants and MS medium with five
different concentrations of growth regulators were
used for callus induction. Table (8) shows the callus
induction for different explants (node, inter node,
leaves. Shoot tips) on different media and supported
with figure (4).
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Abd El. Hamid Ali et al.
4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
Table (3): The mean performance values for five callus induction media and four explants.
Shooting under
callus media
Callusing
Explant
type
Node
Inter node
Leaf
Shoot tips
Callus
induction%
Callus
fresh
weight
(gm)/
explant
Indicator
to callus
quantity
MS1
100
0.1950
+++
MS2
100
0.1455
++
MS3
100
0.1079
++
MS4
100
0.2940
+++
MS5
100
0.1512
++
MS1
62.5
0.0301
+
MS2
75
0.0918
+
MS3
100
0.0128
+
MS4
75
0.1385
++
MS5
50
0.0608
+
MS1
100
0.4325
+++
MS2
100
0.1695
++
MS3
100
0.1858
++
MS4
100
0.4099
+++
MS5
100
0.1259
++
MS1
100
0.3485
+++
MS2
100
0.2116
+++
MS3
100
0.1735
++
MS4
100
0.2458
MS5
100
0.1442
Media
+, Poor callus; ++, medium callus; and +++, profuse callus.
Callus color
Yellow tends
to green
Yellow tends
to green
Mainly
brownish
Yellow
No of shoots/callus
0
0
0
2.3
Yellow
Yellow tends
to green
Yellow tends
to green
Mainly
brownish
Yellow
0
Yellow
Yellow tends
to green
Yellow tends
to green
Yellow tends
to green
Yellow
0
0
+++
Yellow
Yellow tends
to green
Yellow tends
to green
Yellow tends
to green
Yellow
++
Yellow
0
0
0
0
0
0
0
0
0
0
0
0
3
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Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
The data showed that the node segments, leaf
and shoot tip explants were produced high callus induction percent (100%) for all five media.
For callus fresh weight, MS4 showed the high
value (0.2940) followed by MS1 (0.1950), while MS5
showed the lowest value (0.1512) with the node explant.
Regarding the quantity indicator of callus and its
color, MS1, MS2 and MS4 were the best media for
both traits.
The lowest of callus induction percent and callus
fresh weight and its quantity indicator were achieved
by inter node explant.
Regarding callus fresh weight, MS1 and MS4 for
both two explants (leaf and shoot tip) were close to
(A)
(B)
achieve high weight [(0.4325, 0.3485 gm, respectivily) and (0.4099, 0.2458 gm, respectively)].
Regarding the number of shoots per callus regenerated on callus media, the results illustrated that
there were no response to regenerate any shoots under callus induction media except MS4 medium with
node and shoot tip as explants gave good response
(2.3 shoots).
The obtained data indicated that MS4 (callus induction medium) was the best medium for callus induction (100%) and fresh weight. Similar results are
obtained by Moktaduzzaman and Rahman(2009)
found that 1.5 mg L-1 NAA with 1.0 mg L-1 BA (MS4)
was the best for callus induction (91.67%) which
also produced highest fresh weight.
(C)
(D)
MS1
MS2
MS3
MS4
MS5
Fig. (2) Callus growth under different callus induction media using four explants { A(Inter node ), B(shoot tip), C(leaf),
and D(node) }.
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Abd El. Hamid Ali et al.
4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
1.4. Regeneration media.
MS medium with four different combinations of
growth regulator were used for shoot formation. All
calli which produced from callus induction media were
transplanted in different combinations of BA and NAA
to evaluate the number of shoots per callus and the average length of shoots (Table 9).
Table (4): Effect of different hormones combination on shoots number and length of Stevia rebaudiana.
