Application of combination of auxins in callus induction and proliferation of shallot cv Trisula and Sumenep through in vitro technique

Shallot (Allium cepa L. var. aggregatum) is one of a strategic commodity in Indonesia. To increase of production of shallot seed, there needs a good tuber with high productivity tolerant to abiotic stress. One of the obstacles to increase of shallot production with tuber in good quality and tolerant to abiotic stress is not available. Tissue culture technique is one of the methods proved can propagate plant in large amounts and in a shorter time compared to the conventional method. The objectives of this research are to get the best media formulation for callus induction and proliferation. The explant used in this research are shoot tip of shallot from cultivar Trisula and Sumenep. The Media used were MS. The medium was enriched with (0,5 – 3 mg/l) 2,4-D + 2 mg/l picloram (0,5 – 3 mg/l) or NAA + with or without 3 gr/l Casein hydrolysate; and MS + 3 mg/l Picloram + 2 or 3 mg/l NAA + 3 g/l Casein Hydrolysate. The best media formulation for callus induction of Trisula variety was MS + 3 mg/l 2,4D + 2 mg/l picloram + 3 g/l Casein Hydrolysate, whereas the best media formulation for callus induction of Sumenep variety was MS + 0.5 mg/l 2,4-D + 2 mg/l picloram. The best media formulation for callusproliferation was MS + 0.5 mg/l 2,4-D + 2 mg/l picloram both Trisula and Sumenep varieties. These results could be used for plant propagation and breeding program of shallot in the future.


Introduction
Shallot (Allium cepa L. var.aggregatum) have high economic value and various benefits for flavour and health [1].The demand of shallot products has increased every year in accordance to the development of world population [2].Seedlings are one of factors that influence plant productivity; however, the limitation of good quality of seedlings is one of main problems in the effort to increasing of shallot production [3].Seeds of shallot can use for generative propagation; nevertheless, in general tubers is used to conventional propagation of shallot.The obstacle of shallot production using tubers have faced to biotic stress problems from various important disease such as basal rot of tubers disease caused by Fusarium [4].
The technique of in vitro culture has been used to produce large amounts of seedlings that are free from disturbing of environment and pests and diseases.One of a stage of in vitro culture technique for shallot propagation is callus induction.A callus is defined as an unorganized structure.Callus formation is central to many investigative and applied tissue cultures.This callus, it can be used for micropropagation through organogenesis and somatic embryogenesis [5].
In vitro culture of shallot has been used in some research in various cultivars and variety with various aims.Auxin is a plant growth regulator (PGR) that is used to initiate callus such as 2,4-D, NAA, IBA, IAA and picloram.Auxins can be used in a single or combination to get the optimum result [5].
The objective of this research was to obtain the best media composition for callus induction and proliferation.This research was conducted in Trisula and Sumenep varieties.Callus from this research will be used for further research both regeneration by organogenesis and somatic embryogenesis as well for mutagenesis and in vitro selection.

Methods
The research was conducted in Cell Biology and Tissue Culture Laboratory, The Agricultural Instrument Standardization Agency for Biotechnology and Genetic Resources, the Agricultural Instrument Standardization Agency, Ministry of Agriculture of the Republic of Indonesia.This research had two activities, callus induction, and proliferation.The detail of the stages is as follows:

Callus induction
The callus induction stage was the early stage of in vitro technique to get callus formation.Materials used in this activity were shallot from Trisula and Sumenep varieties.The explant used in this research was shoot tip from shallot tubers of eight weeks after harvest.The size of the shoot tip was ± 0.5 cm.
In callus induction activity was used fifteen media compositions.The basic media used was Murashige dan Skoog (MS) (1962) enriched with a plant growth regulator auxin (2,4-D, picloram and NAA) in combination with or without Casein Hydrolysate.The medium compositions were MS medium enriched with (0.5 -3 mg/l) 2,4-D + 2 mg/l picloram (0.5 -3 mg/l) or NAA + with or without 3 gr/l Casein hydrolysate; and MS + 3 mg/l Picloram + 2 or 3 mg/l NAA + 3 g/l Casein Hydrolysate.Every medium contained 30 g/l glucose and 2.5 g/l phytagel.Potential of hydrogen (pH) was 5.8.Containers of culture were bottles 100 ml in size.Every bottle was contained 1 explant and every treatment was repeated 15 times.Bottles of culture were incubated in culture room at 18-22 o C temperature in dark condition.
Growth parameters observed were the percentage of explant to form callus, callus formation score, callus structure and callus texture.The callus scoring was made as follows: 0 = no callus formation 1 = 0-25% callus formation in surface of explant 2 = 25-50% callus formation in surface of explant 3 = 50-75% callus formation in surface of explant 4 = 75-100% callus formation in surface of explant Those data were arranged in a complete randomized design and then analyzed using analysis of variance or if no significantly differences were analyzed descriptively.

