Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

Background Recent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. Results Abiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. Conclusions The present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.


Background
The genus Citrus includes more than 162 species belonging to the order Geraniales, family Rutaceae and subfamily Aurantoideae. Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck are two primitive species of Citrus. Citrus sinensis also known as Sweet-orange or Malta, is the most cultivated Citrus in the world which accounts for about 70% of the total production. Citrus reticulata Blanco is a species of Citrus also known as Mandarin, Tangerine, Unshu orange, Comola etc in the Asian Subcontinent. Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, both are grown in tropical and semi-tropical areas around the globe for its sweet, juicy, and nutritious fruits. Orange farming has been extending rapidly in some regions of Bangladesh like Sylhet, Panchagarh, Chattogram, and Thakurgaon. Recent studies indicate that farmers are facing several problems due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses and would be high yielding. Traditional breeding methods have been used successfully over the years to improve Citrus; however, these methods are limited by slow growth, incompatibility, polyembryony, parthenocarpy etc and traditional breeding takes a long time for the incorporation of desirable traits. In-vitro culture made it easy to improve Citrus against different abiotic stresses, diseases, low-yield through exploiting somaclonal variations, somatic cell hybridization [1,2], transformation of high yielding cultivars [3] and also to conserve important Citrus genotypes. In plants, the Ca 2+ is involved in almost all biological processes. Calcium serves as a ubiquitous secondary messenger and regulates a multitude of physiological and developmental processes, including responses to abiotic stress, pathogen defense, and adjustment of ion homeostasis [4][5][6]. In response to environmental and developmental stimuli, plant cells react with specific temporal changes in cytosolic calcium (Ca 2+ ) concentration. Ca 2+ can serve simultaneously as a messenger and a regulator in so many different processes, that it raises the fundamental question of how specificity in information processing and output determination can be achieved [7][8][9]. The families of calcineurin B-like (CBL) proteins represent a unique group of calcium sensors and contribute to the decoding of calcium transients by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). In higher plants, CBL proteins and CIPKs form a complex signaling network that allows for flexible but specific signal response coupling during environmental adaptation reactions [10]. The CBL-CIPK network helps to maintain proper ion balance when abiotic stresses occur. The CBL and CIPK homolog are present in all green lineages, and phylogenomic analysis suggests their expansion from a single CBL-CIPK pair present in the ancestor of modern plants and algae [10]. Thus, the incorporation of these two key genes into the desired plants would improve the resistance against abiotic stresses. This study was aimed to develop resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation.

Plant materials and media
Orange species Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck was collected from Citrus Research Station, Jaintapur, Sylhet, Bangladesh, and identification and confirmation of collected samples were done by J.C. Sarker, Senior Scientific Officer, Citrus Research Station, Jaintapur, Sylhet , Bangladesh. The healthy and good quality mature seeds from these orange cultivars were collected. Dehusked mature seeds were sterilized with 70% ethanol for 5 mins, 0.1% HgCl 2 and Tween 20 for 2 mins, followed by washing with autoclaved distilled water and then dried on sterilized filter paper. The culture medium used in this study was based on MS [11] basal salts and vitamins ( Table 1). All plant samples handled in this experiment were maintained in specific culture room to avoid any direct contact with environment, and all the used chemicals and plant materials were discared following bio-safety guideline provided by institute.

Agrobacterium strains and culture conditions
The transformation studies were carried out with Agrobacterium tumefaciens LBA4404 harboring the binary vector pBI121/PsCBL and pBI121/PsCIPK containing abiotic stress tolerant PsCBL and PsCIPK genes, respectively, uidA gene encoding β-glucuronidase (GUS) and nptII gene encoding neomycin phosphotransferase II conferring kanamycin resistance [12][13][14]. The single colonies of both strains were cultured in liquid YEP medium containing 100 mg/l kanamycin and grown in a shaker at 200 rpm in dark at 28°C for 48 h until the OD 600 reached between 0.4 and 0.8. The bacterial cells were collected with centrifugation at 8000 rpm for 5 mins and suspended in MS resuspension medium in an appropriate ratio to adjust OD 600 to 0.5 and then different concentrations of acetosyringone (100, 150, and 200 μM) were added.

