ESTABLISHMENT OF AN EFFICIENT REGENERATION SYSTEM AMENABLE TO AGROBACTERIUM MEDIATED TRANSFORMATION OF TWO ELITE INDICA RICE VARIETIES OF NORTH EAST INDIA

An efficient plant regeneration system from embryogenic callus of two elite indica rice (Oryza sativa spp. indica) varieties of Northeast India, Ketokijoha and Monoharsali is established. The effect of auxin, 2,4-dicholorophenoxy acetic acid (2,4-D) on callus induction was optimized. Friable, nodular and creamish-white embryogenic calli were induced from mature seeds on NB medium supplemented with 2.5 mg/l 2,4-D. Plants were regenerated from 40-50 days old embryogenic callus on NB medium containing 0.5 mg/l BAP (6-benzylaminopurine) and 0.25 mg/l ABA (abscisic acid). Regenerated plants with multiple tillers were rooted on half strength MS medium and rooted plants were acclimatized with 94% survival rate. Higher frequency of callus induction as well as plant regeneration was recorded in Ketokijoha as compared to Monoharsali. The calli of both the varieties were found amenable to Agrobacterium-mediated transformation as evident from strong GUS (βglucuronidase) expression. The results may find wide application for genetic improvement for valuable traits these elite indica rice varieties of Northeast India.


Introduction
Rice (Oryza sativa L.) is the main staple food for more than half of the world. In Asia, more than 90% of rice is produced and consumed (Datta 2004). Among the two major subspecies of rice, long-grained indica rice accounts for 80% of cultivated rice. Establishment of a highly efficient regeneration system for indica rice is a prequisite for successful application of genetic engineering tools for improvement of cultivars against biotic and abiotic stress, and value added traits. Genetic transformation of rice in general and indica subspecies in particular is considered to be difficult due to their recalcitrance to in vitro manipulations. However, these constraints have been overcome in part by regenerating transgenic rice plants from transformed embryogenic calli (Hiei et al., 1994) as these tissues are known for their amenability to both gene delivery and plant regeneration. Among the factors that affect frequency of callus induction and plant regeneration in indica rice, genotype, physiological status of the seed scutellum, type and dose of the plant growth regulators among others are very important (Ge et al., 2006). Although there are several reports of successful regeneration of transgenic indica rice from Agrobacterium-mediated transformation of embryogenic calli (Ignacimuthu andArockiasamy 2006, Mohanty et al.,2002), many indica varieties are still recalcitrant to cultural manipulations due to poor callus induction, proliferation and regeneration abilities (Islam et al.,2004, Khaleda and Al-Forkan 2006, Zuraida et al., 2011. Difficulties still exist in establishing a widely used culture system for indica rice applicable to wide range of cultivars. Northeast India is considered to be one of the hot spots of rice genetic resources of the world and rice is extensively cultivated in this region under extremely diverse cultivation conditions (Durai et al., 2009). Among the rice varieties of north-eastern region, aromatic rice of the state Assam, Ketokijoha is most popular due to its aroma, quality and palatability (Das et al.,2010) and Monoharsali is an widely adapted variety and identified as resistant to blast, bacterial leaf blight, sheath blight and brown plant hopper (Rathaiah and Das 1987). However, protocols for high frequency plant regeneration using embryogenic callus cultures have not been reported in these highly popular two local indica rice varieties.
Success in transformation of indica rice using embryogenic callus based regeneration systems depends on the factors that favor the formation of friable and high quality callus in a shorter time competent for shoot regeneration. The potential for callus induction and regeneration have been reported to be varietal dependent limiting efficient regeneration in large number of regional indica rice Research Article varieties for genetic manipulation (Ali et al. 2007). Here, we established an efficient and reproducible protocol for plant regeneration from embyogenic callus induced from seed scutellum amenable to Agrobacterium-mediated transformation in two of the most popular indica rice varieties of Northeast India.

