Toleransi kacang tanah dan tembakau terhadap stres kekeringan dengan over ekspresi gen P5CS- penyandi ensim kunci biosintesis prolina

@ Hak cipta milik

Hak Cicita Dilindungi Undang-Undang

IPB (Institut Pertanian Bogor]

Bogor

ricultura

-

1. Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan dan menyebutkan sumber:

a. Pengutipan hanya untuk kepentingan pendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik atau tinjauan suatu masalah. b. Pengutipan tidak 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

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

s

-

germinated and the TI seedlings were used to identify that the TO plarits were

X

_-.

P

_{s transgenic plants, (ii) to evaluate effect of PSCS transgene expression on growth}

2

-

and biomass yield of tobacco under non-stress conditions, (iii) to determine

.o

3

la g 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

ym

and tolerance of the T2 plants against drought stress.

Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress at

8

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

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4

g F Q 0

9

;g ;g

$

15

-

80

days

( ~ x p .

a)

by

watering

them every 4

-

7

days.

They

were

harvested at

3 3

z ~ g z 75 days ( ~ ~ p . 1) or at maturity (Exp. 11). The other group was grown optimally in

* * 3 1

z

X.

8

a

.

plastic pot up to hmesting period. Leaf proline content was determined at 15

s 3 3 ' z r

g

,

,

(before stress), 32 (after three periods of stress), and 47 (after six periods of stress) ~ 3 g a . a

,z

8 in Exp. I and only at 64 days

after

planting in Exp. 11. The results indicated

3 s 8 g drought stress initiated at vegetative growth stage reduced plant hei*, branches

5

$

number, shoot and root dry weight, and peanut yield. Based on sensitivity index

3"

= Z: 3

5

calculated using seed yields per plant indicated that peanut cv. Jerapah and Singa $ s x e S . edium tolerance while Gajah, Keiinci, Macan, and Simpai were sensitive

8

$

f

G

E

&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

X T ' ?

Q c mcngased was observed in peanut cv. Gajah, Kelinci, Macan and Simpai. Total

g;

2 2 3

g g

c

leaQsugar content of peanut cv. Jerapah, Gajah, Macan and Simpai decreased

acn Z E

2

Q Q a P un&r drought stress while that of Singa and Kelinci were the same as that under

%?.€,3-.

Q &, a.

a opt&al condition. The ability to increase leaf proline content and maintiin total

8

a lea6sugar content under drought stress could be used as indicators of drought

S

g 9 3 rol&nce in peanut cuttivan.

=

2 a,-, Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress

r 3 Q

r

s g

Q at Gnerative Growth Stage, Leaf Proline and Total Sugar Content was presented

9 3 In &apter

N.

The objectives of t h s experiment were to determine the effects of dr&&t stress at generative growth stage on growth and yield of peanut, to

=

$

"' % $

-.

.

= 3 evduate tolerance bf six peanut cultivars against drought stress, and to determine

3

2.

the& leaf proline and total sugar content. One group of peanut plants

were

gown

7 8 in #astic pots and subjected to stress condition during the period of 32

-

64 days

Y _&

$

Z 5

(Ev.

I) or 32

-

80 days (Exp. fI) by watering them every 4

-

7 days. They were

2

g

ha?tested at 75 days (Exp. I) or at maturity (Exp. 11). The other group of peanut

*r

Q E plants was watered normally up to harvest period and was used as control. Lesrf

.u E W proline content was determined at 32, 47, and 64 days in Exp. I and only at 64

5

,+

'

a

'

days after planting in Exp.

IL

The results indicated drought stress at generative

s ?

aowth stage reduced plant height, shoot dry weight, and peanut yield. Based on

X

Y

-. s

2

-.

=

sensitivity index calculated using seed yields per plant-indicated the tested peanut

5

3

cultivars were either medium tolerance or sensitive against drought stress at

m

*

g

generative growth stage. Increased in leaf proline content and absence of

E Fedduction of tots1 leaf sugar content may be uscd to inhcate tolerancc of peanut

4

Q

E c a v a r s against drought stress at generative growth stage.

g

O

Experiment entitled: Regeneration of Transgenic Tobacco Carrying P5CS

-.

