Influence of colchicine concentrations with exposure duration showed diversified responses in various vegetative parameters. Cotyledon characteristics like shape and intensity of green colour is presented in Fig. 2. Early screening according to these attributes found that diploids (control treatment) exhibited with narrow elliptic shape and light green colour whereas the putative polyploids composed with broad elliptic and darker green appearance. Then the hypocotyl thickness was much prominent for the higher concentration (0.5%) with its three exposure durations (Fig. 3). This organ thickness was helpful to make decision about the early screening for polyploid induction.
All the quantitative characters were observed at the vegetative stage of watermelon is disclosed in Table 1. At the beginning, germination response showed the prominent colchicine inhibitory effect. It was noted that for final germination (40 DAS), the highest germination (100%) was found in control at 72h duration and the lowest in the combination of 0.5% colchicine at 48h of time exposure (77.8%). On the other hand, the number of mortal seedlings were abundant in the field. Results revealed that the highest colchicine concentration (0.5%) with the 72h of time exposure expressed the highest influence on mortality percentage (42.2%) and the lowest mortality was registered at the control with the 48h of duration (6.3%). The similar trend of findings were also noticed regarding the speed of germination where higher concentration of colchicine had the less speed of germination. More specifically, it was revealed that the combination of 0.5% colchicine and 24 h of time exposure exerted the lowest speed (466.49). Meanwhile, the highest speed was found at control combined with the 48 h of exposure duration (563.10). The germination speed was found at the range of 547.96 to 5470.24, for the rest of the other treatment combination (Table 1).
In case of leaf blade, these were with some diversified responses in concern of their phenotypic appearance and quantitative attributes (Fig. 4 and Table 1). The leaf green colour intensity was found dark in nature for diploids (control treatments) and tetraploids (control 0.05, 0.1, 05% with three durations) but some of the putative octoploid plants belongs to the 0.5% colchicine with three durations was exhibited as lighter in colour. Interestingly, the degree of lobing like medium, weak and strong was varied according the ploidy level variation of diploid, tetraploid and octoploid (Fig. 4).
Considering the phenotypic appearance of leaf structure in the cultivated duration 0.5% colchicine exhibited the smallest third leaves production whereas the control treatment always appeared with the double sized third leaves. Consequently, the highest leaf length was found at the response of control with 48h after 45 days of transplanting (10.5cm). Statistically the highest leaf width (12.7cm) was found in 0.1% colchicine concentration and 24 h of duration. Also, at C2×D2, C3×D2 in leaf width were found higher viz. 11.9, 11.1 cm respectively. On the contrary, the higher colchicine concentration (0.5%) with three duration were appeared as the representative of lower leaf width viz. 5.3, 6.3, 6.0cm for the C4×D1, C4×D2 and C4×D3 interactions.
Other organs like leaf petiole, stem was also showed some differences. Petiole length fluctuates in between 3.6-6.2cm where the lowest petiole length (3.6 cm) represented by the effect of 0.5% colchicine at 24 h duration and the highest petiole length was found in the interaction of 0.1% colchicine at 24 h of duration. Then, most of the stem diameter showing with the range of 8.3-7.6mm where the highest interaction effect (8.3mm) was observed with the 0.1% colchicine at 24 h of duration. Whereas the lowest effect (4.6mm) was found in the 0.5% at 48h of duration. In fact, the higher concentration with three different durations ranging the stem diameter value lower in tendency (4.6-5.4mm) compared to the others. Besides, the control associated interactions were assessed as massive in growth. Although at 45 DAT, 0.05% and 0.1% colchicine concentration in combination with 24h exposure duration manifested plant height of 73.5 cm of each. While the tallest plant (74.3cm) was registered in 0.1% colchicine when combined with 48h soaking durations.
Male flower exhibited differences after colchicine treatments. Therefore, differences were noticed for the petal number of male flowers. Generally, the petal number of diploid male flowers is 5 (Zygomorphic) in watermelon plant. But in the present study some flowers were found with 6 petals treated with 0.5% colchicine (Fig. 5).
