This study reveals that the polyploid induction of N. oculata was started at a lower concentration of 10 mg L− 1 colchicine which shows the increase of DNA content and cell size. At higher concentration, it increases pigments such as chlorophyll and carotenoid. In general polyploid induction using a mutagenesis agent enhanced biomass production and pigments concentration as shown in this study.
Polyploidy is one method that is widely used to domesticate plants by doubling the entire set of chromosomes (Yildiz & Yildiz, 2013). Polyploidy can be applied to microalgae to obtain microalgae with increased pigment that can be used for food colorant, cosmetics, and pharmaceuticals, as a substitute for synthetic pigments (Begum et al., 2016). Studies by (Le-Feuvre et al., 2021) and (Nezhad & Mansouri, 2019) reported the use of this technique to increase astaxanthin and β-carotene, respectively. In this study, the concentration of colchicine used started from a lower range compared to previous studies, which showed that a concentration of 10 mg L− 1 was able to induce polyploidy in N. oculata. Induction over a longer period of time and at lower concentrations can be used to obtain ploidy organisms (Manzoor et al., 2019). Lower concentration of colchicine would be more favored since it will not causing much cell death from its toxicity.
Increasing the biomass and lipid content of N. oculata was previously tested in the study of (Rezasoltani et al., 2019) with the addition of cyanobacteria Anabaena vaginicola extract. Cyanobacteria in the culture as a substitute for nitrogen fertilizer. Another study used nitrogen deprivation to obtain increased biomass and lipids in N. oculata (Tran et al., 2016). Reducing chlorophyll content is useful for increasing algae biomass and facilitating the lipid extraction process (Hunt et al., 2010). Research by (Cecchin et al., 2020) increased lipid productivity in N. gaditana by lowering nitrogen levels and selection of mutants with pale green pigments which resulted in a 30% reduction in chlorophyll and an 80% increase in lipid productivity. This result is suspected in the N148 group which is polyploid but does not produce more pigment than N348, however it is necessary to measure the lipid content present in this group in further studies.
N. oculata cells autofluorescent in this study was measured by a CLSM. In general, autofluorescent in plants can be used for biochemical, physiological and imaging studies. There are several molecules that exist in the same location in the cells. Chlorophyll autofluorescence is used in laboratory testing and remote sensing applications to evaluate cell health/vigor, as well as being a technique for evaluating plant response to environmental changes (Lamb et al., 2018; Mohammed et al., 2019). Chlorophyll is excited by UV, blue or green light and emits strong red light with emission maxima bimodal at 685 and 720–730 nm (Donaldson, 2020; Lamb et al., 2018). In this study using a PE channel with excitation at a wavelength of 543 nm. The results of this study showed that the strongest red fluorescence was in the N348 group. Carotenoids absorb blue light and transfer that energy to nearby chlorophyll molecules, thereby increasing energy absorption for photosynthesis (Sun et al., 2022). Weak autofluorescence at 560 nm was reported and detected as carotenoids found in green algae (Kleinegris et al., 2010). Green autofluorescence is associated with lignin whose composition is coniferyl, sinapyl or p-coumaryl alcohol, flavonoids, stilbenes, etc. (Donaldson, 2020; Donaldson & Williams, 2018). It is expected that there is an increase of these compounds inside the N. oculata cells after polyploid induction.
Stability of genome size after polyploid induction might have become another problem. This loss is more pronounce in early polyploid generation (Wang et al., 2021). These downsizing of the genome might be advantageous for the cells on photosynthesis, water efficiency and/or nutrient cost. This is thought to occur in the N348 group which has smaller cell size, more pigment and also a better growth rate than the wild type.