Identification of oil palm tissue culture contaminants by using DNA barcodes: preliminary results

The success of oil palm tissue culture is constrained by the microbes contaminating cultures in the laboratory. Accurate identification of contaminant species leads to the right strategies for solving the contamination problem. This research was done to identify contaminant species at the Indonesian Oil Palm Research Institute Tissue Culture Laboratory at its stages by using DNA barcodes. Bacteria identification was done by 16S rRNA sequencing, while fungi identification was done by ITS region sequencing. Preliminary results showed that bacteria and fungi were identified in the laboratory’s stages of the tissue culture process. Four species were identified in the dark room, two in the light room, and four in the advanced light room. The identified bacteria were Bacillus subtilis, Streptococcus parasuis, Priestia megaterium, and Bacillus sp. The identified fungi were Tritirachium candoliense, Penicillium citrinum, Irpex laceratus, Aspergillus reticulatus, and Perenniporia tephropora. More data is needed to determine the right strategies to control the contamination problem.


Introduction
The tissue culture technique has been widely used in oil palm.It can be used as a tool in vegetative propagations of particular germplasm.Besides that, using clonal planting material derived from elite oil palm materials will increase the production of plantation per area, compared to the usage of planting material derived from DxP crosses [1,2,3].
There are some obstacles in oil palm tissue culture.One of them is the contamination of cultures in the laboratory by microbes which will cause high losses if it is not controlled well.Cell totipotency is exploited during the tissue culture process, in which somatic cells are induced to become different tissues [4].The process is held in an aseptic condition, using enriched media with nutrition and plant growth regulators to grow the culture.If the condition of the culture is not aseptic, the media will also be perfect for the growth of microbes.Microbes can harm the culture since they can kill them.They can also be rivals of the culture in getting the nutrition in the media for their growth [5,6].
One of the efforts to control the contamination of the oil palm tissue culture is by identifying the species of the contaminants.The identification can be conducted by morphological observation of the microbes, gram stain, endospore, or biochemical test [7].Another identification technique is by using the information of the DNA barcode.Each technique has its advantages or disadvantages.DNA barcode for species identification is faster than other methods [8].The DNA barcodes could be an Internal Transcribed Spacer (ITS) region [9] and a small subunit ribosomal 16S rRNA gene [10,11].
This research was done to identify contaminant species at Indonesian Oil Palm Research Institute Tissue Culture Laboratory at specific stages at the laboratory by using DNA barcodes.Accurate identification of contaminant species leads to the right strategies for solving the contamination problem.

Sampling, culturing and DNA extraction of contaminant organisms
At every step of the tissue culture process (dark room, light room, advanced light room; consecutively are the rooms in the protocol used) at the IOPRI's tissue culture laboratory, it was taken samples of culture having microbes contaminations.Sampling was done randomly for each microbe (fungus and bacterium).
DNA extraction was done using Isolate II Genomic DNA Kit (Bioline) for the bacteria samples or DNeasy Plant Mini Kit (Qiagen) for fungi samples found in the culture material.If the contaminants could be differentiated morphologically and separated from each other, the DNA extraction could use the contaminant material in the vessel of the culture directly.If the contaminants could not be separated, they were first isolated from the vessel and cultured in Potato Dextrose Agar (PDA) for fungy and Luria Broth (LB) for bacteria.Subsequently, DNA extraction used the cultured material result ing from the isolation.

DNA Amplification and sequencing
The resulting DNA was quantified using Qubit® 2.0 Fluorometer (Invitrogen) and amplified using an 8F/1492 primer pair (for bacteria) [12] or ITS1/ITS4 (for fungi) [9].The Polymerase Chain Reaction (PCR) protocol was done as described by the previous report [13].Direct sequencing was done for the amplicon using the commercial service of Macrogen, Korea.The DNA sequences obtained were then confirmed to National Center for Biotechnology Information (NCBI) database using megablast.

