Callus stage and morphology affect the DNA yield on the DNA extraction from the sugarcane in vitro callus

The main concern with the micropropagation of sugarcane (Saccharum officinarum) with a complex polyploid genome is the frequent occurrence of somaclonal variations. Therefore, genetic stability analysis is often required to ensure the uniformity of the micropropagated sugarcane, as early as in the callus stage. DNA extraction is the initial and important step of the genetic stability analysis. In this experiment, we compared the yield of DNA extraction from the sugarcane embryogenic and non-embryogenic calli. The sugarcane non-embryogenic callus was initiated from immature rolled leaf explants of the 6 months-old plant cane in the callus induction media, that were incubated in the dark room. The embryogenic calli were obtained by routine subcultures of the non-embryogenic calli every six weeks. The results showed that the extraction yield from the non-embryogenic calli was far lower than that from the embryogenic calli, most likely due to the high-water content of the non-embryogenic calli. Furthermore, histological analysis using Safranin and Fast Green staining revealed the low lignin content and nuclei in the non-embryogenic calli cell mass, causing the transparent and watery appearance of the calli and might influence the low DNA extraction yield.


Introduction
Sugarcane is an important crop worldwide as the main sugar-producing plant.Sugarcane is propagated primarily by cuttings of the stalk (seed cane), containing usually two or more nodes with buds.This traditional method however has the disadvantage of being time-consuming [1].Another seedling production technique is plant tissue culture, known as micropropagation.The advantages over the traditional techniques are not needing much manpower, large nurseries, logistic storage, transportation, and irrigation [2].Recently, many commercial plantations have applied micropropagation, especially to produce and propagate their elite sugarcane varieties [3][4][5] 2 Most of the commercially available sugarcanes are complex interspecific hybrids with polyploid genomes, primarily between Saccharum officinarum and Saccharum spontaneum [6].On one side, polyploidy in sugarcane is considered an advantage for breeding programs as it allows the development of sugarcane varieties with multiple desired traits [7][8][9].On the other side, the highly complex chromosomal structure of polyploid sugarcanes may increase the frequency of somaclonal variations (SV) formed during micropropagation, which is unfavourable for seedling production.
SV is the variation that often occurs in plants produced by tissue culture, caused by genetic or epigenetic alterations compared to the mother or donor plants [10][11][12][13].SV is often related to cell exposure to hormones contained in the tissue culture media [14].The occurrence of SV in the sugarcane micropropagation products can be detected by genetic analysis using molecular markers such as amplified fragment length polymorphism (AFLP) [15], random amplified polymorphic DNA (RAPD) [16], target region amplification polymorphism (TRAP) [17], inter simple sequence repeat (ISSR), and microsatellite or simple sequence repeats (SSRs) [18,19].
DNA extraction is a crucial initial step in genetic stability analysis using molecular markers.The DNA yield is determined both by the extraction method [20] and the type or condition of the plant materials [21,22].In the genetic analysis of micropropagated sugarcane, DNA was often extracted from callus or in vitro-generated shoots.In the DNA extraction from callus, an efficient method is especially crucial to ensure adequate quality and amount of genomic DNA since the initial amount of samples is usually limited.Therefore, this study was conducted to evaluate the effect of different stages and morphology of the sugarcane calli on the DNA extraction yield.

