Comparison of three different RNA extraction kits for transcriptome analysis of Indonesian rice (Oryza sativa L) explant for anther culture

Recalcitrancy is the main problem in rice anther culture, especially in an Indica subspecies. It is well known that recalcitrancy is genotype dependent, and the effort to tackle this problem is mostly in the manipulations of media. Uncovering the genes responsible for recalcitrancy will benefit to enhance the success of this technique to accelerate the breeding process of Indonesian rice varieties which mostly belong to the indica subspecies. RNA-Seq is considered one of the best approaches to finding candidate genes responsible for specific traits including recalcitrant in anther culture. The aim of this study was to obtain the high concentration and good quality of RNA extracted methods using various extraction kits. The RNA extraction was carried out using Tripure, Promega, and RibospinTM Plant (Geneall) kit methods. Quality of RNA provided by the Promega kit showed good quality of RNA in all three genotypes tested ranging from 2.06±0.01-2.11±0.00 for A260/280 and 1.86±0.14-1.95±0.10 for A260/230.


Introduction
It has been reported that 7.6 billion people were estimated to increase to 10 billion by 2050 [1].The future of food security will depend on the plant breeding solution, especially in developing countries [2].Improving agriculture productivity through the integration of new technology in plant breeding is considered powerful [3].Previously, the conventional breeding approach through crossing strategy followed by line development played an important role in enhancing agriculture productivity.Pedigree, recurrent selection, back-cross, and mutation induction were the ultimate methods in conventional breeding [4].
In the conventional breeding approach, eight to ten years were needed to develop new varieties, which required crossing, line development, and field testing [5].In the development of new hybrid rice varieties using cytoplasmic male sterility (CMS) to obtain restorer lines, [6] reported that it needed at least three to four years.To provide rice varieties with better quantity and quality, climate-resilient, as well as resistant to pests and diseases in a short period, plant breeding re-orientation is needed to shorten the breeding cycles [7].Haploid breeding offers a rapid method to produce new varieties to provide food for two or three decades ahead [8].
Haploid breeding through anther culture for indica rice is more problematic than the japonica sub-species [9].Indica genotypes were known as recalcitrant and became the main factor in the limitation of the utilization of this technique.Meanwhile, the improvement of the approach through the manipulation of the media and the growing environment is still ongoing.A better understanding of the processes involved in reprogramming microspores to embryogenesis in anther culture is needed [10].To cope with recalcitrancy in indica rice, the utilization of the culturability of parental lines became important.The previous report showed that most of the mega rice varieties in Indonesia are recalcitrant 1255 (2023) 012052 IOP Publishing doi:10.1088/1755-1315/1255/1/012052 2 to anther culture.A few varieties were reported to have high culturability, such as Gadjah Mungkur and Fatmawati [11;12].So cracking recalcitrancy problems through a molecular approach using Indonesian rice varieties is needed.
Uncovering the genes responsible for recalcitrancy will enhance the success of this technique to speed up the breeding process of Indonesian rice varieties belonging to the indica subspecies.This information could be used as fundamental steps to undertake innovation and manipulation to make anther culture more regularly used by breeders in Indonesia.Providing new high-yielding varieties, resistance to pests and disease, and premium rice quality will be faster.
Next-generation sequencing (NGS) is a tool for identifying and characterizing genomic variations associated with agronomic traits.This technique can unravel the recalcitrancy problem in rice anther culture.Moreover, RNA sequencing (RNA-Seq) is considered one of the best approaches to finding candidate genes responsible for specific traits including recalcitrant in anther culture.In addition, transcriptome data from various tissues can be obtained by this method [13].The broadness of the NGS application became a tool for geneticists to identify and characterize genomic variations associated with agronomic traits.Regarding the NGS, RNA-Seq offers functional context to the candidate genes [14].RNA-Seq is considered one of the best approaches to finding candidate genes responsible for specific traits including recalcitrant in anther culture.This study aimed to evaluate RNA extraction methods on explant for rice anther culture for transcriptomic analysis using three different kit extraction.

Plant materials and sample preparations
Three Indonesian rice varieties with different backgrounds of culturability were used namely: Fatmawati (high culturabiliy; new plant type), IR64 (recalcitrant; indica sub-species), and Tarabas (Unknown; japonica sub-species).These genotypes were planted in pots and grown in the greenhouse of the Agroecotechnology study program at the University of Trunojoyo Madura, East Java, Indonesia.Fertilization and the management of plants were done according to [11].
Rice anther was collected at the booting stage, where the distance between the flag leaf and penultimate leaf was 5-10 cm.The samples were then rinsed in running tap water and wrapped with wet tissue paper and a plastic bag.Cold pretreatment was conducted by incubating panicles in cold storage at 5°C of temperature for eight days before RNA extraction.

