Kandungan Hormon Endogen dan Status Nutrisi dalam Buah Tanpa Biji Muda Hasil Induksi Antibiotik pada Anggur

Muscat of Alexandria

Winarso Drajad Widodo

Pt

Embrio

EndospermPTT

Degenerated

Developed

Streptomisin (SM); Spektinomisin (SE); Aquadest

?

4

10

16

22

1 2 3 4 5 6 7 8

Minggu setelah mekar penuh

Dia

met

er b

uah

(mm

)

SM-200SE-200Control

Gambar 1. Pengaruh antibiotika terhadap diameter buah

SM SE

control

-3 -3 +5 HSA-6 -6 +5 HSA

Pertumbuhan awal buah pra fertilisasi: Dipicu oleh hormon yang diproduksi oleh

pollenPertumbuhan buah pasca fertilisasi: Diatur/distimulir oleh hormon-2 yang

disintesis oleh biji muda yang ditranslokasikan ke dinding buah; jaringan nourishing adalah endosperm

tanpa endosperm embrio gagal tumbuh dan

sebaliknya, tanpa embrio, endosperm tidak

berkembang

seedlessseeded

Kandungan hormon endogendan status nutrisi dalambuah muda

4 tahun(30-40) cm x 50 cm x 20 cm

SM 200 mg/lSE 200 mg/l- 3 HSB

Celup

Treatment 6 HSP 10 HSP 13 HSP

ControlSM-200SE-200

728973550

2066a664b621b

1077667588

Kandungan ABA

Rf

Gambar 2. Aktivitas sitokinin

13 HSP

0

2

4

6

8

10

12

14

16

18

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 blank

Ca

llu

s w

eig

ht

(mg

)SM-200

SE-200

Control

13 HSP

-3

0

3

6

9

12

Rf0.1 Rf0.2 Rf0.3 Rf0.4 Rf0.5 Rf0.6 Rf0.7 Rf0.8 Rf0.9 Rf1.0

Exc

ess

of

bla

nk

(%)

Streptomycin

Spectinomycin

Control

ns

Gambar 3. Aktivitas auksin (IAA)

Gambar 4. Aktivitas GA

10 HSP

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Rf0.1 Rf0.2 Rf0.3 Rf0.4 Rf0.5 Rf0.6 Rf0.7 Rf0.8 Rf0.9 Rf1.0 Blank

Ab

sorb

ance

SM

SE

Control

Nutrisi:Sukrosa dan Fruktosa (gula aktif dan reaktif)rendah pada buah dari tandan yang diberiperlakuan antibiotika

Nitrogen: Kandungan dalam buah tanpa biji lebih rendah dibandingkan buah berbiji

Asam amino: glutamin, sistein, asparagin, tirosin, isoleusin, leusin, asam aspartat, asam glutamat dan alanin lebih rendah pada buah tanpa biji dibanding buah berbiji

GA merupakan hormon terpenting dalampertumbuhan tahap awal buah!

Application of Cytokinin and Gibberellin to Promote Enlargement of Muscat of Alexandria Grape

Seedless Berries induced by Streptomycin1)

Winarso Drajad Widodo2

1)A part of the final experiments of : Widodo, W. D. 2000. Seedlessness Induction by Antibiotics and Its Mechanism in Grapes. The Graduate School of Natural Science and Technology, Okayama University - Japan (Doctor Thesis). 2)Secretary of Crop Production Laboratory, Department of Agronomy, Faculty of Agriculture - IPB.

DEPARTMENT OF AGRONOMYFACULTY OF AGRICULTURE

BOGOR AGRICULTURAL UNIVERSITY

Background

• Seedlessness: the important characteristic for table grapes

• Natural seedless cultivars are very few

• Breeding for producing seedless cultivars: - seedless inheritance is unclear - time consuming performance

How to produce seedless berries artificially?

