Toleransi kacang tanah dan tembakau terhadap stres kekeringan dengan over ekspresi gen P5CS- penyandi ensim kunci biosintesis prolina
@ Hak cipta milik IPB (Institut Pertanian Bogor]
Bogor
riculturaI Universi
Hak Cicita Dilindungi
- Undang-Undang
1. Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan dan menyebutkan sumber:
a. Pengutipanhanya untuk kepentinganpendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik atau tinjauan suatu masalah.
b. Pengutipantidak merugikan kepentingan yang wajar IPB.
2 Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis ini dalam bentuk apapun tanpa izin IPB.
SUMMARY
and Tobacco against Drought Stress by
y Enzyme for Proline Biosynthesis.
INNOOR, SUDARSONO, JAJAH
This dissertation was written based on results of experiments directed
rought tolerance among peanut cultivars grown in
sia and determination of the tolerance mechanisms. Subsequently, genetic
ring was employed to develop drought tolerance transgekc plants (peanut
co) through over-expression of PSCS gene encoding the key enzyme for
s
-
X-. Ps
2
-.o 3
la
g
ance in plants was a dynamic character, therefore its
conducted at various growth stages, such as: vegetative and
uation of drought tolerance in plants also needs to be
ontrolled environment, such as in the glass house. Field
cter is hampered by the difficulty of getting homogeneous
and of predicting effects of weather changes (especially
ght stress evaluation may be conducted (i) by reducing
ly in pot experiment or (ii) by drenching with various
ion in hydr%ponic system.
sgenic tobacco fa model plant) and peanut (a
sing PSCS transgene need to be conducted in order
o achieve this objective, genetic transformation
introduce PSCS transgene into tobacco genome
genic plants need to be conducted. In this
of tobacco resulted in five putative TO PSCS
hili (GS-I, GS-2, GS-3, GS-4 and GS-5). The
each of the TO plants. The TO:1 seeds were
germinated and the TI seedlings were used to identify that the TO plarits were
transgenic plants, (ii) to evaluate effect of PSCS transgene expression on growth
and biomass yield of tobacco under non-stress conditions, (iii) to determine
drought stress and (iv) PEG induced stress effects on growth, biorrlass yield and
tolerance against drought stress.
I]O Genetic transformation of peanut resulted in four putative TO PSCS
genic peanut cv. Singa (S-1, S-2, S-3, and S-4). The TO:1 seeds were
from TO plants and they were grown in the glass house to produce T1:2
plants gown from Tl:2 seeds were used (i) to identify that the TO piants
w* transgenic plants and (ii) to evaluate drought stress efiects on growth, peanut
and tolerance of the T2 plants against drought stress.
Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress
at egetative Growth Stage, Leaf Proline and Total Sugar Content was presented
i n p p t e r III. The objectives of this experiment were to determine ihe effects of
dreught stress initiated at vegetative growth skge on yield of peanut, to evaluate
toekuw of six peanut cultivars against drought strcss, and to determine their leaf
p r e e and total sugar content. One group of peanut plants were grown in plastic
and subjected to stress condition during the period of 15 - 47 days (Exp. I) or
ym
8
-.
<
CD
4
I .
u"
9g F;g ;gQ$0
15 - 80 days ( ~ x pa)
. by watering them every 4 - 7 days. They were harvested at
z ~ g z75 days ( ~ ~ 1)por. at maturity (Exp. 11). The other group was grown optimally in
**3 1
3 3
z X.8
.
plastic pot up to hmesting period. Leaf proline content was determined at 15
g , (before stress), 32 (after three periods of stress), and 47 (after six periods of stress)
~ 3 g a . in
a Exp. I and only at 64 days after planting in Exp. 11. The results indicated
,z
8
3 s 8 g drought stress initiated at vegetative growth stage reduced plant hei*,
branches
number,
shoot
and
root
dry
weight,
and
peanut
yield.
Based
on
sensitivity
index
3"5 $ 3 5 calculated using seed yields per plant indicated that peanut cv. Jerapah and Singa
= Z:
$sxeS.
edium tolerance while Gajah, Keiinci, Macan, and Simpai were sensitive
&ought stress initiated at vegetative stage. Higher leaf proline content
B D 5 9 wdfr drou&t stress was observed in peanct cv. Jerapah and Singa, while less
XT'?
mcngased was observed in peanut cv. Gajah, Kelinci, Macan and Simpai. Total
g; 2 2 c
3
leaQsugar content of peanut cv. Jerapah, Gajah, Macan and Simpai decreased
g g c
acn Z E 2
a
P un&r drought stress while that of Singa and Kelinci were the same as that under
%?.€,3-.