Callus induction
The sterilized seeds were inoculated in test tube on MS medium containing 2,4-D at different concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0 mg/l) and incubated at 25 ± 1°C in 2000 lx light. Fifty seeds were inoculated per replication, and experiments were repeated three times. After 3 weeks only yellowish white colored globular calli were selected and cut into small pieces and subcultured onto fresh callus induction medium (Table 1) for infection with Agrobacterium.

Infection with Agrobacterium and co-culture
Fresh calli were taken from previous experiment and subjected to infection by immersing them in the infection medium containing the LBA4404 strain having pBI121/PsCBL sense vector and LBA4404 strain having pBI121/PsCIPK sense vector for 90 mins with intermittent gentle shaking and dried on sterile filter paper for 30 mins. The infected calli were cultured on cocultivation medium (Table 1) for 3 days in dark conditions at 25°C ± 1°C. The co-cultivation medium was fortified with 0, 100, 150, and 200 μM acetosyringone to determine the optimum concentration of acetosyringone to increase the transformation efficiency.

Washing and inoculation in selection medium
After the appearance of slight growth of Agrobacterium around most of the calli were rinsed 8-10 times in sterile distilled water. Followed by washing with sterile distilled water, the calli were washed with the washing solution containing cefotaxime (500, 600, 700, and 800 mg/l) for 5 mins. The antibiotic treated calli were washed with sterile distilled water and dried on sterile filter paper. Then, the calli were transferred onto the selection medium and incubated for 15 days at 25 ± 1°C in dark. After that, the calli were again sub-cultured on the selection media (Table 1) for 7 days and incubated in 25 ± 1°C in dark.

Regeneration of putative transformants and In-vitro evaluation
Fresh and growing calli from the second selection medium were transferred to regeneration medium containing different concentrations and combinations of BA and NAA and were incubated at 25 ± 1°C in white fluorescent light under 16-h photoperiods and subcultured on the same media after 14 days. Shoot proliferation started after 3 weeks and the regenerated shoots were shifted to the rooting media and were maintained at 25 ± 1°C in white fluorescent light under 16-h photoperiods. In-vitro evaluation of kanamycin resistantregenerated plantlets was done in MS medium supplemented with different concentrations of NaCl (50, 100, 150, and 200 mM) and 3 mg/l PEG. Tolerant plants  Infection medium Infection medium was prepared by adding bacterial culture in MS resuspension medium in an appropriate ratio to adjust OD 600 to 0.5.
Selection media MS medium was supplemented with 30 g/l sucrose and 3.0 mg/l 2,4-D followed by adjustment to the required volume with dH 2 O and pH adjustment to 5.8. After autoclave filter sterilized kanamycin 100 mg/l was added.
Regeneration media MS medium supplemented with different concentrations of BA and NAA.
Rooting medium MS medium containing no hormone. were maintained under controlled conditions by subculturing at an interval of 21 days regularly.

GUS histochemical assay of transformed callus
GUS activity was detected as described by Jefferson et al. (1987). Randomly kanamycin-resistant calli of two Citrus species were taken from selection medium and washed with autoclaved distilled water to remove adjacent media. The calli were incubated in X-glucuronide (5-bromo-4-chloro-indoyl β-D glucuronide) staining solution at 37°C for 24 h in dark. The X-glucuronide was broken down by the activity of β-glucuronidase (GUS) gene, which was transferred to the cells of calli by Agrobacterium tumefaciens. The stained tissues were rinsed several times with 75% ethanol. Calli stained with indigogenic dye were scored, and stable GUS expression was tested in kanamycin-resistant calli. The transformation efficiency was calculated by percent of GUS-positive calli.