Callus induction
media
Regeneration media
(BA+NAA) mgl-1
No. of total shoot/
culture
Average length of
shoots/culture(cm)
1.0+0.500
0.00
0.00
MS4
1.5+0.100
1.8+0.120
2.0+0.134
1.0+0.500
1.5+0.100
1.8+0.120
2.0+0.134
1.0+0.500
1.5+0.100
1.8+0.120
2.0+0.134
1.8+0.120
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
7.5
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
4.60
MS5
1.8+0.120
0.00
0.00
MS1
MS2
MS3
The data in table (9) revealed that there were
no response to regenerate any shoots from callus source which produced from each of MS1,
MS2, MS3 and MS5 (callus induction media) under
1.0+0.500, 1.5+0.100, 1.8+0.120 and 2.0+0.134 mg
L-1 BA+NAA ( regeneration media). In contrary,
only calli obtained from MS4 callus induction medium gave the best response to regenerate a sufficient
number of shoots on the medium which consisted of
1.8+0.120 mg L-1 BA+NAA. So, MS4 callus induction medium was recommended to be used as a good
callus induction source for plantlet production on
MS supplemented with 1.8+0.120 mg L-1 BA+NAA
as regeneration medium with two type of explants
(node and shoot tip) which gave the best results.
Similar results are obtained by Moktaduzzaman
and Rahman (2009) they found that the highest
number of shoots and the highest average length of
the shoot per culture was observed at 1.8 mg L-1 of
BA with 0.12 mg L-1 of NAA. In addition, direct
plantlet regeneration was also induced from leaf explants on modified MS medium supplemented with
1.0 mg L-1 BA and 0.1 mg L-1 NAA (Smitha et al.,
2005).
1.5. Root formation.
The mean percentage of root formation, number
of roots per shoot and roots length are listed in table
(10). The data showed that the concentration 1.5mg
L-1 IBA gave the best values (40.00, 11.87 and 2.00
respectively) for these root traits with half MS medium. Results under disscusion are in line with Smitha
et al. (2005) who noticed that successful rooting
(90%) of Stevia rebaudiana was recorded in modified MS medium supplemented with 1.5 mgl-1 IBA.
The roots appeared normal and healthy.
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Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
Table (5): The effect of different concentration of IBA on percentage of root formation, No of roots and roots
length.
Traits
Root
formation%
Half
MS
Full
MS
IBA mg L-1
0
1.5
00.00
40.00
00.00
13.33
Mean
20
6.66
MS strenght
Mean
No of roots
Mean
Half
MS
Full
MS
00.00
26.65
0.00
11.87
0.00
8.50
13.3
5.9
4.25
As mentioned before, the obtained data revealed
that half MS medium with 1.5 IBA mg L-1 gave the
highest number of roots and root formation compared
with full MS medium, these results are conflict with
Mubarak (2009) who mentioned that the maximum
number of roots was achieved by the interaction between 1 mg L-1 of IBA and full MS medium while,
the half MS medium did not give any response for
root formation in stevia.
2. In vitro selection for salinity tolerance using
gamma irradiated plants.
To realize this experiment both of callus induc-
Roots length
(cm)
Mean
Half
MS
Full MS
0.00
6.20
0.00
2.00
0.00
2.00
0.00
2.00
3.1
1
1
1
tion and regeneration media under salinity stress and
gamma rays were used.
2.1. Callus induction.
The data showed that there were highly significant differences between the gamma ray doses for
callus induction and shoot formation on callus induction medium.
Data presented in table (6) showed the mean
percentage of callus induction and shoot formation
traits for the four gamma ray doses and two explants
(node and shoot tip).
Table (6): Mean percentage of callus induction and shoot formation for four gamma irradiation doses and two
explants (node and shoot tip).
Type of explant
No. of
explants
Callus induction%
Shoot formation%
Node
Shoot tip
Node
Shoot tip
Node
Shoot tip
Node
Shoot tip
Node
Shoot tip
5
5
5
5
5
5
5
5
5
5
100
100
100
100
67.5
95
35
35
75.6
82.5
95
100
90
95
40
70
25
35
62.5
75
Mean
79
68.75
LSD 0.05
0.01
0.073
0.102
0.133
0.186
Dose (Rad)
0
750
1500
2250
Average
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Abd El. Hamid Ali et al.