Plant Material and Callus Induction
Mature seeds of rice (Oryza sativa L) varieties, Ketokijoha and Monoharsali were obtained from Krishi Vigyan Kendra Regional Agricultural Research Station Akbarpur, Karimganj, India. Mature seeds were dehusked, surface sterilized with 70% ethanol for 30 s, rinsed with 1% bavistin and 0.2% HgCl2 (w/v) for 5 min each. The seeds were subsequently rinsed five times with sterile double distilled water and cultured on NB medium (Chu et al.,1975) containing proline (500 mg/l), glutamine (500 mg/l), casein hydrolysate (300 mg/l) with varying concentrations of 2,4-D (0, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mg/l) for callus induction. The proliferating calli were subcultured twice onto fresh media at an interval of 30 and 15 days respectively. The frequency of callus induction was determined as the number of seeds producing calli per number of seeds cultured on callus induction media. Each treatment consisted of 30 seeds, and each experiment was repeated thrice. Visual observations of the cultures were made periodically in every week, and the frequency of callus induction was recorded after 30 days. The relative frequency of callus induction was compared between the two rice varieties.

Multiple Shoot Induction and Plant Regeneration
Embryogenic calli, after two subcultures, were transferred to regeneration medium NB medium containing different concentration of BAP (0.5, 1.0 and 1.5 mg/l) in combination with 0.25 mg/l of ABA. The efficiency of multiple shoot induction was evaluated by scoring the mean number of shoots induced from responding embryogenic callus, and measuring the mean shoot length. The regeneration frequency was calculated based on the number of callus responding to regeneration by total number of callus cultured. The regeneration frequency and efficiency of multiple shoot induction were compared among the two cultivars.

Culture Conditions
In all the case, the pH of the medium was adjusted to 5.8 with 1 N NaOH or HCl and 0.8% agar (Hi-Media, Mumbai) was added prior to autoclaving at 121 0 C at 15 psi for 15 min.
All the cultures were maintained at 25±2 0 C under 16-h photoperiod with a photosynthetic photon flux density (PPFD) of 50 μmol m -2 s -1 provided by 40 W cool white fluorescent lamps (Philips, India).Seeds cultured in hormone free media were considered as control.

Rooting and Transplantation
The shoots (5-8 cm) were separated from multiple shoot clumps and transferred individually to MS medium (Murashige and Skoog 1962) devoid of growth regulator for two weeks for root formation. Subsequently plantlets with well-developed root system were washed in tap water and acclimatized in polybags containing soil and vermicompost (1:1), covered with transparent polybags at 27°C and 16 h photoperiod for 14 days. Finally, the acclimatized plantlets were transferred to pots containing soil and established in a green house.

Transformation Procedure and GUS Assay
Agrobacterium tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with β-glucuronidase (gus) interrupted with an intron in the coding region and neomycin phosphotransferase (nptII) genes, and plant selection marker hygromycin phosphotransferase (hptII) driven by CaMV35S promoter (Fig. 1) was used for transformation studies. The bacteria was grown on YEP (10 g/l yeast extract, 10 g/l peptone, 50 g/l NaCl,15 g/l agar and pH 7.0-7.2) solid medium containing 50 mg/l kanamycin and 10 mg/l rifampicin at 28° C. A single bacterial colony was inoculated into 2 ml of liquid AB medium containing 5 mg/l rifampicin and 25 mg/l kanamycin and grown overnight on a rotary shaker at 200 rpm at 28º C. Bacteria were pelleted at 5000 rpm for 5 min and resuspended in liquid NB medium supplemented with 2.5 mg/l 2,4-D, containing 100 μM acetosyringone at a density of OD600=1. Embryogenic callus from 45-d-old calli were used in transformation being immersed in bacterial suspension for 30 min with shaking at 80 rpm at 25º C. Inoculated explants were blotted on sterile filter paper and co-cultivated on solid NB medium supplemented with 2.5 mg/l 2,4-D containing and 100 µM acetosyringone for 3 days at 25º C under dark condition. After co-cultivation, the embryogenic calli were washed three to four times with sterile double distilled water by constant stirring and blotted dry on sterile filter paper. Approximately 20 explants were tested for transient gus expression by histochemical assay using 5-Bromo-4-Chloro-3-Indolyl Glucuronide (X-Gluc) as a substrate (Jefferson 1987). The explants were then visually scored for transient gus activity.