Stenospermic seedless berries: can be induced in seeded cultivars by Gibberellin (GA) simple method free from genetic factors and barriers

Fertilized Ovules Abortion: Stenospermy

Stenospermocarpic formation (pseudo seedless)‘remaining seed’ can not be detected organolep-tically --> stenospermic seedless berries

Certain vinifera grapes - included MOA - are severely induced lignification by GA; berries can not develop normally

Seedless berries can not be produced artificially by GA on seeded Muscat of Alexandria, an excellent cultivar of Japan grapes

The Problems:

Change

An antibiotic, streptomycin (SM) could induce seedlessness in several seeded cultivars, evidenced by:1. Ogasawara (1986) on Muscat Bailey-A grape2. Ishikawa et al. (1996-1998) on Kyoho and Fujiminori grapes3. Pommer et al. (1996) on Italia (a vinifera) grape

The mechanism of seedless berry information was not sufficiently studied yet

The whole research objectives:

1. to investigate seedlessness induction

capability of various antibiotics

2. to investigate the process of seedless

berry formation induced by antibiotics

3. to study the cause of insufficient growth

of antibiotic induced seedless berries by

anatomical and hormonal analysis

4. to seek the method of seedless berry

enlargement, especially Muscat of

Alexandria

1. Seedlessness Induction Capability of Bactericidal and Bacteriostatical Antibiotics

Plant materials: Muscat of Alexandria (MOA), Kyoho and PioneAntibiotics: Bactricide group: Streptomycin and Gentamicin Bacteriostatic group: Tetracycline and Specti- mycinTreatments: 1. Streptomycin: 200 mg/l 2. Tetracycline: 80 mg/l 3. Gentamicin: 60 mg/l 4. Spectinomycin: 200 mg/l vs. Control (distilled water)

Application: Cluster dipping 3 days before full bloom

Seedlessness induction capability of antibiotics

Treatment10-berry weight

(%)

Seeded berries with

Seedless berries3

(%)

normal seed(%)

empty seed(%)

Muscat of AlexandriaControl 23.74a 100.0a 0.0 0.0cSM-200 5.46c 0.0c 0.0 100.0aTC-80 11.26b 57.3b 15.0 26.7bGM-60 13.07b 56.7b 6.7 36.7bSE-200 1.99c 0.0c 0.0 100.0a

Seedlessness induction capability of antibiotics

Treatment10-berry weight

(%)

Seeded berries with

Seedless berries3

(%)

normal seed(%)

empty seed(%)

Kyoho

Control 38.59b 100.0a 0.0 0.0c

SM-200 13.93c 0.0b 39.0 61.0a

TC-80 50.72a 98.0a 0.0 2.0c

GM-60 37.17b 79.0a 21.0 0.0c

SE-200 11.97c 0.0b 70.0 30.0b

Seedlessness induction capability of antibiotics

Treatment10-berry weight

(%)

Seeded berries with

Seedless berries3

(%)

normal seed(%)

empty seed(%)

Pione

Control 39.6a 98.5a 0.3 0.0d

SM-200 27.3b 0.0c 8.0 60.0b

TC-80 40.8a 61.5b 2.0 38.3c

GM-60 39.3a 75.0ab 1.7 16.5d

SE-200 24.6b 0.0c 5.3 73.5a

2. Anatomical analysis

Materials: young berries of Muscat of Alexandria

collected 0, 3, 5, 7, 9, 11 and 14 days

after anthesis (dAA)

Method: paraffin-sectioned pistils, observed un-

der light microscope to determine ovule

width and endosperm nuclei number

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 7 9 11 14

Day(s) after anthesis

Ov

ule

wid

th (

mm

)

Control SM 200 TC 80

GM 60 SE 200

Effect of antibiotics on ovule width

Ovule at 7 days after anthesis (Widodo, W. D. 2000. Seedlessness Induction by Antibiotics and Its Mechanism in Grapes. Doctoral Thesis, The Graduate School of Natural Science and Technology, Okayama University, Japan.)

