Variables distribution and clustering of the genotype-colchicine treatment associations
Normal probability plot correlation coefficients of the variables used to carry out ordination and cluster analysis indicates that the data showed normal distribution (Fig. 2a) with very high correlation coefficients ranging from 0.863 (Surv-16) to 0.9963 (CC) (Fig. 2b). Cluster analysis performed on the genotype-colchicine treatment associations also revealed five distinct clusters with a cophenetic correlation coefficient of 0.7341 (Fig. 3.) based on the physio-morphological traits in response to the mutagenic treatment. The first cluster (cluster I) consisted of 3 colchicine-induced morphotypes (CIMs) which are morpho-variants of G-132 resulting from colchicine treatment of G-132 at low concentrations of 1.26 and 3.76 mM and for long and short durations of 12, 24 and 3 hours respectively. Individuals in this cluster included V2C1-12, V2C1-24 and V2C2-3.
The second cluster (cluster II), however, was made up of 9 CIMs which included mixed genotypes of G-131 and G-132. CIMs of G-131 were seedlings treated with colchicine concentrations of between 3.76–8.76 mM at variable treatment durations ranging from 6–24 hours. CIMs of G-132 included seedlings treated with 3.6 and 8.76 mM colchicine for 24 hours. Individuals in this cluster included V1C2-6, V1C2-12, V1C2-24 V1C3-6, V1C3-12, V1C3-24, V1C4-24, and V2C2-24, V2C4-12. This second cluster was further sub-clustered into 2 groups. The first sub-cluster consisted individual morphotypes derived solely from oil palm genotype G-131 treated with 3.76 mM colchicine for 6 and 12 hours and 6.26 mM colchicine for 6 hours (V1C2-6, V1C2-12 and V1C3-6). The second sub-cluster was made up of G-131 and G-132 treated with between 3.76–8.76 mM colchicine for variable durations ranging from 12–24 hours (V1C2-24, V1C3-12, V1C3-24, V1C4-24, V2C2-24 and V2C4-12).
The third cluster (cluster III) consisted of 12 CIMs derived from the two genotypes G-131 and G-132 as a result of exhibition of trans-morphologies of G-131 and G-132. Individuals in this group were made up predominantly of G-132 genotype treated with between 3.76–8.76 mM colchicine at variable durations 3–24 hours. CIMs of G-131 were seedlings treated with 8.76 mM colchicine for 3, 6 and 12 hours. Members in this third cluster were thus, V1C4-3, V1C4-6, V1C4-12, V2C2-6, V2C2-12, V2C3-3, V2C3-6, V2C3-12, V2C3-24, V2C4-3, V2C4-6 and V2C4-24. Furthermore, 2 sub-clusters were identified within the third cluster and this included V1C4- 3 and V1C4-12 in one sub-group and V1C4-6, V2C2-6, V2C2-12, V2C3-3, V2C3-6, V2C3-12, V2C3-24, V2C4-3, V2C4-6 and V2C4-24 in the other sub-cluster.
The fourth cluster (cluster IV) comprised solely of individuals derived from the genotype G-132 treated with only distilled water as controls for durations ranging from 3–24 hours. Individuals in the fourth cluster were V2C0-3, V2C0-6, V2C0-12 and V2C0-24.
In the fifth cluster (cluster V), 12 CIMs predominantly derived from G-131 were observed. It also included some morphotypes derived from the genotype G-132 treated at very low colchicine concentrations for shorter durations of 3 and 6 hours. Individuals derived from G-131 were mainly seedlings treated with only distilled water for variable durations of 3–24 hours and also seedlings treated with low to relatively high colchicine concentrations (1.26–6.26 mM) for variable treatment durations (3, 6, 12 and 24 hours). Individuals in this cluster were, V1C0-3, V1C0-6, V1C0-12, V1C0-24, V1C1-3, VICI-6, V1C1-12, V1C1-24, V1C2-3, V1C3-3, V2C1-3 and V2C1-6.