RNA isolation methods and total RNA quality and quantity check
Anther explant of three Indonesian rice varieties was collected and total RNA was isolated by using three kits for RNA extraction such as RibospinTM Plant (Geneall), Tripure TM Isolation Reagent (Roche ® Life Science), and SV Total RNA Isolation System (Promega) according to the manufacturer's protocol.The quantity of extracted RNA was examined using spectrophotometer nanodrop ND100 and the quality was analyzed by running in 1% agarose gel electrophoresis and visualizing under a UV transilluminator.

RNA concentration of Indonesian rice varieties in three different RNA extraction kits
The total extracted RNA concentrations are presented in Figure 1.Fatmawati had the highest concentration in the Ribospin kit (139.13 ng/µL), but the Tripure kit had the lowest (188.73 ng/µL).Meanwhile, Tarabas performed the least since produced <100 ng/µL of RNA (72.85 ng/µL).Similar findings were reported by [15], the Ribospin kit generated a concentration of RNA lower than the two other kits.However, in the Promega kit, Tarabas generated the highest concentrations of RNA (224.43ng/µL), compared to other genotypes Meanwhile, IR64 was the lowest (133.51 ng/µL) but it was good enough for reaching a concentration > 100 ng/µL.In Tripure Kit, RNA extracted from Tarabas gave the highest concentration (500.30ng/µL), followed by IR64 (405.40 ng/µL), and the lowest RNA showed by Fatmawati (188.73 ng/µ).Based on these results, each genotype in this study might have a different degree of other substances and affect the concentration of RNA.

Quality and quantity of RNA of three genotypes in three different kits
The RNA concentration and purity were determined using a spectrophotometer nanodrop.Two kits, Ribospin Kit and Promega Kit, performed well except for Tripure (Table 1.) Using the Ribospin kit, even though given the lowest concentration, Tarabas showed the best purity of RNA, seen by A260/280 and A260/230 (2.16±0.0 and 2.06±0.09,respectively).Meanwhile, IR64 had the lowest purity of RNA in the Ribospin kit (2.10 ±0.05 for A260/280 and 1.71 ±0.35 for A260/230).Even though Fatmawati had the highest RNA concentration, the purity of RNA shown by A260/230 was not good since it exceeded 2.20 (2.46 ±0.00 ).The purity of RNA provided by the Promega kit showed good purity of RNA in all three genotypes tested ranging from 2.06 ±0.01 -2.11 ±0.00 for A260/280 and 1.86 ±0.14 -1.95 ±0.10 for A260/230.Overall, all genotypes tested were not performed well in the Tripure kit (Table 1).Tarabas with a concentration of RNA had less than 1.80 in A260/230, and IR64 also had similar results.Fatmawati with Tripure kit performed the worst since both A260/280 and 260/230 were low (less than 1.80).

Figure 2.
Agarose gel electrophoresis of total RNA isolated Figure 2. Shows the results of RNA visualization in gel electrophoresis, Fatmawati performed the best compared to IR64 and Tarabas in all three kits.In the Ribospin kit, Fatmawati had the thickest band compared to other genotypes.This showed that the Fatmawati had the highest RNA concentration and also a high RNA purity compared to the others.This results was supported by concentration of RNA provided by nandodrop electrophoresis.Meanwhile, IR64 had the clearest band compared to other genotypes.Tarabas performed the worst, with a low visible band, which means had the lowest concentration of RNA.In the Tripure kit, it was shown that Tarabas and IR64 had the highest contamination.This was due to the presence of smears in all bands.Meanwhile, Fatmawati performed better, with fewer smears and thick bands.This was also confirmed that the high concentrations of RNA of Tarabas in the Tripure kit were due to a high degree of contamination.
In the Promega kit, Tarabas also performed the least with low visibility of bands compared to the others.Meanwhile, Fatmawati performed the best even though smears also occurred.IR64 performed similarly to Fatmawati but with more smears occurring and visible.Smear in the band also due to a high degree of degradation.Since anther explant was very small and hard to extract from spikelet, working fast and efficiently is the only way to avoid degradation during sample preparation.These findings showed that not only kits determined the purity and quantity of RNA, but genotypes also played a significant role in generating high-quality RNA.

Conclusion
All three Indonesian rice varieties performed differently regarding the quantity and quality of RNA using three different extraction kits in this study.All three kits were able to generate high concentration of RNA (> 100 ng/µL), except for IR64 in Promega kit.Compared to the two other genotypes, Tarabas produced the lowest quality of RNA generated by all three different kit extractions.

Ribospin Kit
Promega Kit

Figure 1 .
Figure 1.The concentration of RNA isolated from 3 rice varieties using different methods a b a s R i b o s p i n K i t F a t m a w a t i R i b o s p i n K i t I R 6 4 R i b o s p i n K i t T a r a b a s P r o m e g a K i t F a t m a w a t i P r o m e g a K i t I R 6 4 P r o m e g a K i t T a r a b a s T r i p u r e K i t F a t m a w a t i T r i p u r e

3. 3
Visualization of RNA in anther explant of Indonesian rice using gel electrophoresis

Table 1 .
Concentration and purity of RNA of Indonesian rice with different culturability