Elimination of systemic viral disease in shallot (Allium ascolonicum L) var. Bima Brebes through unconventional approach

Shallot (Allium ascolonicum L), belonging to the genus Allium are propagated vegetatively through layered bulbs. This research aimed to improve the quality of shallots by eliminating OYDV, SLDV viruses through meristematic tissue combined with the antiviral Ribavirin. The research was conducted in the IVEGRI Tissue Culture Laboratory from April to August 2018. Research materials included the infected shallot bulbs of Bima Brebes variety which were previously confirmed by DAS ELISA serology test. Meristematic tissues were cultured on MS + vitamin, MS + supplement media (sucrose 30 gL−1 + Myo inositol 100 mgL−1 + CaP 2 mgL−1 + NAA 0.15 mgL−1 + Kinetin 2 mgL−1 + GA3 0.1 mgL−1 + Gel gro 2gL−1, pH 5.7). A Completely Randomized Design with ten replications was applied for the combinations between antiviral Ribavirin concentrations i.e., 0.5,10,15, 20, 30 mgL−1, and explant size including meristematic tissue (0.5 mm) and shoot tip (2.5 mm). The results obtained through visual observation showed that the additional antiviral Ribavirin and explant size affected the percentage of growth and development of plantlets. Statistical analysis showed that there were no significant interactions between treatments. DAS Elisa serology test showed that the higher the concentration of antiviral Ribavirin the lower the percentage of virus infection.


Introduction
In vegetatively propagated plants, viral diseases are serious problems.Previous research showed that virus infection reduced production, the number of bulbs, and bulb weight by up to 45% [1,2].The virus in infected bulbs will keep developing and inheriting through generations of shallot.One of the alternative ways to eliminate viral diseases is through meristematic culture, the additional antiviral Ribavirin, or a combination of several treatments that can improve the quality and quantity of shallot yield.
Tissue culture is an unconventional propagation technique by growing a piece of tissue or plant organs in vitro using artificial media aseptically [3,4].Its principle technique is based on the cell theory by Scheiden andSchwann (1929-1939), that the cell is part of the smallest biological unit that can carry out life, reproduction, and growth activities [5][6][7][8].Shallots (Allium ascolonicum L) are propagated through bulbs.In some developed countries shallot seeds have been produced in vitro either for quality improvement or just plant propagation [9][10][11].
According to earlier experiments, isolating explants is one of the keys to success in explant proliferation [6,12].Shallot propagation through an unconventional approach is determined by several factors e.g., the composition of the growing media, genotype, and the source of explants [13].This study aimed to analyze the effect of the additional antiviral Ribavirin and the source of explant on the growth and development (proliferation) of shallot explants of Bima Brebes variety.

Materials and methods
The research was carried out in the tissue culture laboratory of the Indonesia Vegetable Research Institute (IVEGRI) from April to August 2018.ELISA serology method was used in initial testing/detection of OYDV and SYSV virus infected-shallot bulbs of Bima Brebes variety.These infected bulbs were then used as explant materials.Treatments included 1) explant size i.e., meristem = meristematic tissue with 2 leaf primordia and shoot tip = meristematic tissue with 4 -6 leaf primordia, and 2) concentrations of 0, 5, 10, 15, 20 and 30 mgL -1 antiviral Ribavirin.Growth medium consisted of MS (1962) + MS vitamins + myo inositol 100 mgL -1 + CaP 2 mgL -1 + NAA 2 mgL -1 + Kinetin 2 mgL - 1 + GA3 0.1 mgL -1 + GelGro 2 gL -1 , pH 5.7.A Completely Randomized experimental design was used with each treatment consisting of 20 test tubes of 25 x 150 mm, with a volume of 10 ml media for each tube.

Explant preparation
Shallot bulbs that had passed the dormancy stage were washed and peeled.The internal shoots were excised and washed with sterile aquadest 3 -5 times, immersed in 70% alcohol, and then soaked in 25% chlorox solution for 10 minutes, rinsed again with sterile aquadest 3 -5 times, and finally transferred to a sterilized petri dish.Meristem tissue and shoot tip were observed using 40x magnification binoculars.Cultures were incubated in a culture room with a temperature of 22 -24 o C, a photoperiod of 16 hours light, and 8 hours dark.Sub-culture was carried out after the tissue grew and developed or depending on the state of the growth medium.