4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
The data illusrated that the mean percentage of callus
induction and shoot formation decreased gradually with
the increase of gamma ray doses.
Regarding the used explants, the results indicated that
the shoot tip explant gave the highest value (82.5%) for
callus induction as compared with node explant (75.6).
In addition, shoot tip explant achieved high percentage
(75%) for regeneration trait meanwhile, node explant gave
low percentage (62.5%).
0 Rad
1500 Rad
750 Rad
2250 Rad
Fig. (3) Callus induction for four gamma ray doses and two explants { A(Node) and B(Shoot tip)}.
Table (7): Mean number of shoots per ECS for each of the four gamma ray doses under four salinity concentrations.
Doses (rad)
Salinity (ppm)
4×Callus 3*
No. of total shoots
0
0
2000
4000
6000
12
12
12
12
0
2000
4000
6000
12
12
12
12
0
2000
4000
6000
12
12
12
12
95
66
34
31
56.5
34
20
22
13
22.25
30
16
19
6
17.75
No of shoots per
ECS
7.9
5.5
2.83
2.58
4.7
2.8
1.66
1.8
1.1
1.8
2.5
1.3
1.6
0.5
1.5
0
2000
4000
6000
12
12
12
12
27
10
8
6
2.25
0.83
0.66
0.5
12.75
1.1
3.854
5.403
Mean
750
Mean
1500
Mean
2250
Mean
LSD 0.05
0.01
* four calli in three replicats
Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
The results in table (14) showed that the highest
number of shoots were observed for 0.0 rad gamma
rays dose (control) under all salinity concentrations
with the mean value (4.7) and the number of shoots
decreased with the increase of gamma ray doses.
These responses were supported with figure (6).
And the number of shoot decreased by increasing the
salinity concentration.
Concerning the effect of gamma rays and salinity together, there were more sever effects on the
( 105 )
number of shoots compared with the effect of salinity or gamma rays alone.
As mentioned before, the obtained data showed
that 0.0 ppm recorded the highest number of shoots
per ECS with the mean value (7.9) for 0.0 rad, (2.8)
for 750.0 rad, (2.5) for 1500.0 rad and (2.25) for
2250.0 rad. While, 6000 ppm showed the lowest
number of shoots with 1500.0 and 2250.0 rad (0.5).
Similar findings were obtained by ( Epstein et al.
1980; Francois et al. 1986 and Sharp et al. 1990)
0 Rad
750 Rad
1500 Rad
2250 Rad
Fig. (4) Number of shoots per explant callus source for four gamma ray doses and four salinity concentrations { A(0
ppm), B(2000 ppm), C(4000 ppm), and D(6000 ppm)}.
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4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
Generally, production of stevia plants under salinity stress of the concentrations of 4000 and 6000
ppm is considered a big gain for stevia breeders and
the obtained results gave enough amount of salinity tolerant stevia plants which could be increased
through micropropagation and adapted them in the
North Delta or in the both new reclamation and
Northern Cost areas.
4.5. Effect of gamma rays on the growth traits of
micropropagated plants.
Abd El. Hamid Ali et al.
cant differences between doses for all studied characters. According to type of explant, plant length
trait showed significant differences only. On the
other hand, number of leaves, number of shoots,
length of inter node and responded explants traits
showed no significance. Regarding the interaction replicates×doses and replicates×explant, they
both showed no significance for all studied traits.
Meanwhile, the interaction between doses×explant
showed significant differences only for number of
leaves trait.
The data showed that there were highly signifiTable (8): Mean performances of plant length, no. of leaves, no. of shoots, length of inter node and responded
explants for the four gamma ray doses and two explants.
