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Review Article

Garlic Micro-propagation and Polyploidy Induction In Vitro by Colchicine

Plant Breeding and Biotechnology 2021;9(1):1-19.
Published online: March 1, 2021

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*Corresponding author Molla G. Hailu, molla_6218@apps.ipb.ac.id, Tel: +62-85890142566
• Received: October 29, 2020   • Revised: December 25, 2020   • Accepted: December 31, 2020

Copyright © 2021 by the Korean Society of Breeding Science

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Garlic Micro-propagation and Polyploidy Induction In Vitro by Colchicine
Plant Breed. Biotech.. 2021;9(1):1-19.   Published online March 1, 2021
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Garlic Micro-propagation and Polyploidy Induction In Vitro by Colchicine
Image Image Image Image Image
Graphical abstract
Graphical abstract
Graphical abstract
Fig. 1 DNA content and cell cycle.
Fig. 2 Analysis of nuclear DNA content. Adapted from Doležel and Bartoš (2005), Doležel et al. (1994), and Tuwo and Indrianto (2016).
Garlic Micro-propagation and Polyploidy Induction In Vitro by Colchicine

Composition of inorganic salt stalks in Basal Dunstan Short (BDS, 1977).

Chemical compound Conc. (g/L) Vol. (ml/L) Molarity
Calcium chloride (CaCl2·2H2O) 30 20 1.02 mM
Ammonium nitrate (NH4NO3) 126.5 20 25.02 mM
Potassium nitrate (KNO3) 16.008 20 4.0 mM
NH4H2PO4 0.230 20 2.0 mM
(NH4)2 SO4 0.134 1.01 mM
Magnesium sulfate (MgSO4·7H2O) 49.4 20 1.00 mM
Manganous sulfate (MnSO4·4H2O) 2.64 0.045 mM
Zinc sulfate (ZnSO4·7H2O) 0.4 6.95 mM
Cupric sulfate (CuSO4·5H2O) 0.0078 0.1 mM
KI (Potassium iodide) 0.15 20 4.52 mM
Cobalt chloride (CoCl2·6H2O) 0.005 0.105 mM
Boric acid (H3BO3) 0.6 0.049 mM
Sodium molybdate (Na2MoO4·2H2O) 0.05 1.03 mM
NaH2PO4.2H20 0.172 1.04 mM
Ferrous sulfate (FeSO2·7H2O) 0.028 10 0.10 mM
Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA.2H2O) 37.25 0.10 mM
Nicotinic acid 0.1 20 8.1 mM
Thiamine HCl 0.001 0.03 mM
Pyridoxine HCl 0.1 4.9 mM
Meso-Inositol 10 10 1.8 mM
Sucrose 30 0.088 M
2,4-D (2,4-dichlorophenoxyacetic acid) 0.4 mg/L 1.25 mM

Composition of inorganic salt stalks of the Murashige and Skoog (1962).

Chemical compound Conc. (g/L) Vol. (ml/L) ppm (mg/L)
Ammonium nitrate (NH4NO3) 82.5 20 1,650
Potassium nitrate (KNO3) 95 20 1,900
Magnesium sulfate (MgSO4·7H2O) 74 5 370
Manganous sulfate (MnSO4·H2O) 4.46 22.3
Zinc sulfate (ZnSO4·4H2O) 1.72 8.6
Cupric sulfate (CuSO4·7H2O) 0.005 0.025
Calcium chloride (CaCl2·6H2O) 88 5 440
Potassium iodide (Kl) 0.166 0.83
Cobalt chloride (CoCl2·2H2O) 0.005 0.025
Potassium phosphate (KH2PO4) 34 5 170
Boric acid (H3BO3) 1.24 6.2
Sodium molybdate (Na2MoO4·2H2O) 0.05 0.25
Ferrous sulfate (FeSO2·7H2O) 2.78 13.9
Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA.2H2O) 3.73 18.65
Myo-Inositol 10 10 100
Thiamine 0.01 10 0.1
Nicotine 0.05 0.5
Pyridoxine 0.05 0.5
Glycine 0.2 2.0
Sugar 30

Morphological and chemical traits of diploid and polyploid garlic plants.

