nuclear stain solution DAPI (solution B kit) was added to it after a minute to count
devices. At least 5000 cells per sample volume typically measured by the peaks
obtained and were interpreted by software Mode Fit (Otto 1990 ).
Flow cytometry is now used routinely for ploidy analyses and it is regarded as
the most accurate tool for ploidy determination (Loureiro et al. 2005 ).
17.2.6 Analysis of Morphological Characteristics
Major morphological and growth habit characteristics in the plants of confirmed
tetraploidy were compared to those of plants with confirmed diploid status derived
from the 0/0% w/v colchicine treated population. Dimensions measured were: leaf
index (taken from five randomly selected leaves of each individual); growth
parameters (height and fresh weight of leaves and roots taken fromfive plants);
flower diameter; size and density of stomata were measured as described above.
Density of glandular trichomes was also measured on the axial leaf surfaces by nail
varnish technique. The investigation of the morphological characteristics of leaf,
stem, andflower was performed after 4 months.
17.2.7 Phytochemical Measurement
The third leaves of 2-month-old seedlings were used for all analyses. Soluble sugar
content of samples were determined with anthrone reagent based on Roe method
( 1955 ). The total protein of fresh leaves and roots of plants was determined
according to Bradford ( 1976 ). Chromatographic measurement of cannabinoids were
performed as described by Rustichelli et al. ( 1996 ).
17.3 Results
17.3.1 Survival and Growth of Colchicine-Treated
Shoot Tips
The survival rate of shoots was affected by the concentration of colchicine and the
duration of treatment (Table17.1). In general, high concentration and longer
duration reduced survival of the shoot tips. The survival was 100% when shoot tips
were treated with 0.0% w/v colchicine. The survival rate didn’t change when the
duration on 0.1% colchicine was prolonged from 24 to 48 h, but the survival rate
decreased from 89.96% to 73.33% when the concentration of colchicine increased
17 Induction of Polyploidy and Its Effect onCannabis sativaL. 371