PCR analysis of transformed regenerated plantlets
The genomic DNA of kanamycin-resistant regenerated plantlets (putative) and control plants of two Citrus species were extracted from the leaves by using the modified CTAB method [15]. The 600 bp fragments of the GUS gene were amplified using the following set of primer: Forward 5´TTTGCAAGTGGTGAATCCCGACCT-3´and Reverse 5´AGTTTACGCGTTGCTTCCGCC AGT-3 [ 16]. The PCR reaction was carried out in 25-μl mixture containing 2.5 μl 10X Taq buffer, 1.5 μl 25 mM MgCl 2 ,   was also loaded on one side of the gel and electrophoresis was conducted at 75 V for 50 mins. After completion of electrophoresis, the gel was stained in ethidium bromide for 2 h and placed on UV transilluminator in Gel Documentation System. Finally, the photograph was captured (Nikon D5300).

Statistical analysis
All the experimental data were collected at regular intervals for analysis and reckoned under statistical basis.

Mature dehusked seeds from Citrus reticulata Blanco and
Citrus sinensis (L.) Osbeck were sterilized and inoculated on MS medium containing different concentrations of 2, 4-D. After 3 weeks of incubation, yellowish white, light green, and green colored globular calli were generated (Fig. 1). Maximum callus induction frequency of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck 89.33 ± 3.03% and 95.67 ± 4.04%, respectively, was found when MS medium was supplemented with 3 mg/l 2,4-D (Fig. 2). Yellowish white colored globular calli were selected for transformation. The selected calli of both species were pre-cultured on the same media for 3 days. Then precultured calli were infected by immersing them in the infection medium (MS) containing both Agrobacterium strains for 90 min with intermittent gentle shaking and dried on sterile filter paper for 30 minutes. After 3 days of co-culture in dark on co-culture medium, calli were washed with 500 mg/l cefotaxime and transferred onto selection medium for fifteen days and was sub-cultured again on same selection medium (Fig. 1). The concentration of antibiotic was optimized via antibiogram of Agrobacterium strains and plant tissue sensitivity test (Fig.  3). At 500mg/l cefotaxime, clear zones were found and no harmful effect on plant tissue (callus) was observed after 7 days of treatment. After the second sub-culture, the GUShistochemical assay was done and the frequency of GUSpositive calli was 83.33 ± 2.42% and 43 ± 2.00% for both species respectively (Fig. 4). Maximum GUS-positive results were found when 150-μM acetosyringone was added to the co-culture media and incubated for 3 days (Fig. 5).
The fresh kanamycin-resistant calli were then transferred to the regeneration medium and sub-cultured on same media at an interval of 21 days. After 4 weeks of inoculation on regeneration medium, shoot (2-10 shoot per callus) generation started (Fig. 6). The frequencies of shoot induction were 61.11 ± 1.35% and 55.5 ± 1.03% for both species respectively, when MS medium was supplemented with 3.0 mg/l of BA and 2 mg/l of NAA (Fig. 7). The shoots obtained from differentiation of callus were taken and cultured on rooting medium [17]. Roots appeared within 20 days of inoculation. The genomic DNA was isolated from leaves of the putative transformed plantlets and nontransformed control plants and run on a 0.8% agarose gel for each sample (Fig. 8a). Transformation at genomic level was detected by amplifying GUS gene. Specific primers were used in PCR analysis to verify the presence of transgenes (GUS). Transgenic plantlets of both species produced bands of the expected size of 600 bp (Fig. 8b), whereas the corresponding bands were not found in con- The transgenic plantlets of two species remained fresh and survived for more than 4 weeks on MS medium supplemented with 50 mM NaCl, 2 weeks on 100 and 150 mM NaCl, and 1 week on 200 mM NaCl but control plantlets became pale within 5-7 days and died after 10 days (Fig. 9). These results confirm the expression of stress tolerant genes in these Citrus species.

