Agrobacterium tumefaciens-MEDIATED IN PLANTA TRANSFORMATION METHOD FOR THE SoSPS1 GENE IN CITRUS PLANTS (Citrus nobilis L.)

The SoSPS1 gene of sugar cane plants previously subjected to Agrobacterium tumefacienmediated cloning was to be transferred to citrus plants to increase metabolism of sucrose in plant. The T-DNA harbored the SoSPS1 gene under the control of the CaMV 35S promoter from the cauliflower mosaic virus and contained the NPTII gene (kanamycin resistance gene) as a selectable marker for transformant selection. Generally, gene transformation in plants is carried out by tissue culture. However, tissue culture has several disadvantages such as its being time-consuming, its sometimes resulting in somatic mutations and somaclonal variations, and the requirement of sterile conditions in the procedure of gene transfer. In planta transformation is a useful system for those plants that lack tissue culture and regeneration system. The main function of in planta transformation is to recover the advantages of tissue culture as an efficient, quick method, including its ability to produce a large number of transgenic plants and to accumulate a high concentration of total soluble protein in short time. There are two procedures of in planta transformation for the seeds of citrus plants, namely “prick and coat” and “seed tip-cutting and imbibition”. In the prick and coat method, seeds are pricked on their entire surfaces and smeared with a suspension of Agrobacterium tumefaciens. In the seed tip-cutting and imbibition method, on the other hand, seeds are cut at the tip and soaked in a suspension of Agrobacterium tumefaciens. The leaves derived from seeds treatment were taken as samples for DNA extraction and PCR using primers of the NPTII gene (Forward: 5’-GTCATCTCACCTTCCTCCTGCC-3’; Reverse: 5’GTCGCTTGGTCGGTCATTTCG-3’). This research found that only the seed tip-cutting and imbibition plants amplified along the 550-bp band, while those of the prick and coat method did not. Additionally, the T-DNA was successfully integrated into the genome of the plants treated with the seed tip-cutting and imbibition method but not with the prick and coat.


INTRODUCTION
Orange is one of the commodities classified as high-value fruits important to both the domestic and the world markets, both in fresh and processed forms. Based on Agriculture Statistics (2017), in 2016, the national citrus consumption per capita increased by 9.76%, while the export volume decreased by 10.5% due to a decrease in the quality of citrus products in Indonesia. Data showed that orange has a high economic value, necessitating improvement of the quality of the products and the variety in order to achieve high variety, high production, sweet taste, and resistance to biotic and abiotic stresses. stages. Genetic transformation is the process of introducing a gene from one organism to another directly via particle bombardment (Hansen and Wright, 1999), the use of polyethylene glycol (PEG), and electroporation (D'Halluin et al., 1992) and indirectly via the help of Agrobacterium tumafaciens (Ishida et al., 1996;Zhao et al., 1998;Negrotto et al., 2000;Frame et al., 2002;Dwiyani et al., 2016;).
Indirect transformation using A. tumefacienss is better than direct transformation because this method is more effective and efficient in application in that, in indirect transformation, the laboratory practice is simple, and the insertion of the gene more stable, than in direct transformation, resulting in higher stability in the gene expression and less genome alteration of the plant transformant (Hansen & Chilton, 1996;Dai et al., 2001;Nishimura et al., 2006;Rahmawati, 2006;Mohammed and Abalaka, 2011).
The utilization of A. tumefaciens in this study was based on the microbe's ability to infect dicot plants and cause tumors on the trunk (Larebake et al., 1974). A. tumafaciens is also able to transfer recombinant tumor genes into transformation vectors (Dwiyani et al., 2016). Insertion mediated by A.
The in planta method involves soaking by which the pollens of flowering plants are inserted without going through the phase of tissue culture (Feldmann and Marks, 1987;Feldmann, 1992). Several types of plants on which in planta genetic transformation has been successfully conducted are Medicago trunttula (Trieu et al. 2000), Arabidopsis thaliana (Bent 2000), apple, pear, tomato, peach, strawberry (Spolaore et al., 2001, and orange (Ahmad and Mirza, 2005).
According to Bhojwani (1979), orange seeds are capable of polyembryonic seeds development in which one seed can develop into more than one embryo.  After that, each 1 mL of A. were cut at the tip and soaked in a suspension of A. tumafacien for 30 minutes (Bastos et al., 2003;Li et al., 2003) (seed tip-cutting and imbibition); and 3) orange seeds were given no treatment for comparison purpose (control) (Fig.2).
The seeds were planted on growing media (charcoal, husk, soil, and fertilizer) and sterilized at a given temperature to eliminate pathogens. Rearing was carried out, i.e., watering and weeding every day and adding NPK fertilizer once a week. Plants were harvested at the age of 4 weeks, (a height of 20 cm, 20 sheets of leaves).

RESULTS AND DISCUSSION
Before we did the transformation, we confirmed the gene of interest inside the Agrobaterium. Ten (10) single colonies of Agrobacterium were tested. The results can be seen in Fig. 3. In this image, the gene is still harbored by all of the single colonies tested. They are indicated by 550-bp bands amplified using NPTII primers (Forward: 5'-GTCATCTCACCTTCCTCCTGCC-3'; Reverse: 5' GTCGCTTGGTCGGTCATTTCG-3').      (Fig. 4. A), and analysis continued between five replicates (Fig. 4. B tumefaciens suspension is able to go into low-concentration seed cells. Seed imbibition is the spontaneous absorption of liquid by porous, dry material. In polyembryony, the highly porous orange seeds were infiltrated with the suspension through the passive transport of osmosis, with the structure of the pores affecting the rate of the suspension absorption. (Jean et al., 2018).
The factor for the transformation success in this study was the use of TWEEN20, transformational explants, and acetosyringone (AS). Clough and Bent (1998) (Chumakov, 2007).
Based on the data in Table 1