Statistical analysis
Data were subjected to analysis of variance (ANOVA) and mean separation by Duncan's multiple-range test (DMRT) using single-factor completely randomized block design in order to study the effect of different treatments on shoot proliferation and frequencies of transient expression. All experiments were performed at least three times with a minimum of 30-40 calli per treatment.

Results and Discussion
Efficient transformation of rice using Agrobacterium mediated approach and subsequent plant regeneration are dependent on crucial factors such as the choice of explant, hormonal composition of the medium used and nutritional supplements for effective induction of callus and regeneration (Ratnayake and Hettiarachchi 2010). Among the different explants, scutellum derived callus has been reported to be the most amenable explant for Agrobacterium-mediated transformation (Hiei et al., 1994). However, success in transformation of indica rice using scutellum derived callus depends on the factors that favor the formation of friable and high quality callus in a short time competent for shoot regeneration. Moreover, the potential for callus induction and regeneration are often variety-dependent and therefore need establishment of efficient regeneration for specific varieties for genetic manipulation (Ali et al., 2007). Moreover, poor regenerative ability of a cultivar can be a bottleneck in the establishment of an efficient system for genetic transformation (Carsono andYoshida 2006, Nishimura et al., 2005).
In the present study, factors affecting efficient embryogenic callus induction and plant regeneration were presented prior to the investigation of the amenability of explant to Agrobacterium-mediated transformation in two of the most popular indica rice varieties of rice diversity-rich Northeast India.

Effect of 2,4-D on Callus Induction
Most of the seed explants responded to callus induction on medium containing 2,4-D. The callus induction appeared from the scutellar region of the seeds within 7-10 days of culture in 2,4-D containing media in both Ketokijoha and Monoharsali varieties (Fig. 2). However, significant differences were recorded among the 2,4-D concentrations tested with respect to percentage of callus induction (Fig.  2). The highest percentage of callus induction was achieved in medium containing 2.5 mg/l 2,4-D in both Ketokijoha (84%) and Monoharsali (82.87%). Apparently, no significant difference in percentage of callus induction was observed in the two varieties tested (Fig. 2). It was also observed that 2.5 mg/l 2,4-D was ideal concentration for further proliferation of embryogenic callus in the sub cultured medium. Callus induction was poor on medium with lower (1.0, 1.5 and 2.0 mg/l) concentrations of 2,4-D while necrosis of callus was visible with increasing concentration of 2,4-D (3.0 and 3.5 mg/l) (Fig. 2). The browning in most of the initiated callus at higher 2,4-D concentrations affected further proliferation in the subcultured medium. No callus formation was observed in control NB medium devoid of 2,4-D (Fig. 2).
The explants were cultured on callus induction medium for 4 weeks and the calli were sub-cultured onto the same fresh medium thrice. Compact, nodular creamish-white embryogenic calli were formed within 24 days of culture initiation. The high-quality calli was yellow or light yellow in color and was dry, compact, and globular in appearance (Fig. 3a). The stimulatory effect of 2,4-D at 2.5 mg/l on induction of embryogenic callus from seed scutellum has been reported in a number of indica rice genotypes (Karthikeyan et al.,2009, Sabir et al.,2010, Saharan et al.,2004. The 2,4-D at concentrations between 2.0-2.5 mg/l facilitated embryogenic callus induction in somatic tissues of indica and japonica rice varieties (Tyagi et al.,2007).