Doses
(rad)
Explants
O
Shoot tip
Node
Mean
750
Shoot tip
Node
Mean
1500
Mean
2250
Shoot tip
Node
Shoot tip
Node
Mean
Mean
Shoot tip
Node
Plant
length
(cm)
3.5
5.4
4.45
5
6.1
5.55
1.4
1.78
1.59
1.8
1.9
1.85
2.9
3.8
No. of
shoots
No. of
leaves
3
2.9
2.95
3.1
3.2
3.15
1.6
1.9
1.75
1.9
2.1
2
2.4
2.5
48.5
50
49.25
68.3
55
61.65
18.75
26.9
22.82
18.5
19
18.75
38.5
37.7
The results in table (8) revealed that 750.0 rad
was activation dose and gave the highest values for
all characters in both explants. While, the other two
doses (1500.0 and 2250.0 rad ) gave very close results for all traits in both explants, except the length
of inter node trait which gave more response (1 cm)
under 2250.0 rad compared with 1500.0 rad in both
Length of
Responded
inter node
explants
(cm)
1.1
5
1.4
5
1.25
5
1.6
5
1.8
5
1.7
5
0.5
4.7
0.3
4.7
0.4
4.7
1
4.3
1
3.3
1
3.8
1
4.8
1.1
4.5
Average
Mean
12.22
12.94
12.58
16.6
14.22
15.41
5.39
7.11
6.25
5.5
5.46
5.48
9.92
9.92
9.92
shoot tip and node explants (0.5 and 0.3 cm).
The obtained data revealed that, there were
highly significant differences between gamma ray
doses. Similar results were obtained by Mathius et
al. (1995) who found that there were significant differences between the clones and between irradiation
doses within each clone.
Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses
(A)
(B)
( 107 )
2. Ali, A. A. (1998). Genetic evaluation of somaclonal
variants of egyption garlic (Allium sativum L.). J. Agric Sci. Mansoura Univ., 23 (5): 1929-1937.
0 Rad
3. Badria, F.A. and A. Ali (1999) Chemical and Genetic Evaluation of Somaclonal Variants of Egyption
Garlic (Allium sativum L.). Journal of medicinal food.
2(2).
4. Bhosle, S. (2004) Commercial cultivation of Stevia
rebaudiana, Agrobios Newsletter. 3 (2): 43-45.
750 Rad
1500 Rad
5. Chalapathi, M. V. and S. Thimmegowda, (1997).
Natural non-calorie sweetener Stevia ( Stevia rebaudiana Bertoni ) : Afuture Crop of India. Crop Research Hisar. 14, 347-350.
6. Epstein, E.; J.D. Norlyn; D.W. Kingsobury; D.B.
Kelley; G.A. Cunniningham and A.F. Wrona
(1980). Salline culture crops and genetic approach.
Science, 210: 399-404.
7. Evans, N.; D. Foulger; L. Farrer and S. Bright
(1986). Somaclonal variation in explant
2250 Rad
Fig. (5) Some different traits for four gamma ray doses
and the two used explants { A(Node) and B(Shoot tip)}.
It could be concluded that, through the previous experiments, stevia plants have been produced
in sufficient quantity through tissue culture (somaclonal variants) or from inducing mutations using
gamma irradiation or obtained from both techniques.
So, these lines need further testing through cytological, physiological, biochemical and molecular studies to know the behavior of these plants (new strains)
for their tolerance to various environmental stresses
(biotic or abiotic stresses) and select the best strains
in stevia breeding programms.
REFERENCES
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S.L. Schultz, (1990). Somaclonal Variation in riceSubmergence tolerance and other agronomic characters. Physiol. Plant. 80: 647-645.
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quality, vegetive growth and germination of simedourf and durum wheat. Agron. J., 78: 1053-1058.
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Ruas; J.N. Nakajima (1996). Chromosome studies in some Stevia. Cav. (Compositae) species from
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Preto 1996.
10. Katayma, O.; T. Sumida; H. Hayashi and H. Mitsuhashi, (1976). The Practical pplication of Stevia
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Japan. P. 747.
11. Kuntal, D.; D. Raman; S. Khanam; B.G. Shivananda,; P.E. Ajasekharan (2005). In vitro methods
for production of stevioside from Stevia rebaudiana.
Indian-J. Of Natural Products. 2005; 21(1): 14-15.