_Q

s

Q

@nsgene and Analysis of its Expression was presented in chapter V. The 3 o&ctives of this experiment were (1) to regenerate transgenic tobacco cv.

E

4 &be1 Shili (GS) carrying chimera P5CS transgene through AgrbacteriMI- s e i a t e d transformation, (2) to analyze integration of chimera PSCS transgene in

8

8 %genome of transgenic GS tobacco by total nucleic acid

PCR,

(3) to estimate

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 -formation was able to introduce nptn md chimera PSCS transgenes into GS

tiitkicco genome and frve putative kanamycin resistance transgenic GS tobacco ~ z e regenerated. Positive results of total nucleic mid PCR analysis in populahon

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b' *

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 plant-

dry weight compared to tobacco nontr~nsgenic at

-

s p s s and non-stress condition. Higher increased in leaf proline content after

=

$ 3

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2.8

q u g h t stress was observed in all tobacco transgenic, while less increased was 2.

-

Q iJ

2

okerved in tobacco non-transgenic. Increased leaf proline content due to by over-

a

7 8 e8ression of P5CS gene under 5% or 10% PEG induced stress correlated with

I

,,

P

~5 Wught

-

tolerance phenotype in T1 plants derived from transgenic GS tobacco.

$

g y

Experiment entitled: Tolerance of Transgenic Peanut Expressing P5CS

r

2 E Gene against Drought Stress was presented in chapter

IX.

The objectives of this

X

c g

experiments were (i) to determine efEects of drought stress during the period

s

'

'

vegetative growth stage up to pod filling on growth and yield of T2 plants derived

s

!I

s = from TQ generation of transgenic peanut cv. Singa canying PSCS transgenes, (ii)

X

_-.

Y

s

$ & 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

.o

3

g

leaf proline content. Peanut plants were grown in plastic pots and subjected to

E stress condition during the period of 15

-

80 days after planting @AP) by

8

ing water only every 4 - 7 days. The plants were harvested at maturity. A

E

4 er of peanut plants were supplied with water up to field capacity from the a.

n

E of the experiment up to maturity, and t h s population was used as control.

9 prolina content were determined from leaf samples harvested at 55 DAP E _{6x stress period). The results of the experiment indicated the T2 peanut}

5

ts were transgenic plants carrying PSCS transgene since it accumulated more

2

lii m l i n e contents in their leaf as compared to non-transgenic plants under non-

F

= m 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'

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2

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b' * g a p P $;g;g$ 5 : ~ :

a s s

8

gf

8

5

AHMAn RIDli4N. Toleransi Kacang Tanah dm Tembakau terhadap Stres

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Kekefingan den, Over-Ekspresi Gen PSCS

-

Penyandi Ensim Kunci

S...SSQ

2

k 9

3

Biosintesis Prolina. Komisi Pembimbing : EIAJRIAL ASWIDINNOOR,

5

f

_{g SUDARSONO, J.4JAH KOSWARA, dan DJOKO SANTOSO.}

QcPa3 Q

Disertasi ini ditulis berdasarkaa hasil-hasil percobaan yang mengarah pada i beberapa kultivar unggul nasional dan mekanisme tolemsi h a n g tanah

stres kekeringan pada fase vegetatif dan generatif. Berdasarkan hasil rnekanisme tersebut dilakukan teknik rekayasa genetika untuk tamman yang toleran kekeringan melalui mekanisme over-ekspresi

ensim kunci biosintesis prolina-

Q 5 Toleransi tanaman terhadap stres kekeringan bersifat dinamis, sehingga

FS., a.

