2.1. Plant Materials

The plant materials used in this study were obtained from a previous study conducted by Eng et al., [11]. Fully developed young leaves from both the control plantlets and the plantlets treated with colchicine were collected for ploidy level determination. The untreated plantlets served as the control group, while Glycine max Merr. “Polanka” was used as an external standard in the FCM analysis, following the method described by Doležel et al. [12].

2.2. FCM Analysis

To optimize the FCM study for N. cadamba, two types of leaves were assessed. Leaf samples for the FCM optimization study were obtained from ex vitro and in vitro N. cadamba plantlets. The ex vitro N. cadamba leaf samples were harvested at least 6 h before their transportation to the Advanced Technology and Breeding Center at the Malaysian palm oil board (MPOB) in Bangi, Selangor. During transportation, the harvested leaves were placed in a moistened ziplock bag. On the other hand, the in vitro plantlets were transported from UNIMAS to MPOB in culture vessels. The in vitro leaves were harvested at MPOB right before the FCM study.

Two lysis buffers, LB01 and Otto’s buffers, were evaluated to optimize the DNA extraction for FCM. LB01 buffer consisted of 80 mM KCl, 20 mM NaCl, 15 mM Tris, 15 mM mercaptoethanol, 2 mM Na2EDTA, 0.5 mM spermidine, 0.1% Triton X-100, pH 7.5, 50 μg propidium iodide, and 50 μg RNase [12]. Otto’s buffer comprised 0.1 M citric acid, 0.5% Tween-20, 50 μg propidium iodide, and 50 μg RNase [13]. For each sample, approximately 1 cm² of a fresh young leaf was chopped in 1 mL of either LB01 buffer or Otto’s buffer using a scalpel on a disposable Petri dish. The finely chopped leaf pieces were individually filtered through a 50-μm cell strainer into a 5 mL FCM tube. The filtrate of each sample was then analyzed using a FACSCalibur flow cytometer (Becton Dickinson). Histograms were obtained for each sample across 1024 channels.

2.3. Data Analysis

The FCM-generated histograms were used to determine (a) the ploidy level and (b) the DNA content of a sample. The formulas employed for these calculations are as follows:

To determine the ploidy level, the following formula by Guo et al., [14] was used:

S_PL = (S _(G0/G1)/R_(G0/G1)) × R_PL

where S_PL represents the ploidy level of the colchicine-treated sample, R_PL denotes the ploidy level of the control sample, S_(G0/G1) indicates the mean position of the peak in the colchicine-treated sample, and R_(G0/G1) signifies the mean position of the peak in the control sample.

To determine the DNA content, the formula developed by Doležel et al., [4] was employed:

S_PL = (S _(G0/G1)/R_(G0/G1))×R_PL

where S_PL corresponds to the 2Cx value of the colchicine-treated sample, R_PL represents the 2C value of the control sample (specifically, Glycine max Merr. “Polanka” with a value of 2.50), S_(G0/G1) indicates the mean position of the colchicine-treated sample, and R_(G0/G1) signifies the mean position of the peak in the control sample.

3.1. FCM Optimization of N. cadamba

The analysis of leaf samples obtained from ex vitro N. cadamba plants using a flow cytometer [Figure 1a] revealed poor results in the FCM analysis. The low count of events, only 1515, can be attributed to the rapid degradation of DNA in leaves within hours after harvesting. The low event number count in this study indicated that a significant number of cells were not analyzed, thus failing to represent the entire cell population of the plantlet. However, when the in vitro leaf samples were analyzed, the number of event counts significantly increased to 10,980, which was 10 times higher [Figure 1b]. This discrepancy indicated that utilizing leaves from ex vitro plants harvested at least 6 h before analysis resulted in minimal viable DNA that the cytometer could detect effectively.

Figure 1: Flow cytometer analysis of Neolamarckia cadamba plantlets. (a). The ex vitro control plant using Otto’s buffer (event counts 1,515; CV 9.19) and (b). The in vitro control plantlet using Otto’s buffer (event counts 10,980; CV 5.20). [Click here to view]

In this study, the plant samples from UNIMAS (Sarawak) and the flow cytometer located at MPOB (Selangor) were separated by a distance of 1000 km, and transporting plant samples were a major obstacle. To overcome this logistical hurdle, tissue-cultured N. cadamba plantlets cultivated in glass jars, which are more convenient for packing and transportation, were utilized. Since tissue-cultured plants are free from diseases, pests, and contaminants, they pose minimal phytosanitary risks. The presence of a horizontal laminar flow system in the laboratory where the cytometer is located facilitated the procurement of leaves without contamination. After analysis, the plantlets were transported back to UNIMAS for further maintenance and multiplication. Using in vitro plantlets as samples for FCM is a non-destructive method as they remain protected and viable throughout transportation and analysis. The vigor of the plantlets is therefore maintained.