Two sub-clusters were further identified under cluster V which included individuals of G-132 treated at very low colchicine concentrations (1.26 mM) for 3 and 6 hours in one sub-group (V2C1-3 and V2C1-6) and those of G-131 treated with only distilled water for between 3–24 hours and seedling treated at between 1.26–6.26 mM for variable durations of between 3–24 hours in the other sub-group (V1C0-3, V1C0-6, V1C0-12, V1C0-24, V1C1-3, VICI-6, V1C1-12, V1C1-24, V1C2-3, V1C3-3).
Variables with the widest range and variability and therefore contributed significantly to variations observed among the colchicine-mutagenized oil palm populations in order of decreasing strength include stomatal conductance (SC), chlorophyll content (CC), leaf area (LA) and plant height (PH) (Fig. 4 ).
Frequency of aberrancy in the colchicine-mutagenized oil palm population.
Seedlings with aberrant (off-type) phenotypes or morphologies such as thick and reduced leaf sizes and plant height of approximately 50% of the control were also studied and statistically significant, (p ≤ 0.05) variation was found between the oil palm genotypes (Fig. 5). Genotype x concentration, concentration x treatment duration and genotype x treatment duration interactions were also statistically significant (p ≤ 0.05). At higher colchicine concentrations (6.26 and 8.76 mM) the genotype G-131 produced significantly higher proportions of aberrant seedling types than G-132. No aberrant seedlings were recorded among the control seedlings and seedlings treated with 1.26 mM colchicine for 3, 6, 12 and 24 hours respectively in both genotypes. Furthermore, G-132 did not record any aberrant seedling at a lower concentration of 3.76 mM applied to the seedlings for 3 hours.
Seedlings with aberrant morphologies increased significantly (p ≤ 0.05) with increasing level of colchicine and treatment durations. In G-131, seedlings with aberrant phenotypes ranged from 0–22.23, 0–40.0, 0 -57.80 and 0–66.67% for treatment durations of 3, 6, 12 and 24 hours, respectively. G-132, however, recorded between 0–20.0, 0–28.90, 0–37.80 and 0–35.57% for colchicine treatment durations of 3, 6, 12 and 24 hours, respectively. In G-131, the highest proportion of seedlings with aberrant phenotypes (66.7%) was recorded among seedlings treated with 6.26 mM for 24 hours. In G-132, nevertheless, the highest proportion of seedlings with aberrant phenotypes was observed among seedlings treated with 8.76 mM for 12 hours. The off-type seedlings in G-132 ranged from 0–20.0, 0–28.90, 0–37.80, 0–37.80 and 0–35.60% in seedlings treated with colchicine for 3, 6, 12 and 24 hours respectively.
Principal component analysis
The principal components determined on the 12 physio-morphological traits in the oil palm genotypes subjected to the colchicine mutagenic treatment together with the eigen values and the proportion of the variance explained by each of the principal components are presented in Table 1. The analysis revealed 12 principal axes but only 4 which had proportion of the eigen values greater than 1% were retained. The scree plot (Fig. 6a) also showed that after the first 4 principal components the percentage contribution of the remaining axes were negligible. Together the 4 principal components explained 99.95% of the total variation observed in the colchicine-induced response variables. The principal component 1 (PC1) explained 65.89% of the total variation while the second, third and fourth principal components, PC2, PC3 and PC4 accounted for 27.40% 5.59% and 1.09% of the total variation observed, respectively. The most variable parameters on the first two principal axes PC1 and PC2 were SC, LA, CC and PH (Fig. 6b).