= G ID rr un& rnenguji tingkat toleransi terhadap stres kekeringan dilakukan pada berbagai

2

s

1 9

t i n e t pertumbuhan (fa vegetatif dan generatif). Pengujian tingkat toleransi

s

!JJ...X

g 9 3 m a n terhadap stres kekeringan juga dilakkan

di

bawah kondisi terkontrol

di

b J run& kaca karena kondisi di lapang terkendda-~kh sulitnya mendapatkan

5

_{2 s}

a

k g

koeisi stres kekeringan yang homogen dan sulitnya men&ga gangguan cuaca

ya$ terjadi terhadap percobaan. Metoda pengujian yang digunakan

(i)

stres

'

=

k e w n g a n denngan pengurangan air pda medium tanah di dalam pot, (ii) stres

$ 3

ii'

-. d e v n penyiraman berbagai konsentrasi larutan

PEG

6000 pada medium coco

2.

-

2.

8

p e a dan arang sekam (1 : 1).

a

F < Dari transformasi gen PSCS pada ehplan daun tembaku berhasil didapat

l i d nornor calon tanaman tembakau cv. Gombel Shili (GS) transgenik PSCS

P

< =

5

gen&asi TO &I, dberi kode GS-1, GS-2, GS-3, G S 4 dan GS-5. Dari masing-

a- _{masing tanaman TO dihasilkan benih T0:l yang setelah ditumbuhkan menjadi}

X

tanaman TI digunakan untuk (i) pernbuktian bahwa tanaman TO yang didapat .

e

3

merupakan tanaman t m g e n i k yang mengintegrasikan gen P5CS. (ii) penGjian

El _{damp& stres kekeringan,}

dan

_{(iii) stres akibat penyiraman larutan PEG terhadap} s =

8.

!

pertumbuhan dan hasil biornasa serta toleransi tanarnan Ti -at ternbakau GS

$ &

-

'

transgenik PSCS terhadap stres. Sedangkan pada tanaman kacang tanah didapat empat nomor calon tanaman kacang tanah c v Singa transgenik P5CS generasi TO

'

5 dm diberi kode S-1, $2, S-3 dan M. Dari masing-masing tanaman TO

2

QHZblkan sejurnlah benih TO: 1. Setelah benh TO: 1 ditanam, dari masing-masing

s

4 T I dihasilkan sejurnlah benih

TI:?

yang setelah ditumbuhkan rnenjadi a.

Q T2 digunakan untuk (i) pembuktian bahwa tanan~an TO yang didapat

s

g merapakan tanarnan transgenik yang mengekspresikan gen P5CS dan (ii) pe&jian dam* stres kekeringan terhadap perhrmbuhan dan hasil A-acang tanah

s e q toleransi tanaman

T2

zuriat kacang tanah cv. Singa transgenik PSCS terbdap sees kekeringaa

lii

F

s

Percobaan yang be rjudul : Toleransi Kultivar Kacang Tanah terhadap Stres

= _{~ e p g a n pada Fase Vegetatif s a t s Kandungan Prolina dm Gula Total Dam}

dis kan dalam BAB 111. Penelitian bertujuan untuk menentukan pengaruh atres ke

&

'ngan pada fase pertumbuhan vegetatif terhadap pertumbuhan

dan

hasil

k a e g tanah, serta mengevaluasi toleransi, kandungan prolina dan gula total

daun

damenam kultivar kacang tanah &lam kondisi stres tersebut. Kacang tanah

7 -

(D

3

-.

@ Hak cipta milik

Hak Cicita Dilindungi Undang-Undang

IPB (Institut Pertanian Bogor]

Bogor

ricultura

-

1. Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan dan menyebutkan sumber:

a. Pengutipan hanya untuk kepentingan pendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik atau tinjauan suatu masalah. b. Pengutipan tidak 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

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

s

-

germinated and the TI seedlings were used to identify that the TO plarits were

X

_-.

P

_{s transgenic plants, (ii) to evaluate effect of PSCS transgene expression on growth}

2

-

and biomass yield of tobacco under non-stress conditions, (iii) to determine

.o

3

la g 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

ym

and tolerance of the T2 plants against drought stress.

Experiment entitled: Tolerance of Peanut Cultivars against Drought Stress at

8

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

-.

<

CD

4