Q &
, a.
opt&al condition. The ability to increase leaf proline content and maintiin total
a
lea6sugar
content under drought stress could be used as indicators of drought
8a
S
g 9 3 rol&nce in peanut cuttivan.
= a,-,
Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress
2r
3 Q
at Gnerative Growth Stage, Leaf Proline and Total Sugar Content was presented
r s g
Q
In &apter N.The objectives of t h s experiment were to determine the effects of
93
dr&&t
stress at generative growth stage on growth and yield of peanut, to
$
"'
-. % $ evduate tolerance bf six peanut cultivars against drought stress, and to determine
. =3
the& leaf proline and total sugar content. One group of peanut plants were gown
3
78
in #astic pots and subjected to stress condition during the period of 32 - 64 days
Y & (Ev.
I) or 32 - 80 days (Exp. fI) by watering them every 4 - 7 days. They were
$ Z 5 ha?tested
at 75 days (Exp. I) or at maturity (Exp. 11). The other group of peanut
2g
*r
Q
E
plants was watered normally up to harvest period and was used as control. Lesrf
proline content was determined at 32, 47, and 64 days in Exp. I and only at 64
.u
EW
days after planting in Exp. IL The results indicated drought stress at generative
sX5,+ ?Ya
aowth stage reduced plant height, shoot dry weight, and peanut yield. Based on
-. s
sensitivity index calculated using seed yields per plant-indicatedthe tested peanut
2 -.
cultivars
were either medium tolerance or sensitive against drought stress at
5 3
m
generative growth stage. Increased in leaf proline content and absence of
* g
Fedduction
of tots1 leaf sugar content may be uscd to inhcate tolerancc of peanut
E
4
c a v a r s against drought stress at generative growth stage.
E
Q
O Experiment entitled: Regeneration of Transgenic Tobacco Carrying P5CS
-.Qg
@nsgene and Analysis of its Expression was presented in chapter V. The
Q
s
o&ctives of this experiment were (1) to regenerate transgenic tobacco cv.
3
E
&be1 Shili (GS) carrying chimera P5CS transgene through AgrbacteriMI4
s
e i a t e d transformation, (2) to analyze integration of chimera PSCS transgene in
8
%genome of transgenic GS tobacco by total nucleic acid PCR,(3) to estimate
8
h
tlE number of functional nptU loci integrated in the genome of transgenic GS
=
t@wo by progeny analysis, and (4) to analyze proline accumulation in leaf
ues of the transgenic GS tobacco. Results of the experiment indicated although
of success was only 3.7%, the Agrobncterium-mediated genetics
was able to introduce nptn md chimera PSCS transgenes into GS
-formation
tiitkicco genome and frve putative kanamycin resistance transgenic GS tobacco
~ z regenerated.
e
Positive results of total nucleic mid PCR analysis in populahon
,
a
s33'zr
8$ f G
E
Q
Q
Q
=
2.
''
=
CD
-
3
-.
u^
*
b'
-=
-.
"
2.
$ 3
2.8
-
,,
Q
iJ 2
a
I 78
P ~5
$r g y
2
'X c!Ig'
s
ss
=
X-. Ys
$ &
-.o 3
g
E
8
E
4
a
.
n
E
9
E
5
2lii
F
=
E
Glycol (PEG) was presented in chapter
were (i) to determine the effects of stress
treatment the period of 15 - 60 days after
plants derived from transgenic GS tobacco
T1 plants derived from five TO transgenic GS
stress, and (iii) to determine their leaf proline
under PEG induced stress conditions and their
correlation to stress responses the T1 seedlings derived from five TO transgenic
were grown individually in plastic pot (500 ml) containing a
re of rice hull: coco pit medium (1:l). After 15 DAP, seedlings were
to stress conditions by drenching them with solution of PEG 6000 at
10% concentration for the period of 15 - 60 DAP, the plants were
61 DAP. Leaf samples for proline content analysis were collected at
indicated stress due to PEG treatment (5% or
number, total leaf width, leaf, shoot, biomass, and
plants. Stress sensitivity index calculated using leaf
grouped the TI plants derived fiom PSCS transgenic GS
medium tolerance and sensitive against PEG induced stress
GS tobacco was medium tolerance. T~FTT
$ants
tobacco showed better growth and higher plantdry weight compared to tobacco nontr~nsgenicat
s p s s and non-stress condition. Higher increased in leaf proline content after
q u g h t stress was observed in all tobacco transgenic, while less increased was
okerved in tobacco non-transgenic. Increased leaf proline content due to by overe8ression of P5CS gene under 5% or 10% PEG induced stress correlated with
Wught
- tolerance phenotype in T1 plants derived from transgenic GS tobacco.