Callus proliferation
Callus proliferation stage was propagated by the callus formed from the induction stage.Materials used in this activity were shallot from Trisula and Sumenep varieties.The explants used in this research were calli gained from the callus induction stage of eight weeks of age.The size of callus was ± 1 cm.
In callus proliferation activity was used three media compositions that indicated the best performance callus from inductive activity would be used for this stage (yellowish in color, globular structure, friable texture and embryogenic).Embryogenic calli were the type of calli that had the opportunity to regenerate and become shoot.The medium used were MS fortified with 2 mg/l 2,4-D + 2 mg/l NAA + 3 g/l casein hydrolysate; 0.5 mg/l 2,4-D + 2 mg/l picloram; and 2 mg/l 2,4-D + 2 mg/l picloram.pH media was 5.8.Containers of culture were bottles 100 ml in size.Every bottle was contained 1 explant and every treatment repeated 15 times.Bottles of culture were incubated in the culture room at 18-22 o C temperature in dark conditions.
Growth parameters observed were explant callus color, callus diameter, and callus texture.Data were arranged in a completely randomized design and if there are no significantly different those data were descriptively analyzed.

Callus induction
Callus induction stage has been conducted in fifteen media compositions.PGR used in the stages had auxin, 2,4-D, in combination with picloram or NAA either with casein hydrolysate or without.Callus induction was observed for 8 weeks.After the data collected were analyzed using analyses of variance there was no significant difference (computed F value smaller than F tabular value of 5%), however, analysis data used descriptive analysis.The responses from fifteen media used were different.However, the best media for callus induction in Trisula varieties was 2 mg/l 2,4-D + 2 mg/l NAA added 3 mg/l casein hydrolysate with a callus score was 0.47 ± 1.33.Meanwhile, the best media for callus induction of Sumenep was 1 mg/l 2,4-D + 1 mg/l NAA+ 3 g/l Casein Hydrolysate 1.71 ± 1.80.From the result showed that the more time explant to contact with media (Plant Growth Regulator) the more cell to divide, so the score of calli increased [6].These results have showed on the two of varieties, Trisula and Sumenep (table 1 and 2, figure 1 and 2).
Meanwhile, the shallot cv Sumenep at eight weeks of culture has showed the highest percentage of callus induction at week 8 at 80% from media 2,4D0.5Pic2.However, the best callus formation has been obtained from media 2,4D1N1CH3 with callus score of 1.71 ± 1.80 (table 2).Nevertheless, the friability of the callus in media 2,4D0.5Pic2was more friable than the callus in media 2,4D1N1CH3 (figure 2).The research results have showed that optimum media formulation of callus induction stage from each shallot variety were different.These depended on the genotype of the variety used [7].Therefore, understanding of callus induction and proliferation method of different variety of plant was very needed.

Callus proliferation
The stage of callus proliferation has used three media gained from callus induction stage media that showed the best performance.The best media was chosen from the percentage of callus formation, score of callus formation, callus structure and callus texture.The three of media were 2,4D2N2CH3; 2,4D0.5Pic2;dan 2,4D2Pic2.
The callus proliferation stage showed that the growth of callus formation in Trisula obtained from media 2,4D0.5Pic2with callus diameter of 1.25 ± 6.18 cm and Callus texture was friable until 60.71%, however 67.86% of callus have yellow redness in color (table 3, figure 3).The redness of color could be caused by the long period of culture and the redness color could cause the dead of callus [8].To overcome this problem, the media need to add PolyvinylPyrrolidone (PVP) in future research [9].In table 4 above showed that 2,4D0.5 Pic2 media gave callus the best result of callus proliferation of Sumenep variety.This result could be seen from the diameter (2.16 ± 5.15 cm), Friable texture of callus (83.33%) and the yellowish color of callus (100%).The high of percentage of callus proliferation with Friable texture and yellowish color were a good result for the other steps of this research where the callus would be used as explant for mutagenesis study to get mutan population and in vitro selection [10,11].Besides that, callus could be regenerated become a plantlet, and the plantlet could be used for propagate or used for other research like in vitro selection.Of the two varieties used showed that the response of Sumenep variety to media of callus formation was better than the response from Trisula variety to the same media (figure 3 and 4).

Acknowledgments
Thanks to National Research and Innovation Agency, Indonesia (BRIN) was for funding this research.

Figure 1 .
Figure 1.Callus induction in Trisula variety in media of 2 mg/l 2,4-D + 2 mg/l NAA + 3 g/l casein hydrolysate at eight weeks of culture

Figure 2 .
Figure 2. Callus induction in Sumenep variety in media of 0.5 mg/l 2,4-D + 2 mg/l Picloram at eight weeks of culture

Table 1 .
Callus induction stage of shallot cv Trisula at eight weeks of culture

Table 2 .
Callus induction stage of shallot Sumenep variety at eight weeks of culture