a. normal b. early degenerated c. late degenerated

Integument

Nucellus

Endosperm

Embryo

3. The small berry formation

3.1. Cell size and cell number observation

Plant material: Muscat of Alexandria

Antibiotics: Streptomycin (SM) and

Spectinomycin (SE) at the concentration

of 200 mg/l applied 3 days before full bloom

Effect of Streptomycin and Spectinomycin on cellwidth of ovule wall

TreatmentOuter wall cell width (m) Inner wall cell width (m)

RadialLongitudin

alTangential Radial

Longitudinal

Tangential

7 days after anthesis

Control 22.1 25.6 31.5 32.2a 43.7 62.4

SM 18.9 28.8 30.6 26.8ab 52.5 50.7

SE 20.2 26.1 30.1 21.8b 51.1 54.5

15 days after anthesis

Control 20.4 41.3 62.4 54.2a 85.2 39.6

SM 18.5 50.8 50.7 30.5c 96.2 46.9

SE 15.6 56.8 54.5 41.9b 90.4 40.2

Effect of antibiotic treatment on cell numbers along pericarp area

Treatment

No. cell of outer wall No. cell of inner wall No. of cell layer

Longitu-dinal tangential longitu-dinal tangentialouter wall

inner wall

0 day after treatment

116 187 110 157 5 56 days after treatment

Control 190a 246 177a 161 8.7a 8.5bSM-200 183ab 233 150b 161 8.7a 9.0aSE-200 177b 228 146b 148 8.0b 7.0c

10 days after treatmentControl 292 313a 217a 242a 15.3a 15.4abSM-200 267 278b 160b 237a 13.4b 16.2aSE-200 266 290b 151b 194b 13.2b 13.2b

17 days after treatment

Control 483a 390a 246 214 19.8a 18.4aSM-200 366c 359b 228 212 17.6b 15.2cSE-200 421b 352b 258 246 17.6b 16.5b

3.2. Hormone analysis

1. ABA: Gas Chromatography not significant

2. Auxins: Avena Coleoptile Straight-growth Test

Not significant

3. Cytokinins: Soybean Hypocotile Culture Test

significant

4. Gibberellins: Barley Endosperm Amylase

Activity Assay significant

4. Berry enlargement experiments

• GAs is the important hormones for berry

enlargement

• GA (as GA3) was tested in low level con-

centration to enlarge MOA-seedless

berries induced by Streptomycin; as

well as Forchlor-fenthuron (CPPU) a

synthetic cytokinin

CPPU

0

2

4

6

8

10

12

14

16

18

20

3 4 5 6 7 8

Weeks after Full Bloom

Ber

ry d

iam

eter

(m

m)

control 5 ppm, 10 dAB10 ppm, 5 dAB 10 ppm, 10 dAB10 ppm, 15 dAB 15 ppm, 10 dAB

Effect of CPPU on berry growth of seedless berry induced by streptomycin

GA3

0

5

10

15

20

25

3 4 5 6 7 8

Weeks after Full Bloom

Ber

ry d

iam

eter

(m

m)

control 1 ppm, 10 dAB5 ppm, 10 dAB 5 ppm, 15 dAB10 ppm, 5 dAB 10 ppm, 10 dAB

Effect of GA3 on berry growth of seedless berry induced by streptomycin

GA

CPPU

GACPPU

GA: A= 5 ppm, 10 dAA D= 5ppm , 15 dAA B= 10 ppm, 5 dAA E= 10 ppm, 10 dAAC= 1 ppm, 10 dAA

CPPU: A= 10 ppm, 5 dAA D= 10 ppm, 10 dAAB= 10 ppm, 15 dAA E= 15 ppm, 10 dAAC= 5 ppm, 10 dAA

CPPU or GA treatments as the second treatments could enlarge the small-sized seedless berries induced by Streptomycin

Conclusion