Preparing the Sugarcane Callus
The sugarcane variety PS 094 released by the Indonesian Sugar Research Institute (ISRI), Pasuruan, East Java, was used as a plant material for this study.Callus was initiated from immature rolled leaf explants of the 6 months-old plant cane.Explants were cleaned with ethanol wipes, followed by further sterilization in the Laminar Air Flow (LAF) cabinet.Sterilization was conducted by spraying the explant with 70% ethanol then flaming it with a burner.The sterile explant was then sectioned crosswise with 0.5 cm of thickness.
Pieces of explants were then inoculated into jar bottles containing sugarcane callus induction media.Sugarcane callus induction media contained Murashige & Skoog's (MS) basal minerals added with 30 g L -1 sucrose, 3 mg L -1 2,4-Dicholorophenoxy acetic acid (2,4-D), and 3.5 g L -1 media solidified agent, and was previously sterilized in the autoclave with 1 atm of pressure, 121 °C of temperature, for 15 minutes.Explants cultures in the bottles were kept in the dark room with 25 °C of temperature until callus emerged from the explants, in about 2 -3 months after inoculation.The initial callus was the non-embryogenic callus.It was then subjected into routine subcultures every 6 weeks using the same media composition.After 3 -4 times subcultures it achieved the embryogenic callus.
Determination of the non-embryogenic calli and embryogenic calli was conducted based on the morphology and histological observation.The calli water content was also measured, as the percentage of the difference between the calli fresh weight and dry weight.The dried callus for dry weight measurement was prepared by collecting and placing about 5 gram of the non-embryogenic calli and embryogenic calli in the aluminium foil.The calli were then kept in an oven at 60 °C for 48 h.Afterwards, the dry weight was measured by using a digital balance.

Histological Analysis of the Sugarcane Callus
The sugarcane callus was fixated in the buffered formaldehyde (FAA), followed with dehydration, infiltration, embedding steps in paraffin.The samples embedded in the paraffin bloks were then sectioned into slices (± 15 uM of thickness) using microtome, and the slices were put into object glasses, followed by staining with Safranin 1% and Fast Green 1% (Merck, Jerman).Afterwards, the cover glass was added onto the glass object, and the samples on the glass object were observed under digital stereo microscope.

Standard Callus DNA Extraction
DNA was extracted from the sugarcane embryogenic calli and non-embryogenic calli, following the method of Orozco-Castillo et al. [27].The initial weights of the callus sample used for extraction were 50, 100, 200, and 400 mg.The calli were ground with micropestels in a 1.5 mL tube.500 uL of buffer containing CTAB (2% CTAB, 0.2 M EDTA pH 8.0, 1 M Tris-HCl pH 8.0, 1.2 M NaCl, 1% ß-Mercaptoethanol) was added and the mixture is incubated in a water bath at 65 °C for 30 min, mixed occasionally and cooled to room temperature.Afterwards, 500 uL of chloroform: isoamyl alcohol 1:1 (v/v) was added and mixed by inverting.The mixture was centrifuged at 11,000 rpm for 15 minutes at room temperature.The aqueous phase is moved to a new tube and mixed with cold isopropanol 1:1 (v/v) and incubated at 4 °C for 30 min.Further centrifugation was carried out at 11,000 rpm for 10 min (4 °C).The pellet was separated and mixed with Tris-EDTA (10 mM of Tris-HCl, 1 mM of EDTA pH 8), CH3COONa 3 M pH 5.2, and 1:10 (v/v) ethanol, and was then incubated at -20 °C for 2 h.The mixture was then centrifuged at 11,000 rpm for 10 min (4 °C) and the pellet was washed with 70% ethanol.The dried pellet was then resuspended in 50 µl TrisEDTA buffer.Thereafter, the quantity of the purified DNA was measured with a NanoDrop spectrophotometer (Thermofisher), and the quality was analysed using gel electrophoresis.The purified DNA was then used for PCR.

Rapid Callus DNA Extraction
DNA was extracted from the sugarcane embryogenic calli and non-embryogenic calli.About 100 of calli were ground with micropestels in a 1.5 mL tube.500 uL of buffer containing CTAB (2% CTAB, 0.2 M EDTA pH 8.0, 1 M Tris-HCl pH 8.0, 1.2 M NaCl, 1% ß-Mercaptoethanol) was added and the mixture is incubated in a water bath at 65 °C for 30 min, mixed occasionally and cooled to room temperature.Following that, 500 uL of chloroform: isoamyl alcohol 1:1 (v/v) was then added and mixed by inverting.The mixture was centrifuged at 11,000 rpm for 15 minutes at room temperature.The aqueous phase is moved to a new tube and diluted 10 times with water before used for PCR.The quantity of the DNA were measured with a NanoDrop spectrophotometer (Thermofisher), and the quality was analysed using gel electrophoresis.

