Obtaining of transgenic potato (Solanum tuberosum L.) cultivar IPB CP3 containing LYZ-C gene resistant to bacterial wilt disease

Bacterial wilt caused by Ralstonia solanacearum is one of the most important bacterial diseases in potato production. This study aimed to obtain the transgenic potato (Solanum tuberosum L.) cultivar IPB CP3, containing LYZ‐C gene encoding for lysozyme type C, resistant to bacterial disease caused by R. solanacearum. Genetic transformation using Agrobacterium tumefaciens LBA4404 to 124 internode explants resulted in the transformation efficiency of about 47.58% with a regeneration efficiency of approximately 30.51%. Gene integration analysis showed that 16 clones were confirmed as transgenic clones containing the LYZ‐C gene. Analysis of resistance to R. solanacearum of three transgenic clones showed that all three transgenic clones were more resistant than a non‐transgenic one. This result showed that the LYZ‐C gene integrated in the genome of transgenic potato increased the resistance of potato plants to R. solanacearum. We obtained two transgenic clones considered resistant to bacterial wilt disease.


Introduction
Potato is the third major food crop based on total consump tion in the world after rice and wheat (FAOSTAT 2020). In Indonesia, consumption of potatoes tends to continue to in crease, especially in the form of processed potatoes such as french fries and chips. However, potato production still faces several obstacles, mainly due to the availability of lowquality potato seeds, disease infections, and unfavor able environmental conditions. Potato disease can quickly spread and develop since potato is propagated vegetatively (Davidson and Xie 2014). Bacterial wilt disease caused by Ralstonia solanacearum is the most common disease to at tack potato plants. R. solanacearum is reported to attack more than 250 plant species; most of the hosts belong to the Solanaceae and Musaceae families (Charkowski et al. 2019).
Possible actions to control the infection and the spread of pathogens in potato are to use diseaseresistant cultivars through the fusion of protoplasts between species (Fock et al. 2000) and the introduction of disease resistance genes (R genes) to induce innate immune responses (Gururani et al. 2012). Lysozyme is an enzyme with bacteriolytic activity capable of degrading peptidoglycan, a constituent of bacterial cell walls (YonKahn 1996). A gene encod ing for lysozyme has been incorporated into many plants to increase the resistance to pathogenic infections. The in troduction of the T4 lysozyme gene into tall fescue grass (Festuca arundinacea Schreb.) succeeded in obtaining transgenic grass plants resistant to Rhizoctonia solani and Magnaporthe grisea (Dong et al. 2008). The LYZC gene encoding ctype lysozyme was also introduced to potato cultivar Desiree, and the obtained transgenic potato plants were resistant to Erwinia carotovora subsp. atroseptica (Serrano et al. 2000).
Transgenic potato cv Jala Ipam harboring LYZC gene under the control of 35S CaMV promoter was also resis tant to R. solanacearum in vitro (Senjaya 2017) and the field condition (Alfian et al. 2020). Although these culti vars are very promising to be applied, potato cv Jala Ipam is sterile and tetraploid. Therefore this clone is not able to be a donor of LYZC gene to other potato cultivars through conventional breeding by sexual crossing. Thus, this study aimed to introduce the LYZC gene under the control of 35S CaMV promoter into another superior potato cultivar, IPB CP3, to increase its resistance to R. solanacearum as a causal agent of bacterial wilt disease.

Plants Materials and Agrobacterium tumefaciens Propagation
IPB CP3 cultivar was propagated in vitro on an MSbased medium (Murashige and Skoog 1962). The cuttings were grown in a culture room at 2425°C for 34 weeks with a photoperiod of 18/6 h and 2,0003,000 lux light intensity. Agrobacterium tumefaciens strain LBA4404 carrying the recombinant plasmid pCXSNLyz Figure 1 was used to transform the potato genetically. A. tumefaciens was cul tured in LB (Luria Bertani) liquid medium supplemented with 50 mg/L of kanamycin, 50 mg/L of hygromycin, and 100 mg/L of streptomycin in the dark condition at room temperature for 1518 h until the optic density at 600 nm was about 0.5. The suspension of A. tumefaciens was cen trifuged at 10,000 rpm for 10 min. The pellet was resus pended in inoculation medium (MS medium containing 16.0 g/L of glucose, 2.0 mg/L of 2,4D, 0.8 mg/L of trans Zeatin) until OD 600 was about 0.3.

