International Journal of Horticulture, 2015, Vol.5, No.21, 1-45
15
Figure 2 Amplification of parent and mutant cultivars of rose using primer P4 (A), P40 (B) and P31 (C)
Note: Lane 1: ‘Contempo’ (P), 2: ‘Contempo New’, 3: ‘Contempo Pink’, 4: ‘Contempo Stripe’, 5: ‘Contempo Tangerine’, 6:
‘Contempo Yellow’, 7: ‘Imperator’ (P), 8: ‘Imperator Stripe’, 9: ‘Imperator Pink’ 10: ‘First prize’ (P), 11: ‘First Prize Lighter’, 12:
‘American’s Junior Miss’, 13: ‘Sukumari’, 14: ‘Sylvia’ (P), 15: ‘Sylvia white’ 16: ‘Mrinalini’(P), 17: ‘Mrinalini lighter’, 18:
‘Mrinalini stripe’. M: DNA molecular marker
1.2.3 Pollen morphology
Pollen grains were collected soon after anthesis and pollen slides were made for the study of pollen sterility, size
and ornamentations both under light microscope and SEM (Datta, 1998).
1.2.4 Spectrophotometric Analysis of Pigments
For spectrophotometric analysis of phenolic compounds 200 mg of all above mentioned explants were extracted
in 50 ml methanol containing 1% HCl. The extracts were scanned from 200-800 nm region of wave length in
Utltroscope 2000, Pharmacia Biotech. Where concentration of phenolic compounds were high, the extracts were
further diluted (Datta, 1986).
1.2.5 TLC of Phenolic Compounds
For the study of phenolic compounds, mature leaf and petal were extracted in methanol containing 1% HCl. The
chromatograms were developed on aluminium plates (6.3 X 10 cm) coated with silica gel emulsion. The plates
were run 8 cm in a mixture of benzene: propionic acid:H
2
O (20:40:10 v/v). They were then dried in air and the
spots were observed and marked under nacked eye and under UV. The plates were then sprayed with flavone
reagent (diphenylboric acid ethanolamine complex) and again marked under UV. The colour reaction of each spot
and their Rf values were determined from six good chromatograms. These were then transformed into hRf (Rf X
100) values (Datta, 1987).
1.2.6 DNAExtraction
Total genomic DNA was extracted from young leaves of rose cultivars by CTAB procedure (Saghai-Maroof et al.,
1984) with some modifications. Extraction in chloroform: isoamyl alcohol (24:1) followed by centrifugation twice
at 14,000 g helped to remove polysaccharides. RNA contaminants in all the samples were digested with 100
mg/mL RNase A for 30 min at 37
℃
, extracted once with phenol: chloroform: isoamyl alcohol (25:24:1). After
ethanol precipitation, DNAwas resuspended in 100 mL of TE (10 mM Tris–Cl+1 mM EDTA) buffer (pH 8.0).
Average yield was calculated by a spectrophotometer (Ultraspec 2000, Pharmacia Biotech) and DNA samples
were stored at -20
℃
.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
A
B
500bp
500bp
500bp
C