Results and Discussion
Sugarcane micropropagation was commonly conducted via organogenesis or somatic embryogenesis involving callus stages.Callus is a desirable material for micropropagation since it easy to be multiplied or treated.There are at least two types of the sugarcane callus, the nonembryogenic callus and embryogenic callus.The embryogenic and non-embryogenic calli differ in their structures and properties.
Morphological analysis of the non-embryogenic calli showed a soft watery structure and translucent colour (Figure 1a), while the embryogenic calli were shown to be dry friable and less translucent than the non-embryogenic calli (Figure 1b).Consistently, histological analysis of the calli using Safranin and Fast-Green staining revealed the low lignin content and nuclei in the nonembryonic calli cell mass (Figure 1c), while the embryogenic calli was possibly rich of nuclei, indicated by the prominent red colour, and the cytoplasm rich of cellulose and protein, indicated by the dense blue colour (Figure 1d). Figure 1d in the box also revealed asymmetric cell division of the embryogenic calli.Safranin stained red any acidic features of cells or tissue such as lignified cell walls, secondary cell wall surrounding vascular system, and nuclei [23].Whereas, Fast-Green stained cellulose, protein, and cytoplasm blue.The lack of lignified cell walls and cellulose/protein rich cytoplasm in the non-embryogenic calli (Figure 1b) might cause the transparent and watery appearance of the non-embryogenic calli (Figure 1b).Moreover, measurement of the calli water content revealed a higher water content in the non-embryogenic calli (Figure 2a).Other studies also characterized the embryogenic callus with having relatively smaller vacuole, and bigger nucleus with very dense nucleus, while the opposite appear in the non-embryogenic callus [24,25].Having bigger nucleus and lower water content is expected to result in higher DNA amount.Agreed with the hypothesis, the DNA yield from the non-embryogenic sugarcane calli was significantly lower (about one-twentieth) than the DNA yield from the embryogenic calli (Figure 2b), with the standard callus DNA extraction method.The total amount of DNA obtained from 100 mg embryogenic and non-embryogenic calli were 6.732 ng and 314 ng, respectively.The result also agreed with the previous study from Betekhtin et al. [25], reporting lower relative DNA content from the non-embryogenic calli compared to the embryogenic calli, by a flow cytometry analysis.The study also revealed that the embryogenic calli has greater genome stability compared to the non-embryogenic calli, as the non-embryogenic calli showed significantly higher level of DNA damage on the TUNEL test, which might be also associated with the low DNA yield from the non-embryogenic calli.
A greater amount of calli and a more efficient extraction method are needed to obtain a higher amount of genomic DNA from the non-embryogenic calli.Luk et al. [26] reported that the cells concentration also influences DNA extraction yield. Figure 3 showed that a higher amount of calli resulted in higher DNA yield.A maximum of 1,200 ng DNA could be obtained from 400 mg of sugarcane calli, while with 200 mg calli, it could be obtained 800 ng DNA.However, 200 mg of calli is considered sufficient to obtain an adequate amount of genomic DNA for further genetic analysis.A rapid callus DNA extraction method involving only lysis and chlorofom-isoamyl extraction steps were utilized to extract DNA from both types of calli.The diluted lysate showed DNA band in the gel electrophoresis analysis (Figure 4), and while still contains impurities, it can be used as templates in PCR analysis (data not shown).This rapid method allows DNA extraction and analysis from a smaller amount of non-embryogenic calli.

Conclusion
Sugarcane embryogenic callus is a better source of genomic DNA as it gives a higher extraction yield.A sufficient amount of DNA for analysis could be obtained from 200 mg of calli, yielding a good quality DNA for genetic stability analysis.

Figure 1 .
Figure 1.Morphology (a,b) and histological (c,d) figures of the sugarcane embryogenic and nonembryogenic calli

Figure 2 .
Figure 2. Water content (a) and DNA yield (b) from the sugarcane embryogenic and nonembryogenic calli

Figure 3 .
Figure 3.DNA yield from the sugarcane non-embryogenic calli with different initial concentrations of the callus sample

Figure 4 .
Figure 4. DNA yield from the sugarcane non-embryogenic calli with different initial concentrations of the callus sample