Experiment entitled: Tolerance of Transgenic Peanut Expressing P5CS
Gene against Drought Stress was presented in chapter IX. The objectives of this
experiments were (i) to determine efEects of drought stress during the period
vegetative growth stage up to pod filling on growth and yield of T2 plants derived
from TQ generation of transgenic peanut cv. Singa canying PSCS transgenes, (ii)
to evaluate the tolerance of threety two families of T2 plants derived from TO
generation of transgenic peanut against drought stress, and (iil) to evaluate their
leaf proline content. Peanut plants were grown in plastic pots and subjected to
stress condition during the period of 15 - 80 days after planting @AP) by
ing water only every 4 - 7 days. The plants were harvested at maturity. A
er of peanut plants were supplied with water up to field capacity from the
of the experiment up to maturity, and t h s population was used as control.
prolina content were determined from leaf samples harvested at 55 DAP
6x stress period). The results of the experiment indicated the T2 peanut
ts were transgenic plants carrying PSCS transgene since it accumulated more
m l i n e contents in their leaf as compared to non-transgenic plants under nonm s s condition. However, all peanut plants accumulated similar amount of Ieaf
S l i n e under drought stress condition. Drought stress conchtiom reduced plant && shoot dry weight, root length and dry weight, and yield per plant ba;is on
a&xaniltgenotypes tested Based on sensitivity index calculated using parameter
@ e e d dry weight, non-transgenic peanut cv. Singa was grouped into sensitive
a i l e T2 plmts defived fiom transgenic peanut cv. Singa were grouped into
7'
0
2
-.
u^
*
P
$;g;g$
5:~:
a s s
b'
gap
AHMAn RIDli4N. Toleransi Kacang Tanah dm Tembakau terhadap Stres
8 gf" 8 5 Kekefingan
den,
Over-Ekspresi Gen PSCS - Penyandi Ensim Kunci
0
S...SSQ
2 k9 3
Biosintesis Prolina. Komisi Pembimbing : EIAJRIAL ASWIDINNOOR,
5 f g SUDARSONO, J.4JAH KOSWARA, dan DJOKO SANTOSO.
QcPa3 Q
Q
FS.,
= G ID a.
rr
2s !JJ...X
s1 9
'
93
b
2 Js
ii'
-
g
5
a
kg
=-.
$ 3
2.
a
P
5a-
2.8
F<
Bogor
riculturaI Universi
Hak Cicita Dilindungi
- Undang-Undang
1. Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan dan menyebutkan sumber:
a. Pengutipanhanya untuk kepentinganpendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik atau tinjauan suatu masalah.
b. Pengutipantidak merugikan kepentingan yang wajar IPB.
2 Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis ini dalam bentuk apapun tanpa izin IPB.
SUMMARY
and Tobacco against Drought Stress by
y Enzyme for Proline Biosynthesis.
INNOOR, SUDARSONO, JAJAH
This dissertation was written based on results of experiments directed
rought tolerance among peanut cultivars grown in
sia and determination of the tolerance mechanisms. Subsequently, genetic
ring was employed to develop drought tolerance transgekc plants (peanut
co) through over-expression of PSCS gene encoding the key enzyme for
s
-
X-. Ps
2
-.o 3
la
g
ance in plants was a dynamic character, therefore its
conducted at various growth stages, such as: vegetative and
uation of drought tolerance in plants also needs to be
ontrolled environment, such as in the glass house. Field
cter is hampered by the difficulty of getting homogeneous
and of predicting effects of weather changes (especially
ght stress evaluation may be conducted (i) by reducing
ly in pot experiment or (ii) by drenching with various
ion in hydr%ponic system.
sgenic tobacco fa model plant) and peanut (a
sing PSCS transgene need to be conducted in order
o achieve this objective, genetic transformation
introduce PSCS transgene into tobacco genome
genic plants need to be conducted. In this
of tobacco resulted in five putative TO PSCS
hili (GS-I, GS-2, GS-3, GS-4 and GS-5). The
each of the TO plants. The TO:1 seeds were
germinated and the TI seedlings were used to identify that the TO plarits were
transgenic plants, (ii) to evaluate effect of PSCS transgene expression on growth
and biomass yield of tobacco under non-stress conditions, (iii) to determine
drought stress and (iv) PEG induced stress effects on growth, biorrlass yield and
tolerance against drought stress.