Data collection
The percentage of growth (proliferation), percentage of contamination, percentage of normal and abnormal growth, the average number of shoots, and the number of leaves were visually observed, whereas the percentage of plantlets infected with OYDV and SYSV was counted based on ELISA Test.

Results and discussion
The shoot formation in shallot tissue culture is affected by various factors [16], such as the composition of explant growth media.In our study which used MS media with the additional supplement, the additional antiviral Ribavirin inhibited the growth of shoots both from meristem explants and shoot tips (Table 1).It also showed that the regeneration of shallot explants of the Bima Brebes variety is affected by various factors such as the composition of the growth medium used, genotype, and the source of the explant [14,17].The failure of explants to differentiate can be resulted from physiological, and genetic factors that are not totipotent [18].Visual observation on 8 WAP (Weeks After Planting) on Table 1 showed that higher antiviral Ribavirin concentration caused a lower number of leaves.The lower average number of leaves was because some cultures were contaminated with bacteria or fungi.These results agreed to previous research that contamination is one of the obstacles in in vitro propagation [14].In the tissue culture technique, different responses of explants depend on the culture conditions (composition of growth media and plant growth hormone) and explant type (variety, size, and treatment on explants).Simultaneous or partial application of combination of several components is needed to increase explant growth [15][16][17].Figure 1 showed that the growth of meristematic tissue and shoot tips of Bima Brebes was 55-90%.The additional antiviral Ribavirin to the growth media and the type of explant affected the percentage of growth/proliferation. Genotype and explant type have a different responses to the additional antiviral ribavirin in MS media [18].Explants from meristematic tissue can grow well since they produce primordia leaves that release auxin and cytokinin for proliferation [19,20].In-vitro propagation offers several advantages, including an aseptic and controlled growing environment, high-speed propagation, and the production of healthy seeds from infected mother plants.Other study also showed that the selection of explant material is critical for successful regeneration and proliferation [21].The percentage of contamination and inactive plantlets at the age of 8 WAP was 15 -55 %.It showed that the higher concentration of antiviral Ribavirin the higher percentage of contamination.The meristematic tissue produced higher contamination than shoot tips.Contaminants can be from the external or internal tissue (endogenous) of the explant.The contaminant-free explant is an important factor in tissue culture.Contaminants can be in the form of fungi or bacteria.Explants covered with contaminants will eventually not develop.In tissue culture techniques, contaminants can hinder the growth and development of the plantlets [22].In Figure 3, the visual observation of the percentage of normal plantlets is always lower than abnormal plants for both meristematic and shoot-tip explants.Some of the main factors that are key to success for in vitro culture are (a) explant source, (b) media composition and growth hormone, (c)  [12].In addition to these factors, plant genotype affects the rate of successful culture.Plant propagation with unconventional techniques is also influenced by the response of varieties/cultivars, explant type, the treatment of explants, and the media composition.It is also stated that the source of explant in tissue culture has an important role in successful explant regeneration [21,23].Carla virus, Potty virus, and Allexi virus are the common viruses on Allium family in Indonesia.SLV (Shallot Latent Virus), OYDV (Onion Yellow Dwarf Virus), and SYSV (Onion Yellow Strips Virus) are the main viruses in shallot.Previous works showed that the ELISA serology method successfully detected OYDV and SYSV, as well as the other combination of viruses [24][25][26].Table 2 showed that the ELISA test on normal plants/plantlets showed that the percentage of OYDV and SYSV viruses was still above 30%.According to the previous studies, when viruses are still detected, it indicates that the additional Ribavirin antiviral in Murashige and Skoog media is not optimal for eliminating viruses [26,27].Even though, the higher concentration of antiviral Ribavirin the lower percentage of virus infection [28].Our study showed that in normal plantlets, the viral disease was still detected.It indicated that the treatment is not optimal to eliminate the virus, thus, when the explant proliferates, the virus particles are still carried.

Conclusion
The growth of shallot plantlets of Bima Brebes variety from shoot tip and meristematic explants infected with OYDV, or SYSV were not significantly different and there is no interaction between explant source and the concentration of antiviral Ribavirin on Murashige and Skoog media.It also showed that the increasing concentration of antiviral Ribavirin will affect the explant growth.Eight weeks after planting, DAS ELISA serology test detected percentage of infected cultures was 20 -33.33%. 1