Potato Transformation
Internodes with size of 0.51 cm were used as explants and grown on solid preculture medium (PC) (MS medium con taining 2 mg/L 2,4D, 0.8 mg/L transZeatin, 40.0 mg/L acetosyringone) for 2 days. Explants were put into a liquid inoculation medium containing A. tumefaciens and shake softly for 10 min at room temperature. The explants were then dried on sterile tissue paper for 5 min and grown on solid cocultivation medium (CO) (MS medium contain ing 16.0 g/L of glucose, 2.0 mg/L of 2,4D, 0.8 mg/L of transZeatin) for three days in the darkroom. The explants were rinsed three times by sterile distilled water contain ing 200 mg/L cefotaxime then dried on sterile tissue pa per for 5 min. Furthermore, explants were grown on solid callus induction medium (CI) (MS medium supplemented by 1.0 mg/L of IAA, 0.5 mg/L of GA3, 0.8 mg/L of trans Zeatin, 100.0 mg/L of cefotaxime) for three weeks. Callus was subcultured every two weeks in a selection medium containing 10 mg/L hygromycin until it regenerated the shoots. The shoots were further propagated by subcul turing every three weeks in MS medium containing hy gromycin with a gradual increase as much as 10 mg/L, 20 mg/L, and 30 mg/L.

Molecular Analyses of Transformed Potato Plants
Genomic DNA of potato was isolated by the CTAB method (Suharsono 2002) using a 2% CTAB buffer. In tegrated transgenes in transgenic plants were analyzed by PCR using primer pair of Lyz114F (5'TAT GAA GCG TCA CGG ACT TG 3') and NosT2R (5'GAA TCC TGT TGC CGG TCT TGC G3'). The PCR was carried out by the condition of predenaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, an nealing at 54°C for 45 s, and extension 72°C for 1 min, and postPCR at 72°C for 5 min. PCR results were elec trophoresed in 1% (w/v) agarose gel, 100 V for 28 min. 1 kb DNA ladder (Thermo Fisher Scientific, USA) was used as a molecular weight marker.

Bacterial Inoculation Assays
In vitro inoculation assays were carried out using the bac teria R. solanacearum as described by Habe (2018). Plants were grown in the jar containing 40 mL vermiculite and 30 mL MS liquid medium. Four weeks old plants were inoculated by 200 µL of bacterial suspension with a con centration of 2x10 9 CFU/mL. Incubation was carried out at 2528°C. Disease symptoms were observed at 10 d af ter inoculation. The frequency of disease was calculated using a formula as follows.

Transformation of Potato Plants
The LYZC gene was successfully inserted into the genome of the potato cultivar IPB CP3 with Agrobacterium mediated transformation. A. tumefaciens is widely used to introduce genes because naturally, it can efficiently trans fer and integrate stably DNA fragments contained in the TDNA region into the genome of the host plant (Hwang et al. 2017). The process of genetic transformation of potato cv IPB CP3 is presented in Figure 2.
The stem segment explants ( Figure 2a) used in this study began to form a callus at two weeks after growing in CI medium. Callus was formed starting from the ends of the stem segments and it grew to cover the entire surface of the explants (Figure 2b). Callus began to regenerate to form shoots at 4 weeks of age. The stemsegment explants developed callus and then regenerated to form shoots on a medium containing 1 mg/L of IAA hormone, 3 mg/L of transZeatin hormone, and 0.5 mg/L of GA3 hormone. Masekesa et al. (2016) explained that the success of cal lus induction and regeneration depends on the genotype of the explants and the right combination of concentrations the number of hormones used. The cytokinin hormone in the form of transZeatin is able to induce both callus and shoot formation (Park et al. 2003).
This study used in vitro precultured explants of intern odes (Figure 2). During growth in PC medium, most of the explants developed to tissue swelling. McHughen et al. (1989) showed that preculture prior to inoculation with A. tumefaciens increase the production of transgenic plants. Furthermore, Sangwan et al. (1992) showed that precul ture treatment increased the number of competent cells to be transformed by A. tumefaciens. The treatment of ex plants on PC medium played a role in adjusting the stress conditions due to cocultivation with Agrobacterium. To induce the process of gene transfer from A. tumefaciens into plant explants, the cocultivation medium was supple mented with acetosyringone 40 mg/mL. Acetosyringone phenolic compounds increased the efficiency of transfor mation in plants with Agrobacteriummediated transfor  mation, such as in ornamental plants Eustoma grandi florum (Nakano 2017), and in plant species of Melas toma malabathricum and Tibouchina semidecandra plants (Yong et al. 2006). The results showed that some explants were able to keep growing to form a callus and regenerate to develop shoots on a selection medium containing 10 mg/L hy gromycin. However, some explants also slowly turned into brown color (browning) and underwent necrosis. In general, browning is caused by the phenolic compound se creted by the wounded tissue (Leng et al. 2009). From three experiments, the transformation efficiency based on the selection using 10 mg/L hygromycin was ranged be tween 41.73% and 51.63%, with an average of 47.58% (Table 1). Based on the number of hygromycinresistant calli, the efficiency of regeneration ranged from 20.63% and 40.98%, with an average of 30.51% (Table 1). The transformation efficiency in this study was higher than those of transformation of potato cultivar Jala Ipam by MmPMA gene (Farhanah et al. 2017) and potato cultivar IPB CP1 cultivar by Hd3a gene (Gea et al. 2017). The explants infected with A. tumefaciens but not containing pCXSNLyz, enabled to form callus in the medium without hygromycin. However, these calli were not able to survive in the selection medium containing 10 mg/L hygromycin. This phenomenon shows that 10 mg/L hygromycin was ef fective for the selection of transgenic calli. The efficiency of regeneration in this study was higher than the regenera tion efficiency obtained by Gea et al. (2017) and Farhanah et al. (2017).
Shoots regenerated from callus grew on the selection medium (Figure 2c), separated from the callus in order to form roots and for propagation (Figure 2d). A total of 31 independent shoots regenerated from resistant calli. These shoots were multiplied in a selection medium where the hygromycin concentration was gradually increased to 30 mg/L. These shoots are called putative transgenic clones. Among these 31 clones, we chose 16 transgenic clones that had the fastest growth to be analysed.