I]O Genetic transformation of peanut resulted in four putative TO PSCS
genic peanut cv. Singa (S-1, S-2, S-3, and S-4). The TO:1 seeds were
from TO plants and they were grown in the glass house to produce T1:2
plants gown from Tl:2 seeds were used (i) to identify that the TO piants
w* transgenic plants and (ii) to evaluate drought stress efiects on growth, peanut
and tolerance of the T2 plants against drought stress.
Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress
at egetative Growth Stage, Leaf Proline and Total Sugar Content was presented
i n p p t e r III. The objectives of this experiment were to determine ihe effects of
dreught stress initiated at vegetative growth skge on yield of peanut, to evaluate
toekuw of six peanut cultivars against drought strcss, and to determine their leaf
p r e e and total sugar content. One group of peanut plants were grown in plastic
and subjected to stress condition during the period of 15 - 47 days (Exp. I) or
ym
8
-.
<
CD
4
I .
u"
9g F;g ;gQ$0
15 - 80 days ( ~ x pa)
. by watering them every 4 - 7 days. They were harvested at
z ~ g z75 days ( ~ ~ 1)por. at maturity (Exp. 11). The other group was grown optimally in
**3 1
3 3
z X.8
.
plastic pot up to hmesting period. Leaf proline content was determined at 15
g , (before stress), 32 (after three periods of stress), and 47 (after six periods of stress)
~ 3 g a . in
a Exp. I and only at 64 days after planting in Exp. 11. The results indicated
,z
8
3 s 8 g drought stress initiated at vegetative growth stage reduced plant hei*,
branches
number,
shoot
and
root
dry
weight,
and
peanut
yield.
Based
on
sensitivity
index
3"5 $ 3 5 calculated using seed yields per plant indicated that peanut cv. Jerapah and Singa
= Z:
$sxeS.
edium tolerance while Gajah, Keiinci, Macan, and Simpai were sensitive
&ought stress initiated at vegetative stage. Higher leaf proline content
B D 5 9 wdfr drou&t stress was observed in peanct cv. Jerapah and Singa, while less
XT'?
mcngased was observed in peanut cv. Gajah, Kelinci, Macan and Simpai. Total
g; 2 2 c
3
leaQsugar content of peanut cv. Jerapah, Gajah, Macan and Simpai decreased
g g c
acn Z E 2
a
P un&r drought stress while that of Singa and Kelinci were the same as that under
%?.€,3-.
Q &
, a.
opt&al condition. The ability to increase leaf proline content and maintiin total
a
lea6sugar
content under drought stress could be used as indicators of drought
8a
S
g 9 3 rol&nce in peanut cuttivan.
= a,-,
Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress
2r
3 Q
at Gnerative Growth Stage, Leaf Proline and Total Sugar Content was presented
r s g
Q
In &apter N.The objectives of t h s experiment were to determine the effects of
93
dr&&t
stress at generative growth stage on growth and yield of peanut, to
$
"'
-. % $ evduate tolerance bf six peanut cultivars against drought stress, and to determine
. =3
the& leaf proline and total sugar content. One group of peanut plants were gown
3
78
in #astic pots and subjected to stress condition during the period of 32 - 64 days
Y & (Ev.
I) or 32 - 80 days (Exp. fI) by watering them every 4 - 7 days. They were
$ Z 5 ha?tested
at 75 days (Exp. I) or at maturity (Exp. 11). The other group of peanut
2g
*r
Q
E
plants was watered normally up to harvest period and was used as control. Lesrf
proline content was determined at 32, 47, and 64 days in Exp. I and only at 64
.u
EW
days after planting in Exp. IL The results indicated drought stress at generative
sX5,+ ?Ya
aowth stage reduced plant height, shoot dry weight, and peanut yield. Based on
-. s
sensitivity index calculated using seed yields per plant-indicatedthe tested peanut
2 -.
cultivars
were either medium tolerance or sensitive against drought stress at
5 3
m
generative growth stage. Increased in leaf proline content and absence of
* g
Fedduction
of tots1 leaf sugar content may be uscd to inhcate tolerancc of peanut
E
4
c a v a r s against drought stress at generative growth stage.
E
Q
O Experiment entitled: Regeneration of Transgenic Tobacco Carrying P5CS
-.Qg
@nsgene and Analysis of its Expression was presented in chapter V. The
Q
s
o&ctives of this experiment were (1) to regenerate transgenic tobacco cv.