Plant Regeneration
In order to evaluate the efficacy of regeneration medium differing in concentrations of BAP on differentiation of shoots, the embryogenic calli were shifted to regeneration medium after 40-50 days of callus initiation. The preoembryogenic clumps on calli turned green in colour within 3 weeks of culture on regeneration medium. The emergence of shoot buds appeared from the green clumps within 3-4 weeks of subculture on fresh medium (Fig. 3b). We found that the concentration of BAP in regeneration medium containing 0.25 mg/l of ABA significantly influenced the regeneration frequency. Among the different concentrations of BAP tested, medium fortified with 0.5 mg/l BAP showed highest regeneration frequency in both Ketokijoha (84%) and Monoharsali (71%). The results revealed significant difference in regeneration frequency between the two indica varieties, Ketokijoha and Monoharsali tested (Fig. 3 and 4) indicating the effect of interaction of genotype and regeneration medium on regeneration frequency. Within indica subspecies, significant variations of in vitro culture response have been reported in different genotypes (Seraj et al., 1997). Regeneration medium containing 2.5 mg/l BAP resulted in an average of 4.33 and 4.21 shoots from embryogenic callus cultures of Ketokijoha and Monoharsali respectively (Table  1; Fig. 3c and 4c).  The stimulatory effect of BAP has been reported to facilitate regeneration from embryogenic callus cultures in rice (Hoque andMansfield 2004, Rachmawati andAnzai 2006). On the contrary, BAP in combination with NAA have been reported to induce differentiation of shoots from embryogenic callus cultures (Karthikeyan et al., 2009). Most of the shoot buds developed to shoots within 15-18 days and complete plants with multiple tillers (Fig. 3d and  4d).
Upon separation and transfer of individual shoots to half strength MS medium, numerous roots were developed within 2 weeks of culture ( Fig. 3d and 4e). Over 94% of rooted plantlets survived when established in soil ( Fig. 3e  and 4f). The plants resumed growth in greenhouse reaching maturity and represented no phenotypic variation or sterility, irrespective of two cultivars tested.

Transformation and GUS Expression
It is generally known that genotype remains the major limiting factor restricting successful transformation in indica rice using seed scutellum derived callus (Ge et al., 2006). In order to determine the competency of the regeneration competent scutellum derived callus of two varieties, Ketokijoha and Monoharsali to Agrobacteriummediated genetic transformation, experiments were carried out to inoculate explants with A. tumefaciens. Strong transient gus expression was detected in the scutellum derived callus in both the varieties, Ketokijoha and Monoharsali (Fig. 5). The endogenous GUS activity (color) was not detected in non-transformed (control) explants. GUS activity at the regenerating sites indicated the amenability of explants to Agrobacterium mediated transformation. Transformation efficiency was determined by measuring the number of GUS positive calli per total calli inoculated. Highest transient gus expression efficiency was recorded in variety Ketokijoha (92%) whereas 74% transient GUS expression efficiency was recorded in Monoharsali (Fig. 6). Calluses derived from mature seeds have been effectively used as target for Agrobacterium-mediated transformation and regeneration of transgenic plants in a number of indica rice varieties (Hiei et al., 1994, Ignacimuthu and Arockiasamy 2006, Kumar et al., 2005, Ozawa et al., 2009, Tyagi et al., 2007.

Conclusion
Production of embryogenic callus with high regeneration capacity is a prerequisite for highly efficient genetic transformation in rice. Our findings established an efficient protocol for in vitro plant regeneration from mature seed derived callus in two of the most popular indica rice varieties of Northeast India, Ketokijoha and Monoharsali. Competency of plant tissues in Agrobacterium infection is an important factor in Agrobacterium-mediated transformation. Our results demonstrated that regeneration competent scutellum-derived callus of both the indica varieties are amenable to Agrobacterium mediated transformation and combined with their shoot proliferation ability could lead to regeneration of stable transgenic plants.
The established method may find immense application as a practical tool for genetic improvement for valuable traits in two of these popular indica rice varieties of Northeast India.