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Optimization of in vitro asymbiotic seed germination protocol for Serapias vomeracea

Arda Acemi
Arda Acemi
 y 
Fazıl Özen
Fazıl Özen
   | 25 jul 2019
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Article Category: Research Article
Publicado en línea: 25 jul 2019
Páginas: 143 - 151
DOI: https://doi.org/10.2478/ebtj-2019-0017
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© 2019 Arda Acemi, Fazıl Özen, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Figure 1

Stereomicroscope view of Serapias vomeracea seeds. A: 16x magnification, the scale bar represents 0.5 mm, B: 80x magnification, the scale bar represents 0.1 mm.
Stereomicroscope view of Serapias vomeracea seeds. A: 16x magnification, the scale bar represents 0.5 mm, B: 80x magnification, the scale bar represents 0.1 mm.

Figure 2

Germination stages of Serapias vomeracea seeds. A: Stage 1 seed in solid KN medium, B: Stage 2 seed at the beginning of germination in solid KN medium, C: Stage 3 seed from solid VW medium, D: General view of stage 3 seeds in liquid KN medium, E: Stunted rhizoids of the seed in liquid medium, F: Elongated rhizoids on solid VW medium.
Germination stages of Serapias vomeracea seeds. A: Stage 1 seed in solid KN medium, B: Stage 2 seed at the beginning of germination in solid KN medium, C: Stage 3 seed from solid VW medium, D: General view of stage 3 seeds in liquid KN medium, E: Stunted rhizoids of the seed in liquid medium, F: Elongated rhizoids on solid VW medium.

Figure 3

Comparison of in vitro germination rates of Serapias vomeracea seeds on solid and liquid media. Data represent mean ± SD. Means having the same superscript letters in a column were not significantly different by Duncan’s multiple range test (p < 0.05).
Comparison of in vitro germination rates of Serapias vomeracea seeds on solid and liquid media. Data represent mean ± SD. Means having the same superscript letters in a column were not significantly different by Duncan’s multiple range test (p < 0.05).

Media compositions tested in in vitro germination of Serapias vomeracea seeds

ContentQuantity (mg L-1)
MacroelementsKNVWLN
KNO3525.000
NH4NO3500.000
NH4H2PO4500.000
(NH4)2SO4500.0001000.000
Ca3(PO4)2200.000
Ca(NO3)2347.200347.200
MgSO4122.125122.10058.620
KH2PO4250.000250.000135.000
KCl250.0001050.000
Microelements
AlCl3·6H2O0.056
NiCl2·6H2O0.031
MnSO·HO5.6825.6000.052
42KI0.099
H3BO31.014
ZnSO4·7H2O0.565
CuSO4·5H2O0.019
Iron source
C6H5FeO74.400
FeSO4·7H2O25.00027.800
Complexing agent
Na2EDTA·2H2O37.260
Vitamins
Nicotinic acid1.000
Pyridoxine-HCl1.000
Thiamin-HCl0.40010.000
myo-Inositol100.000
Aminoacids
Glycine2.000
Carbon source
Sucrose20000.00020000.00020000.000

Effects of different solid media on in vitro germination of Serapias vomeracea seeds

Seed germination stages (%)
MediumStage 0Stage 1Stage 2Stage 3Stage 4
KN15.04 ± 2.68a26.19 ± 5.32d58.77 ± 5.51a0.00 ± 0.00d0.00 ± 0.00b
½KN13.04 ± 2.16ab35.46 ± 4.40c51.51 ± 3.13a0.00 ± 0.00d0.00 ± 0.00b
VW9.75 ± 0.98bc46.00 ± 5.16b40.82 ± 1.92b2.83 ± 0.30a0.79 ± 0.10a
½VW7.45 ± 0.47c58.71 ± 7.69a33.71 ± 7.11bc0.13 ± 0.05d0.00 ± 0.00b
LN13.86 ± 3.25a57.64 ± 2.16a27.47 ± 4.28cd1.03 ± 0.16c0.00 ± 0.00b
½LN13.01 ± 1.59ab61.22 ± 4.95a23.55 ± 5.23d2.21 ± 0.30b0.00 ± 0.00b

Seed surface sterilization treatments tested on Serapias vomeracea seeds

TreatmentConcentration (%)Time (min)
NaOCl0.58
NaOCl1.08
EtOH + NaOCl70 + 0.50.5 + 8
EtOH + NaOCl70 + 1.00.5 + 8

Effects of different liquid media on in vitro germination of Serapias vomeracea seeds

Seed germination stages (%)
MediumStage 0Stage 1Stage 2Stage 3Stage 4
KN11.85 ± 1.36a34.35 ± 3.55c44.61 ± 1.50b8.46 ± 0.88a0.72 ± 0.14a
½KN9.43 ± 1.80abc41.38 ± 3.14b46.75 ± 1.31b2.44 ± 0.92b0.00 ± 0.00b
VW6.29 ± 1.37c41.91 ± 0.10b51.62 ± 1.12a0.17 ± 0.03c0.00 ± 0.00b
½VW7.94 ± 1.52bc36.91 ± 3.42bc52.41 ± 2.34a2.74 ± 0.25b0.00 ± 0.00b
LN10.98 ± 2.07ab48.19 ± 1.91a40.24 ± 1.88c0.60 ± 0.06c0.00 ± 0.00b
½LN10.55 ± 2.26ab50.60 ± 2.57a38.66 ± 2.77c0.19 ± 0.00c0.00 ± 0.00b

Stages of orchid seed germination according to Yamazaki and Miyoshi (2006)

Germination stageIndicators
Stage 0‘No germination’ stage. No growth of embryo occurs.
Stage 1‘Pre-germination’ stage. Embryo swells to fill the seed coat.
Stage 2‘Germination’ stage. Embryo emerges from the seed coat.
Stage 3‘Protocorm’ stage. Embryo is completely discharged from the seed coat.
Stage 4‘Rhizoid’ stage. Rhizoids are formed on the protocorm surface.
Stage 5‘Shoot’ stage. Shoot is diferentiated from the protocorm.
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