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