The loadings derived to determine the most relevant traits, which influenced the 4 principal component axes are shown in Table 2. Principal axis 1 (PC1) correlated positively with stomatal conductance and chlorophyll content and negatively with leaf area. On PC2, the most relevant traits were, number of leaves per plant, leaf emission rate, plant height, proportion of aberrant seedlings, leaf area ratio, and plant height ratio. PC3 was defined mainly by chlorophyll content, leaf area, stem girth, plant height and leaf area ratio. PC4 correlated with leaf thickness, survival of seedlings/ and frequency of seedlings with aberrant morphologies
Table 1
Vector loadings of the principal components of the colchicine-induced response variables determined on the colchicine treated oil palm genotypes
Trait | PC1 | PC2 | PC3 | PC4 |
SUV | -0.010 | 0.0057 | 0.0114 | 0.0233 |
SC | 0.7123 | 0.6806 | -0.1087 | 0.1325 |
LT | 0.0001 | -0.0002 | 0.0003 | 0.0004 |
CC | 0.4531 | -0.2768 | 0.7438 | -0.4036 |
LA | -0.4666 | 0.5430 | 0.6216 | 0.2255 |
SG | -0.0022 | 0.0030 | 0.0035 | 0.0007 |
NL | -0.0148 | 0.0184 | 0.0172 | -0.0064 |
LER | -0.0037 | 0.0046 | 0.0041 | -0.0016 |
PH | -0.1118 | 0.1166 | 0.1181 | 0.1166 |
LAR | -0.0041 | 0.0045 | 0.0047 | 0.0014 |
PHR | -0.0028 | 0.0028 | 0.0027 | 0.0012 |
PAB | 0.2381 | -0.3890 | 0.1848 | 0.8687 |
Eigen value | 3596.88 | 1495.64 | 304.918 | 59.3642 |
% Variation | 65.885 | 27.396 | 5.5853 | 1.0874 |
Cumulative % | 65.885 | 93.281 | 98.8663 | 99.9537 |
SUV = seedling survival rate; SC = stomatal conductance; LT = leaf thickness; CC = chlorophyll content; LA = leaf area; SG = seedling stem girth; LER = leaf emission rate; NL = number of leaves; LAR = Leaf area ratio; PH = plant height; PHR = plant height ratio; PAB = proportion (frequency) of aberrant individuals. Most relevant traits that contributed to most of the variations on each principal component axis are printed in bold.
Ploidy level determination by flow cytometry in the colchicine-mutagenized population.
Variations in chromosome doubling frequency in the oil palm seedlings treated with colchicine are presented in Fig. 7. In determining the ploidy level of the genotypes, the commercial tenera (control, un-treated) with a diploid ploidy status (Figs. 8 and 9) was used as an internal reference standard. Variation observed in the chromosome doubling frequency between the genotypes was found to be statistically significant (p ≤ 0.05). Concentration, treatment duration as well as genotype x concentration, genotype x duration and concentration x duration interactions were also statistically significant (p ≤ 0.05). Genotype x concentration x duration interaction was, however, not statistically significant. The untreated seedlings of both genotypes, G-131 and G-132 did not record any doubling of chromosome at all durations of soaking in sterile distilled water. Nevertheless, in both genotypes, the colchicine treatment induced several morphological variations (Fig. 10) and genome doubling frequency generally increased significantly (p ≤ 0.05) with increasing colchicine concentration and duration of treatment. In G-131, doubling frequency for treated seedlings ranged from 0–6.7, 6.7–22.2, 8.9–28.3 and 13.6–28.9% for treatment durations of 3, 6, 12 and 24 hours respectively. In G-132 however, between 0–6.7, 0–8.9, 0–13.3 and 6.7–17.8% chromosome doubling frequency were observed for treatment durations of 3, 6, 12 and 24 hours respectively. At a relatively mild colchicine concentration of 1.26 mM, G-131 and G-132 began to experience doubling in ploidy level but at a lower frequency of 6.7% at treatment durations of 6 and 24 hours respectively. Seedlings treated with 8.76 m M colchicine for 24 hours recorded the highest doubling frequency in ploidy level in G-131 (28.9%) and G-132 (17.8%).