Selecting fresh, pest-free, and contaminant-free explants are vital to isolate the intact nuclei. In this study, in vitro plantlets were chosen as FCM samples as they met these criteria. This precautionary measure prevents FCM from generating inaccurate data by detecting foreign or damaged DNA. DNA in plant samples begins to degrade after being excised from the plant, but the rate of deterioration varies depending on the species and environmental conditions [15,16]. This limitation has posed a significant challenge in FCM usage, especially when fresh plant materials are logistically or geographically distant from the flow cytometer. In addition, the flow cytometer is a large and sensitive machine, making it nearly impossible to relocate easily. Consequently, logistical issues arise when conducting field studies, as the samples are often situated in remote locations.

Seeds can be used as samples in FCM since they can be stored for extended periods without DNA degradation and lack cytosols [6]. Leaves, on the other hand, contain cytosols that may hinder the staining process using fluorochromes, thereby affecting FCM readings. However, data derived from seeds should be interpreted cautiously since they exhibit genetic variation due to the combination of male and female gametes. In the present study, we encountered difficulties in the FCM analysis when using fresh leaves obtained from acclimatized N. cadamba plants, which had undergone 6 h of packing and transportation, resulting in significantly low nuclear event counts during FCM. This failure indicated two potential causes, either propidium iodide stains were ineffective in staining deteriorated DNA, or cytosol oxidation-prevented DNA staining.

Several alternative methods have been developed to overcome the limitations of using fresh materials in FCM, but they are still considered inferior. These methods involve fixation, freezing, or dehydration [17]. Chemical-based fixatives, such as acid methanol fixation [18], glycerol [19], and formaldehyde preservative [20], have been employed to preserve plant samples before FCM analysis. Compared to non-fixed materials, these fixatives can enhance fluorochrome staining and increase nucleus yield. Dehydration of plant materials is another utilized method [21,22]. However, non-fresh plant material cannot be recommended for estimating DNA content in absolute units unless the protocols undergo further investigation and demonstrate reliability [4].

Both histograms [Figure 1a and b] exhibited broad bases, indicating a high coefficient of variation (CV) and reduced data reliability. Numerous secondary peaks, representing unwanted events or noise in the DNA sample, were observed alongside the dominant peak. The ex vitro sample in Otto’s buffer showed a CV of 9.19 [Figure 1a], while the in vitro sample in the same buffer displayed a CV of 5.20 [Figure 1b]. This suggests that Otto’s buffer is unsuitable as a lysis buffer for N. cadamba leaf samples as it produced a high CV value. To enhance the FCM data and reduce the CV value, Otto’s buffer was replaced with LB01 buffer. The use of LB01 buffer produced histograms with a CV value below five [Figure 2a], while also significantly reducing excessive noise between the dominant peaks. Again, in this study, a higher CV value was obtained with the ex vitro sample. The doubling of the CV value in the ex vitro sample indicates the deterioration of leaf freshness, which affects sample quality. The evidence from both FCM studies of N. cadamba demonstrates that only fresh leaves combined with LB01 buffer can generate clean histograms with sharper peaks, fewer noises, and low CV values.

Figure 2: Flow cytometer analysis of Neolamarckia cadamba plantlets derived from (a) nodal segment treated with distilled water (control) indicating tetraploid plant (2n = 4x), (b) nodal segment treated with colchicine indicating tetraploid plant (2n = 4x), (c) nodal segment treated with colchicine indicating mixoploid plant (2n + 4n = 4x + 8x), and (d) nodal segment treated with colchicine indicating octoploid plant (4n = 8x). [Click here to view]

Lysis buffers were formulated to preserve nucleus integrity, prevent DNA degradation, and enhance stoichiometric staining. Commonly used non-commercial lysis buffers include Galbraith [7], LB01 [12], Otto’s [13], and Tris.MgCl2 [23]. LB01 and Otto’s buffers have been identified as the most effective [24]. Modifying lysis buffers by adjusting the pH and concentration of existing components or incorporating additional chemicals has been a common practice to enhance their effectiveness [25]. However, certain commercial buffers are customized explicitly for specific cytometer models the vendor produces, and their compositions are often undisclosed. Therefore, carefully reading the cytometer instructions and maintaining close communication with the vendor is crucial when encountering any obstacles.

Plants often contain cytosolic compounds that interfere with the ability of fluorochromes to stain DNA. DNA staining aims to achieve sufficient fluorescence intensity for detection by the cytometer. The presence of cytosols can lead to inaccurate DNA content estimation [4]. To overcome the effect of cytosols, a reducing agent called β-mercaptoethanol is added to the buffers [26]. Spermidine, an oxygen quencher, can prevent oxidative damage to DNA while maintaining its integrity [27]. Additional additives such as PVP-10 and PVP-40 can bind phenolic compounds in plants [4]. However, according to Jedrzejczyk and Sliwinska [28], the inclusion of antioxidants such as β-mercaptoethanol and PVP has not been sufficient to overcome the effects of cytosols in DNA content estimation of 11 Rosaceae woody species. In this study, the LB01 buffer was supplemented with β-mercaptoethanol and spermidine to ensure reliable and accurate DNA content estimation of N. cadamba.