Discussion
In this research, Agrobacterium-mediated PsCBL and PsCIPK genes were transferred to citrus species, Citrus reticulata Blanco, and Citrus sinensis (L.) Osbeck using mature seed-derived calli. The previous study [18] reported that mature seed-derived callus was the most amenable explants for Agrobacterium-mediated transformation. Differences between various hormone supplementations were observed in callus induction. The efficiency of the Agrobacterium-mediated transformation method was inspected through vigilant observations of the effects of several parameters considered to be critical. The success of transformation was assessed by the percentage of blue spots signifying transient expression of GUS gene and the PCR detection of GUS gene. The density of Agrobacterium directly affects transformation efficiency since gene transfer only occurs with proper Agrobacterium attachment to plant cells. Therefore, high Agrobacterium concentration increased the number of plant cells being infected [19]. In this study, the highest number of GUS staining was observed in Agrobacterium suspension OD 600 at 0.5. More concentrated Agrobacterium suspension (when OD 600 was 0.8-1.0); however, significantly reduced the number of transformed cells due to the fact that intense Agrobacterium infection caused severe damage to the plant cells. The previous study [19] reported that bacteria concentration at OD 600 0.6 to 0.8 was the most efficient in sweet potato embryogenic callus Agrobacterium-mediated transformation. Karami, 2008 [20] also reported that Agrobacterium concentration for transformation is dependent on multi-factors including Agrobacterium strain and viability, plant species, and cultivar and type of tissue used. The age of the callus is a crucial factor for transformation efficiency. Hiei and Komari, 2008 [21] reported that fresh and healthy immature embryos ensure the successful transformation. Young embryogenic callus is also favorable due to its higher regeneration ability as compared to old calli [22,23]. Sharawat, 2007 defined the pre-culture period as the period that starts at the moment when immature embryos are first isolated and cultured immediately before Agrobacterium infection [24].
Zuraida et al. 2011 [25] showed that pre-culture period for more than 3 days improved transformation frequency. Four-day pre-cultured calli were used for Agrobacterium infection. During infection, the pre-culture calli were immersed in the infection medium for 90 mins with intermittent gentle shaking. The infected calli were dried for 30 mins on sterilized filter paper. Eighty-five transformation rate was recorded when calli were infected for 90 mins and dried for 30 mins [25]. In this study, infected calli were co-cultured for 3 days [25]. Zuraida et. al., 2011 stated a significant increase of transient GUS expressions were observed in 4 days of co-cultured calli but we found maximum GUS expression when co-cultured for 3 days. The influence of various acetosyringone concentrations (0, 100, 150, and 200 μM) in the co-culture medium were evaluated and significant GUS staining was achieved at the addition of 150 μM acetosyringone. A similar result was obtained in the transformation of sweet potato when the effect of acetosyringone concentration in cocultivation medium was investigated [26]. After 3 days of co-cultured at the dark condition, the calli were washed with a washing solution containing 500 mg/l cefotaxime. The antibiotic concentration was selected through antibiogram of Agrobacterium and plant cell sensitivity test. Transient GUS gene expression was determined by GUS assay and PCR amplification of the GUS gene [27]. Almeida et al. 2003 stated that 81.5% GUS-positive result found in the Agrobacterium-mediated transformation of "Hamlin" sweet orange which is nearly similar to our result. Media composition, mainly the hormonal balance is an important factor influencing in-vitro culture initiation and plant regeneration from callus [28]. MS medium supplemented with 3 mg/l of 6-benzylaminopurine (BA) showed maximum regeneration efficiency for both Citrus species [29]. reported that the MS medium supplemented with 3 mg L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. Shoots were rooted on MS medium without supplementation of hormone [29][30][31][32] and the rooted plantlets were evaluated on stress conditions. The transgenic plantlets showed moderate resistance against abiotic stresses like salt.

Conclusion
The demand of Citrus (orange) is increasing day by day in Bangladesh. A huge amount of foreign currency is being spent for importing orange. It is possible to grow Citrus commercially, fulfill the national demand, and save foreign currency by eliminating the problems by developing good varieties. In this research, a reliable and efficient transformation system for two Citrus species was developed and transgenic plants with stable integration of target genes were recovered. This study will help for the development of Citrus varieties with desired traits.