Results and discussions
Five bacterium colonies were randomly taken from the dark room and two bacterium colonies from the advanced light room for species identification using the16S rRNA gene.One fungus colony was taken from the dark room, and two fungus colonies from the light room and advanced light room, respect ively, for species identification using the ITS region.Sequencing of the 16S rRNA gene has been used to identify contaminant bacteria species of plant tissue culture [14,15], and the ITS region was used to identify fungi [16,17].Results showed that bacteria and fungi were identified in the laboratory's steps of the tissue culture process: Streptococcus parasuis, Bacillus subtilis, Priestia megaterium, Tritirachium candoliense, Penicillium citrinum, Irpex laceratus, Aspergillus reticulatus and Perenniporia tephropora (Table 1).Microbial contamination may originate from the source of the explant, equipment, environment, or persons who handle the in vitro culture operation.Table 2 shows that the number of bacterium species was higher in dark room than in light rooms.Three genera were found, which were Bacillus, Streptococcus, and Priesta.Bacillus and Streptococcus were also found as microbial contaminants associated with plant tissue culture and the laboratory environment [18], while Bacillus was found as contaminants of plant in vitro culture [7,15].
At the laboratory, the dark room is the first step of in vitro culture, in which the explant is used to initiate the callus.It shows that the source of bacterial contamination was predicted from the explant used.There are obstacles in sterilizing explants used for tissue culture since difficult to sterilize some epiphytes and endophytic bacteria.They can be locked or localized at tissues difficult to reach by disinfectants [19,20].Endophytic bacteria, which were previously reported to have plant growthpromoting effects, can support the success of plant tissue culture [20].However, in some cases, they harm the in vitro culture [22].From the three genera of bacteria found in this study, at least, it was found previously that Bacillus subtilis was an endophytic bacterium of in vitro culture of Dendrocalamus asper and Bambusa oldhamii, and Bacillus megaterium was an endophytic bacterium of plants [23].
Streptococcus parasuis, a zoonotic pathogen that causes a few pig and human diseases [24], was found in the dark room and the advanced light room.It suggests human errors in handling the tissue culture process, which should be reviewed and improved.
The number of found fungi was higher in the light rooms than in the dark room (Table 2), suggesting a problem during handling in vitro culture incubation.Aspergillus sp. was reported as a contaminant in oil palm tissue culture [25].While Tritirachium oryzae was found as an endophyte of Pinus wallichiana [26] and Syzygium malaccense [27].Penicillium citrinum was reported as an endophyte of Boehmeria nivea [28], and Perenniporia tephropora was reported as an endophyte of Centella asiatica [29].These preliminary results suggest recommendations to control the laboratory contamination problem.Firstly, there should be research to provide explant sterilization protocols that could inhibit the propagation of harmful bacteria during the callus initiation.Secondly, working conditions should be improved so that the aseptic environment requirement in handling the tissue culture process is fulfilled [7].Identification of contaminant species shows the sources of contamination and the strategies to control the problem.However, this study needs proper sampling to understand the sources of contamination comprehensively.The sampling should be done in the culture rooms and the other areas of the laboratory (such as the laminar air flow room, preparation room, and corridors).High throughput sequencing, which allows sequencing of DNA fragments of many species in parallel, will support the analysis efficiently.

Conclusion
The preliminary result proves that microbial contaminant identification can be conducted using ITS region and 16S rRNA sequencing.The identified contaminant species were Streptococcus parasuis, Bacillus subtilis, Priestia megaterium, Tritirachium candoliense, Penicillium citrinum, Irpex laceratus, Aspergillus reticulatus, and Perenniporia tephropora.However, more data is still needed to comprehensively understand and determine the right strategies for preventing and controlling the contamination problem by using an improved sampling method and identification technique.

Table 1 .
List of species identification results based on blast of barcode sequence to NCBI database.

Table 2 .
List of identified species in different culture stages