Propidium iodide was used as the fluorochrome in this study to determine the ploidy level and DNA content of regenerants in N. cadamba. The obtained DNA content of N. cadamba in this study aligns with the data reported by Ohri et al., [29]. The successful determination of N. cadamba DNA content can be attributed to the use of propidium iodide as the fluorochrome, as this stain intercalates into double-helix DNA without selectively binding to specific DNA bases. The inclusion of RNase is necessary to remove RNA and prevent the fluorochrome from binding to RNA.

The choice of DNA stains depends on the excitation source used in the cytometer. In this study, laser excitation was employed, specifically an argon ion laser (15 mW) at 488 nm. Intercalating fluorochromes are more commonly used compared to base-specific fluorochromes. In a survey of three articles by Bennett and Leitch [30,31] and Bennett et al., [32], it was found that out of 892 data points for DNA content, 789 were obtained using intercalating fluorochromes, with propidium iodide being used in 689 out of the 789 cases. On the other hand, DAPI and Hoechst 33258 are used to stain DNA in flow cytometers equipped with an arc lamp. One advantage of using these stains is that RNase is not required [4]. However, these stains preferentially bind to the A-T base pair in DNA, and they are less accurate in estimating DNA content. As a result, DNA content estimation cannot be interpreted as absolute units but as relative units to the control. Therefore, when absolute units of DNA content estimation are required for a study, it is advisable to avoid using a flow cytometer equipped with an arc lamp that utilizes DAPI and Hoechst 33258 as stains, as the estimation will be in relative units compared to the control.

3.2. Ploidy Level and DNA Content Determination of N. cadamba

The histogram generated by FCM analysis revealed distinct patterns in DNA content for different plant types. Both control and tetraploid plants exhibited a single dominant peak, with the mean position corresponding to 4x [Figure 2a and b]. In contrast, mixoploid plants showed two separate dominant peaks in the histogram. The first peak corresponded to 4x, similar to tetraploids, while the second peak corresponded to 8x [Figure 2c]. Octoploid plants displayed a single dominant peak at 8x [Figure 2d]. Notably, the mean position of octoploid cells was twice that of tetraploid cells.

FCM is a valuable tool for detecting mixoploids and quickly analyzing large cell populations. Previous studies on polyploidization, conducted without using FCM, reported high percentages of polyploidization without detecting mixoploids. For instance, percentages of 41.7% for Vitis sp. shoot tips [33], 42.3% for Gossypium arboreum seeds [34], and 100% for Pogostemon cablin leaves [35] were determined through the chromosome counting method. To avoid mixing mixoploids with induced polyploids or the original ploidy category, FCM should be employed in these studies to identify mixoploids accurately.

Mixoploids, consisting of cells with different ploidy levels, represent genetic variants. However, the extent to which the composition of mixoploids changes over time in terms of the proportions of original cell ploidy and polyploid cell ploidy remains uncertain. According to Regalado et al., [36], mixoploid plants are unstable due to competition between the original cells and the polyploid cells, leading to the elimination of the latter. As a result, the mixoploid status reverts back to its original ploidy level. Due to the complexities involved, mixoploids have often been excluded from further characterization or study on their detection [3,37-39]. While generating biodiversity and genotype variation through polyploidization is well-known, the study of mixoploids has been limited, resulting in a scarcity of published literature on the subject. There is currently a lack of empirical evidence regarding the unfavorable aspects of mixoploidy in plant breeding. Therefore, this area deserves more attention and further investigation in the future, including aspects such as productivity and growth, fertility testing through the crossing, adaptation to different environments, and the extent of genotype stability.

The analysis of FCM data revealed that N. cadamba tetraploids (4C DNA) had an estimated DNA content of 2.59 ± 0.09 pg, while octoploids showed a twofold increase in DNA content (8C DNA) at 5.35 ± 0.24 pg. These findings are consistent with the results reported by Ohri et al., [29]. The estimated 4C DNA content for Anthocephalus cadamba (synonyms: N. cadamba), according to Ohri et al., [29], is 2.77 ± 0.27 pg using the M86 Vickers microdensitometer. These data have been published and archived in the C-value Kew Garden database (http://www.kew.org/cval/homepage.html), where the values are reported as estimated 1C DNA, equivalent to 0.69 pg. These findings confirm that N. cadamba, the species under investigation in our study, is a tetraploid. Based on these data, our estimated 4C DNA content of N. cadamba falls within the published range at 2.59 ± 0.09 pg. In addition, our study observed that the estimated 8C DNA content of octoploids was 5.35 ± 0.24 pg. In our experimental setup, the use of colchicine, a chromosome-doubling agent, resulted in doubling the DNA content of N. cadamba octoploids. Estimated DNA content serves various purposes beyond indicating changes in ploidy levels, including molecular investigations, evolution, phylogenetics, phenotypic studies, phenological analyses, ecological and environmental indicators, as well as paleobiological trends [32].