12. Lee, M. and R.L. Phillips (1988). The chromosomal
basis of somclonal variation. Annv. Rev. Plant Physiol. Plant Mol. Biol. 39: 413-437.
13. Liu, J. and S.F.Y. Li (1995). Separation and determination of Stevia sweeteners by capillary electrophoresis and high performance liquid chromatography. J.
Liquid chromatography. 18, 1703-1719.
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14. Manjula, S. M.; M.S. Kuruvinashetti, and Chandrashekhar, C. H. (2000). Regeneration, establishment and evolution of somaclones in Sorghum bicolor (L.) Moench. Euphytica, 115 (3): 173-180.
15. Mathius, T.N.; T. Pratiwi and T. Hutabarat (1995).
Somaclonal variations in Stevia rebaudiana Bertoni
irradiated with Co-60 gamma rays. Menara-Perkebunan,63(2): 33-42.(En.Abst.).
16. Moktaduzzaman, M.D.; S.M.M. Rahman (2009).
Regeneration of Stevia rebaudiana and analysis of somaclonal variation by RAPD. Biotechnology, 2009.
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17. Mubarak, M.H.; A.H Belal; I.H. EL Geddawy;
E.I. EL Sarag and M.I Nasr (2008). Micropropagation of Stevia rebaudiana in vitro. Meeting the Challenges of Sugar Crops and Integrated Industries in
Developing Countries, Al Arish, Egypt, 293-298.
18. Mubarak, M.H. (2009). Studies on Stevia Plants
Stevia rebaudiana Using Tissue Culture Technique.
M.Sc thesis, Plant Production Dep., Faculty of Environmental Agriculture Sciences.
19. Murashige, T. and F. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol., Plant., 15: 473-497.
20. Patil, V.; K.S. Ashwini; P.C. Reddy; M.G. Purushotham; T.G. Prasad and M. Udayakumar (1996).
In vitro multiplication of Stevia rebaudiana. Current
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Physiol., Univ. Agric. Sci., GKVK Campus, Bangalore 560 065, India. http://www.newcrops.uq.edu.au/
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Growth of the maize primary root at low water potentials. Plant physiology, 93, 1337-1346.
22. Smitha, P.S.; P.A. Nazeem; J. Thomas; R. Keshavachandran and D.Girija (2005). Micropropagation for mass multiplication of the important medicinal sweet herb - Stevia rebaudiana. J. of-Medicinaland-Aromatic-Plant-Sciences. 27(2): 247-252.
23. Soejarto, D.D.; A.D. Kinghorn,; N.R. Fransworth,
(1982). Potential sweetening agents of plant origin. J.
Nat. Prod. 45:590-599.
24. Toyoda, K. and H. Matsui (1997). Assessment of
the carcinogenicity of stevioside in F344 rats. Food
Chem. Toxicol., 35 (6): 597-603.
25. Van den B.R.W.; H.J.M. Loffer; W.H. Lindhout
And M. Koorneef (1990). Somaclonal variation
in tomato: Effect of explant source and comparison
chemical mutagensis. Theoretical and applied Genetics, 80 (6): 817-825.
26. Yamazaki, T.; H. E. Flores; K. Shimomura and K.
Yoshihira (1991). Examination of steviol glucosides
production by hairy root and shoot cultures of Stevia
rebaudiana. J. Natural Products, 54 (4): 986-992.