Besides, quantitative characteristics varies with the different level of colchicine concentration at their exposure durations (Table 2). So, from the Table 2 it was distinguishable that 17.0mm petal length with the 0.1% colchicine at 24 h of duration and however 0.5% colchicine at 24 h & 48 h did not produce any flowers during the whole season of flowering. Moreover, at 0.5% of colchicine with 72 h duration also had the lowest (8.5mm) interaction effect among rest of the interaction effects. The interaction between the control with the three durations shows the 6.4-6.7mm of petal breadth that is near to the lowest (5.2mm) of 0.5% concentration with the 72h duration treatment. Rest of the treatments ranging the average petal breadth in between 9.0-10.5mm where 10.5mm for the 0.05% colchicine with 24h of duration. The highest value of average node number for male flower initiation is near 5, that is 4.8 for the control at 24h, 0.1% at 48h; on the contrary the lowest (3.0) was found with treatment of 0.5% colchicine at 72h. Early days of flowering (lowest days required) was noticed in the plants treated with 0.1% at 72h of duration (20.7) and 0.5% colchicine at 72h of duration (21.8). On the other side, rest of the treatments required 42.7 to 46.0 days to initiate first male flower at 0.5% colchicine with 72h.
Pollen features like viability, diameter gave the precise outcomes to distinguish the diploid to polyploids (Fig. 6). From the Fig. 6, it has been illustrated the pollen appearances where smaller and larger pollens was considered as the diploids and tetraploids respectively; whereas mixture of different size of pollens was considered as the mixoploid. Subsequently, significant differences were observed for pollen diameter where the highest (5.6 µm) pollen diameter was measured at 0.5% colchicine concentration with 72h of duration and rest of the interactions were found to had statistically similar pollen diameter as that of control associated treatments ranged from 4-5µm. Furthermore, the lowest viability (57.0%) was observed for the control at 48h of duration. However, the control treatment with 72h and 0.1% concentration with 48h treatment also showed higher percentage of viability viz. 95.2% and 94.4%, respectively.
Watermelon plant always mature with the tendency of less female flowers. In this study, no female flower was observed in various treatments like control with 24 h, 0.1% colchicine with 72 h duration and 0.5% colchicine with all the durations. Besides, the other treatments also contain less flowers that’s why the effects of colchicine and duration was found the less effective individually derived from data analysis. Less flower production also appeared in the field throughout the season; some treatments showed no flower production in both male and female. Therefore, four treatments effect showed higher in their average node number for the first female flowering viz. 8.3 for 0.05% at 72h duration, 5.5 for 0.05% at 48h of duration and control with 72h duration, 5.2 for the 0.1% with 24h. Rest of the interactions showed average node number of ≤ 3.0. The maximum days (61.0) requirement was found at 0.05% of colchicine with 72h of duration; whereas lesser duration for the first female flowering was found at the 0.1% with the 24h, control with 72h and 0.05% colchicine with 48h of exposure duration viz. 39.2, 38.7, 35.3 respectively. The other treatments showed the days requirement ranged between 17.5-20.3.
Anatomical attributes to identify polyploids
The diploid plants have 8-9 chloroplasts were found within the stomatal guard cells of leaf blade that is observed in control treated plant. However, putative mixoploid contained 10-12 chloroplasts within the stomata and sometimes fused together within the guard cell and were not clearly visible. Interestingly, putative polyploids (tetraploid, octoploid) generally had 14-18 chloroplasts. (Fig. 7) Usually, the 0.1% and 0.5% colchicine with their three exposure durations possessed the 14-18 chloroplasts.
Quantitative anatomical characterization related to stomatal attributes are visualized in Fig. 8. These attributes were found to be beneficial for PIE% confirmation. At first, for stomatal length the lowest (15.9 µm) response was found in 0.05% colchicine at 24h (C2×D1) and the highest for 0.5% colchicine with 48h duration (C4×D2). Most of the interaction response were above 20 µm (C1×D2, C2×D3, C3×D1, C3×D2, C3×D3, C4×D1, C1×D3). Other treatments effect was below or near 20 µm (C1×D1, C1×D3, C2×D2). Then the highest concentration 0.5% with three durations (C4×D1, C4×D2, C4×D3) were found the average diameter ≥13.0 µm. Rest of the interactions effect were found between 10.6-12.8 µm where the lowest 10.6 µm diameter belonged to the control with 72h (C1×D3) of duration. The 0.5% colchicine concentration showed higher in average area response viz. for 48 hour 171.2 µm2, 72h 152.8 µm2, 24 h 136.4 µm2. Rest of the area maintained the value below 130 µm2. Also, the lowest one was found in control at 72h (C1×D3) below 100 µm2 viz. 88.6 µm2. So, with higher colchicine concentrations the stomata appeared as larger in size. In case of distance between two guard cells there are some distinguishable responses were found viz. highest (4.0 µm) response for the 0.5% colchicine at 72h of duration (C4×D3); whereas the lowest (1.8 µm) was found in both control at 24h duration and 0.05% colchicine at 24h duration (C1×D1, C2×D1). Most of the response were found within the range of 1.9-2.8 µm. It also indicates the stomatal enlargement by colchicine mutagen.