Molecular Analysis of the Transgenic Potato Plants
PCR analysis of 16 clones of putative transgenic plants with Lyz114F and NosT2R primers showed that all clones contained LYZC transgene and nopaline synthase terminator (tNOS) with a fragment size of 574 bp ( Figure  3). Similarly, the amplification results were obtained by using the pCXSNLyz plasmid as a DNA template. This plasmid was used as a positive control. Conversely, the nontransgenic (NT) plant did not produce these amplicons as expected because it was used as the negative control ( Figure 3). This result proved that the LYZC gene was successfully integrated into the transgenic plants.

Bacterial Resistance Analysis of the Transgenic Potato Plants in vitro
The resistance assay against R. solanacearum was car ried out on three selected clones with the fastest growth, namely CP3lyz1, CP3lyz2, and CP3lyz6 clones from a to tal of 16 clones of transgenic plants. One nontransgenic plant cultivar IPB CP3 was included in this experiment as a control. After inoculation by R. solanacearum, the trans genic clones could survive with different levels of resis tance, whereas the nontransgenic one showed the symp tom of wilt disease (Figure 4). The sign of the disease was indicated by the change of the color of leaves, from green to yellow, then to brown, followed by a broken stem. The wilt in plants infected by R. solanacearum is caused by a disfunction of the plant's vascular system. R. solanacearum reproduces and colonizes the xylem vessels before blocking the water and mineral transport (Lowe Power et al. 2018) and causing plants to wilt. The bacterial wilt disease frequency of clones was ranged from 16.7% to 100.0%. Based on Thaveechai et al. (1989), CP3lyz1 and CP3lyz6 clones were resis tant to R. solanacearum, whereas CP3lyz2 clones were moderately resistant to R. solanacearum. Meanwhile, all nontransgenic potato cultivar IPB CP3 plants were in fected by R. solanacearum, therefore this variety was sen sitive to R. solanacearum (Table 2). Transgenic clones are more resistant to R. solanacearum than the nontransgenic one, possibly because transgenic clones are able to synthe size lysozyme, while nontransgenic clones are unable to synthesize lysozyme. CP3lyz1 and CP3lyz6 clones were resistant to R. solanacearum, possibly because the two clones were able to overexpress the LYZC gene. Both clones, CP3lyz1 and CP3lyz6, were more resistant to R. solanacearum than the other transgenic clone, possibly be cause the expression of the LYZC gene in both clones was higher than in the other clone. Quantitative LYZC gene expression in the transgenic clones has to be investigated to know the expression level of LYZC gene in transgenic clones.
The lysozyme encoded by the LYZC gene is capa ble to cleave the β1,4glycosidic bond between Nacetyl Dmuramic acid (MurNAc) and NacetylDglucosamine (GlcNAc) of peptidoglycan, the cell wall bacteria compo nent (YonKahn 1996). Therefore lysozyme can kill the bacteria, including R. solanacearum, by degrading their cell wall.
The difference in the resistance levels of transgenic plants containing the LYZC gene under the control of the same 35S CaMV promoter could be caused by the dif ferent expression level of LYZC gene. Differences in transgene expression level in transgenic plants containing the same transgene are common phenomena (Kohli et al. 2006). The level of gene expression among clones of the transgenic potato cultivar Jala Ipam containing the same LYZC gene varied and the level of the resistance against R. solanacearum was correlated to the expression level of LYZC gene (Alfian et al. 2020). Differences in transgene expression levels can be caused by the copy number of transgene insertion, the site of transgene insertion, RNA silencing, and the regulators for transgene expression (Bu taye et al. 2005). TDNA can be integrated into the plant genome randomly and distributed throughout all parts of the chromosome (Ko et al. 2018). Therefore it can be in serted in the encoding and noncoding regions. Random insertion can affect the level of gene expression in trans genic plants due to the effect of copy number insertion and the impact of transgene position.

Conclusions
LYZC gene was successfully integrated into the genome of transgenic potato cv IPB CP3. In vitro resistance assay to R. solanacearum of three transgenic clones showed that transgenic clones were more resistant to R. solanacearum than nontransgenic ones. These results indicate that the LYZC gene integrated in the transgenic potato cultivar CP3 IPB could increase the resistance to bacterial wilt dis ease caused by R. solanacearum.