27. Yoshida, S. (1986). Studies on the production of
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‫‪Biotechnological Studies on Gamma Irradiated Stevia (Stevia Rebaudiana) Plant Under Abiotic Stresses‬‬
‫‪ 17 - 13‬أكتوبر ‪ - 2014‬طابا ‪ -‬مصر‬
‫ص ‪119 : 105‬‬
‫دراسات بيوتكنولوجية ووراثية على نبات االستيفيا املعامل بأشعة جاما‬
‫عبد احلميد عبد احلميد علي‪ – 1‬علي أمحد أبو شوشه‪ – 1‬حممد قاسم حممد قاسم‪ – 2‬أميان إبراهيم حممود الضبعاوي‬
‫‪1‬‬
‫أوضحت النتائج أن متوسط نسبة إنتاج الكالس ومعدل إنتاج اجملاميع اخلضرية يقل تدرجييا‬
‫بزيادة اجلرعات اإلشعاعية ‪ .‬بالنسبة إلنتاج الكالس فإن اجلرعات اإلشعاعية (صفر و ‪ 750‬راد ) أعطوا‬
‫أعلى قيمة مع البادئني النباتيني املستخدمني (‪ .)%100‬يتبعهم اجلرعة اإلشعاعية ‪ 1500‬راد مع القمة‬
‫النامية كبادئ نباتي (‪ .)%95‬بينما اجلرعة اإلشعاعية ‪ 2250‬راد أعطت أقل قيمة (‪ )%35‬مع البادئني‬
‫النباتيني املستخدمني‪ .‬بالنسبة إلنتاج اجملاميع اخلضرية على الكالس املتكون من كل بادىء فان عدد‬
‫قليل من النبيتات مت تكوينه على بيئة إنتاج الكالس يرتاوح من (صفر‪ 2-‬نبتة) ‪ .‬كذلك فإن القمة‬
‫النامية كبادىء أعطت أعلى قيمة ‪ % 82.5‬إلنتاج الكالس مقارنة بالعقدة كبادئ نباتي حيث أعطت‬
‫قيمة ‪ .%75.6‬كما أن القمة النامية أعطت أعلى نسبة لتكوين اجملاميع اخلضرية على الكالس املتكون‬
‫(‪ (%75‬بينما أعطت العقدة أقل نسبة (‪ )%62.5‬من عدد البوادىء املستعملة‪ .‬أوضح حتليل التباين انه توجد‬
‫اختالفات عالية املعنوية بالنسبة لعدد اجملاميع اخلضرية بني اجلرعات اإلشعاعية وبني تركيزات‬
‫امللوحة وبني التفاعل بينهما ‪.‬‬
‫أوضحت النتائج أن أعلى عدد من اجملاميع اخلضرية مت مالحظته يف الكنرتول‪ .‬مبتوسط قيمة ‪4.7‬‬
‫وأن عدد اجملاميع اخلضرية يقل تدرجييا مع زيادة اجلرعات اإلشعاعية ‪.‬‬
‫بالنسبة لرتكيزات امللوحة ‪ ,‬فان صفر جزء يف املليون أعطى أعلى عدد من اجملاميع اخلضرية ‪7.9‬‬
‫مع الكنرتول (‪ .)rad 0‬بينما بالرغم من أن اجهاد امللوحة بالرتكيز ‪ 6000‬جزء يف املليون أعطى أقل عدد‬
‫من اجملاميع اخلضرية مع اجلرعات اإلشعاعية ‪ 1500‬و ‪ 2250‬راد (‪ )0.5‬إال أن هذا االجهاد امللحى (‪ 6000‬جزء‬
‫فى املليون) سجل قيمة مميزة حتت جرعة صفر اشعاع لتكوين ‪ 31‬نبات متحمل للملوحة بنسبة ‪2.58‬‬
‫‪ %‬أيضا أعطى ‪ 34 ,66‬نبات متحمل للملوحة لكل من الرتكيزين ‪ 4000 , 2000‬جزء فى املليون على الرتتيب‪.‬‬
‫يتضح من النتائج ان تأثري أشعة جاما وامللوحة معا أدى إىل تقليل عدد اجملاميع اخلضرية بشكل‬
‫قاسى وهذا باملقارنة بتأثري اإلشعاع فقط أو امللوحة فقط ‪.‬‬
‫‪.1‬‬
‫‪.2‬‬
‫‪1‬قسم الوراثة – كليه الزراعة – جامعة كفر الشيخ – مصر‬
‫‪2‬قسم البحوث النباتية – مركز البحوث النووية – هيئة الطاقة الذرية ‪ -‬مصر‬
( 110 )
4th Int. Con. Rad. Res. Appl. Sci., Taba, Egypt (2014)
Abd El. Hamid Ali et al.