Finally, PIE% was assured on the basis of all the morphological and anatomical parameters (Fig. 9). From the findings it has been revealed that 0.5% colchicine with three durations showed the higher induction efficiency viz. C4×D3, C4×D2, C4×D1 followed by 14.5, 11.3, 9.7% efficiency. The colchicine treatment at 0.1% with its three durations maintaining the order of PIE% as C3×D3>C3×D1>C3×D2 were 9.4>7.8>5.2%. Besides, the other treatment combinations like C2×D3, C2×D2 and C2×D1 showed the lowest in PIE% viz. 4.0, 1.8, 1.5% respectively. So, the putative polyploids were more detected in the higher colchicine concentration (0.5%) with the highest duration (72h).
Pearson’s correlation analysis and dendrogram cluster
Polyploid induction demands the changes in various parameters with evident interrelations. Pearson’s correlation matrix showed the extent of both positive and negative correlation among the 20 studied variables. The Figure 9A are distributed from negative to positive values indicated by the red to blue-colored circles. Vacant cells were considered as nonsignificant relationship among the variables at 5% level of significance. From this analysis it has been revealed that most of the studied variables related to vegetative and reproductive morphology positively correlated to each other. But stomatal attributes like SPM, SD negatively correlated to SL. Also, reproductive variables FFM, NNM, PL and PW had the negative correlations. Besides, PIE% had the negative correlation with FFM, LL, PH, PLC, SD (at p < 0.05). That means, if one variable response increases, then the other one will be increased too for positive correlation and vice versa for negative correlation. Big sized circle denotes the stronger correlation between the two respective variables.
Pheatmap with dendrogram cluster prepared using the 20 studied dependent variables depicted 2 major clusters. Cluster 1 showed the characters like SL, PIE, GCD and MF closely related to each other. Less difference was discoverable among them in the concern of their corralation showing tendency with other parameters. For cluster 2 SPM, NNF, FFF, GF, GS, LL, PH, PLC, LW, PV, PD, PL, PW and SD, node number of first male flower initiation (NNM) and duration of first male flowering (FFM) characters were included. Although, the SPM and GS was found in a subcluster which was seperated from the other characters and rest of the characters also form several small clusters. These multiple clusters in single major cluster indicates the variation among the characters even they were similarly correlated to each other at some extent
Principal component analysis (PCA)
The data presented in Table 3 revealed that first two principal components (PC) contributed enough to explain maximum (about 59%) of the pattern variations. Individually, only PC1 and PC2 can explained 46.68% and 12.09% of the total variations. Therefore, from the PCA (Table 4) it is evident that FFM, LL, PH, LW, PLC, SD, NNM, PL, PW have the highest positive loadings on PC1. On the other hand, for PC2 only LL comprises the highest positive loadings. Negative loadings suggested that, SL have the highest value for PC1 and MF, PIE, GCD consists most of the negative loadings. Hence, for biplot-PCA cluster analysis using colchicine concentrations illuminated that 0.5% (C4) concentration form the distinct cluster by including those variables which had the negative loading values (Fig. 11A). Also 0.1% (C3) can influence these parameters to some extent with having rest of the parameters. So, these dependent variables definitely get the dependency by the 0.5% and 0.1% concentration. Rest of the two concentrations not influence negatively loaded parameters including PIE. Therefore, it is clearly evident that among the three studied concentrations specifically 0.5% concentration have the remarkable influence in PIE. Similarly, for the duration effect 72h (D3) have the distinct cluster than the other having most of the parameters and most importantly PIE or other variables shown negative loadings (Fig. 11B).