3
E
&be1 Shili (GS) carrying chimera P5CS transgene through AgrbacteriMI4
s
e i a t e d transformation, (2) to analyze integration of chimera PSCS transgene in
8
%genome of transgenic GS tobacco by total nucleic acid PCR,(3) to estimate
8
h
tlE number of functional nptU loci integrated in the genome of transgenic GS
=
t@wo by progeny analysis, and (4) to analyze proline accumulation in leaf
ues of the transgenic GS tobacco. Results of the experiment indicated although
of success was only 3.7%, the Agrobncterium-mediated genetics
was able to introduce nptn md chimera PSCS transgenes into GS
-formation
tiitkicco genome and frve putative kanamycin resistance transgenic GS tobacco
~ z regenerated.
e
Positive results of total nucleic mid PCR analysis in populahon
,
a
s33'zr
8$ f G
E
Q
Q
Q
=
2.
''
=
CD
-
3
-.
u^
*
b'
-=
-.
"
2.
$ 3
2.8
-
,,
Q
iJ 2
a
I 78
P ~5
$r g y
2
'X c!Ig'
s
ss
=
X-. Ys
$ &
-.o 3
g
E
8
E
4
a
.
n
E
9
E
5
2lii
F
=
E
Glycol (PEG) was presented in chapter
were (i) to determine the effects of stress
treatment the period of 15 - 60 days after
plants derived from transgenic GS tobacco
T1 plants derived from five TO transgenic GS
stress, and (iii) to determine their leaf proline
under PEG induced stress conditions and their
correlation to stress responses the T1 seedlings derived from five TO transgenic
were grown individually in plastic pot (500 ml) containing a
re of rice hull: coco pit medium (1:l). After 15 DAP, seedlings were
to stress conditions by drenching them with solution of PEG 6000 at
10% concentration for the period of 15 - 60 DAP, the plants were
61 DAP. Leaf samples for proline content analysis were collected at
indicated stress due to PEG treatment (5% or
number, total leaf width, leaf, shoot, biomass, and
plants. Stress sensitivity index calculated using leaf
grouped the TI plants derived fiom PSCS transgenic GS
medium tolerance and sensitive against PEG induced stress
GS tobacco was medium tolerance. T~FTT
$ants
tobacco showed better growth and higher plantdry weight compared to tobacco nontr~nsgenicat
s p s s and non-stress condition. Higher increased in leaf proline content after
q u g h t stress was observed in all tobacco transgenic, while less increased was
okerved in tobacco non-transgenic. Increased leaf proline content due to by overe8ression of P5CS gene under 5% or 10% PEG induced stress correlated with
Wught
- tolerance phenotype in T1 plants derived from transgenic GS tobacco.
Experiment entitled: Tolerance of Transgenic Peanut Expressing P5CS
Gene against Drought Stress was presented in chapter IX. The objectives of this
experiments were (i) to determine efEects of drought stress during the period
vegetative growth stage up to pod filling on growth and yield of T2 plants derived
from TQ generation of transgenic peanut cv. Singa canying PSCS transgenes, (ii)
to evaluate the tolerance of threety two families of T2 plants derived from TO
generation of transgenic peanut against drought stress, and (iil) to evaluate their
leaf proline content. Peanut plants were grown in plastic pots and subjected to
stress condition during the period of 15 - 80 days after planting @AP) by
ing water only every 4 - 7 days. The plants were harvested at maturity. A
er of peanut plants were supplied with water up to field capacity from the
of the experiment up to maturity, and t h s population was used as control.
prolina content were determined from leaf samples harvested at 55 DAP
6x stress period). The results of the experiment indicated the T2 peanut
ts were transgenic plants carrying PSCS transgene since it accumulated more
m l i n e contents in their leaf as compared to non-transgenic plants under nonm s s condition. However, all peanut plants accumulated similar amount of Ieaf
S l i n e under drought stress condition. Drought stress conchtiom reduced plant && shoot dry weight, root length and dry weight, and yield per plant ba;is on
a&xaniltgenotypes tested Based on sensitivity index calculated using parameter
@ e e d dry weight, non-transgenic peanut cv. Singa was grouped into sensitive
a i l e T2 plmts defived fiom transgenic peanut cv. Singa were grouped into
7'
0
2
-.
u^
*
P
$;g;g$
5:~:
a s s
b'
gap
AHMAn RIDli4N. Toleransi Kacang Tanah dm Tembakau terhadap Stres
8 gf" 8 5 Kekefingan
den,
Over-Ekspresi Gen PSCS - Penyandi Ensim Kunci
0
S...SSQ
2 k9 3
Biosintesis Prolina. Komisi Pembimbing : EIAJRIAL ASWIDINNOOR,
5 f g SUDARSONO, J.4JAH KOSWARA, dan DJOKO SANTOSO.
QcPa3 Q
Q
FS.,
= G ID a.
rr
2s !JJ...X
s1 9
'
93
b
2 Js
ii'
-
g
5
a
kg
=-.
$ 3
2.
a
P
5a-
2.8
F<