Colchicine-treated plants Leaf area index Stomatal length (mm) Stomatal width (mm) Stomatal area (mm2) Total soluble sugars (g g–1 FW)
Diploid (Control) (2n = 16) 3.01 ± 0.05b 28.20 ± 0.20b 20.90 ± 0.11b 589.28 ± 1.89b 0.33 ± 0.01b
Tetraploid (4n = 32) 9.02 ± 0.49a 56.86 ± 0.26a 33.16 ± 0.09a 1,884.65 ± 9.388a 0.48 ± 0.05a
LSD (P ≤ 0.05) 1.61 5.56 3.23 20.4 0.03

Effect of different concentrations of colchicine on morphological charactersitics and chromosome counts in garlic (Allium sativum).

Morphological character Isolate/clone Chromosome count (2n) Size of stomata (relative to diploid control)* Colchicine concentration (w/v)
Dark-green leaves (DG) DG 1 16 S 0.25
DG 2 16 D 0.25
DG 3 16 S 0.25
DG 4 24 D 0.25
DG 5 16 S 0.25
DG 6 16 S 0.25
DG 7 18 D 0.50
DG 8 16 S 0.50
DG 9 Multiple sets I 0.50
DG 10 Multiple sets I 0.50
DG 11 16 S 0.50
DG 12 16 S 0.50
DG 13 16 S 0.50
DG 14 Multiple sets D 0.50
DG 15 Multiple sets I 0.50
DG 16 16 S 0.75
DG 17 16 S 0.75
DG 18 16 S 0.75
DG 19 Multiple sets I 0.75
DG 20 16 S 0.75
DG 21 16 S 0.75
Reduced growth (RG) RG 1 8 D 0.25
RG 2 8 D 0.25
Green and thick leaves (GT) GT 1 32 I 0.50
GT 2 32 I 0.50
GT 3 32 I 0.50
GT 4 32 I 0.50
GT 5 32 I 0.50
GT 6 32 I 0.50
GT 7 32 I 0.50
GT 8 32 I 0.50
GT 9 32 I 0.50
Curled leaves (CL) CL 1 Multiple sets D 0.75
CL 2 Multiple sets D 0.75
CL 3 Multiple sets D 0.75
CL 4 Multiple sets D 0.75

Common fluorescent dyes for nuclear DNA staining.

Fluorochrome Primary binding mode Wavelength (nm)*

Excitation Emission
Ethidium bromide** Intercalation 530 605
Propidium iodide** Intercalation 540 615
Hoechst 33258 AT-binding 365 465
Hoechst 33342 AT-binding 360 460
DAPI (4,6-diamidino-2-phenylindole dihydrochloride) AT-binding 365 450
DIPI (4’,6-bis (2’-imidazolinyl-4H.5H)-2-phenylindole) AT-binding 365 450
Chromomycin A3 GC-binding 445 570
Mithramycin GC-binding 445 575
Olivomycin GC-binding 440 560
Table 1 Composition of inorganic salt stalks in Basal Dunstan Short (BDS, 1977).
Table 2 Composition of inorganic salt stalks of the Murashige and Skoog (1962).
Table 3 Morphological and chemical traits of diploid and polyploid garlic plants.

Adapted from Dixit and Chaudhary (2014): The Leaf area index is calculated using a leaf area meter. Total soluble sugar concentrations (TSSC) are measured in fully-mature garlic bulbs.

Table 4 Effect of different concentrations of colchicine on morphological charactersitics and chromosome counts in garlic (Allium sativum).

*Adapted from Dixit and Chaudhary (2014): Stomatal measurements were made on the lower epidermis of the second fully-expanded leaf. S: Similar sized stomata to control diploid plant. D: Decreased stomatal size compared to diploid control. I: Increased stomatal size compared to diploid control.

Table 5 Common fluorescent dyes for nuclear DNA staining.

*Dye-DNA complex. **Binds also to double-stranded RNA.