Background

Various in vitro culture techniques are being applied for varietal development of cereal crops among which matured dehusked seed culture is often used in rice for creation of novel genetic variants (somaclonal variation). In this context, addition of chemical mutagen (at very low concentration) to the medium still expands the spectrum of genetic variation in culture. But, in vitro culture of seeds is limited by plant genotype and hormonal composition of medium (Jain, 1997) to sustain growth of calli, subsequent plant regeneration and survival as fertile plants. It is often difficult to induce embryogenic calli and to regenerate plants from these callus cultures specially those belonging to Indica subspecies (Jain, 1997). The recalcitrant nature of this subspecies has, in fact, been a major limiting factor in transfer of valuable genes (Toenniessen, 1991). An efficient callus induction and reproducible rapid regeneration system can achieve the success. Therefore, the present experiment was undertaken to develop an efficient callusing and rapid regeneration system in a popular upland rice variety ‘Khandagiri’.

1 Results and Discussion

1.1 Media response for callus induction

Success in callus induction of rice dated back to 1964 using nodes of young seedlings on Hellers medium (Fujiwara and Yatazama, 1964) and later Nishi et al. (1968) achieved plantlet regeneration via callus culture from seeds. Others used young inflorescence (Chaudhury and Qu, 2000), leaves (Mishra and Khurana, 2003; Gandonou et al., 2005), roots (Hoque and Mansfield, 2004), mature embryos (Zale et al., 2004) and mature seeds (Rachmawati and Anzai, 2006) as explants. However, mature seeds have the advantage as they can be obtained year-round (Alam, 1994).

MS medium induced good callusing response from mature kernels and callusing was initiated as early as 9th day of primary culture. LS medium was equivalent to MS, but BM medium had shown delayed and poor callusing response and callus growth (Table 1).

Root initiation and development of necrosis hinders pace of plantlet regeneration even in the highly responsive regeneration media. Once morphogenetic developmental path turns to direct regeneration of rooting, the calli hardly reverse back to their normal regenerability for plantlet development. Necrosis (spread from centre) is associated with depletion of the carbon source in the medium in addition to other media recipes. In the present investigation, 85-95% calli in BM and B5 medium turned necrotic indicating improper media composition. All media were purposefully supplemented with a reasonable concentration of 2, 4-D (2.0 mg/l) – a known inducer of somatic embryos in the primary calli. MS induced better somatic embryogenic response (30.4%) than LS medium, whereas both BM and B5 did not induced somatic embryos. Therefore, MS was selected for follow-up optimization of in vitro culture in rice.

1.2 Hormonal recipe for callus induction

Totipotency of cells relies on cellular development that involves induction of callus followed by morphoenesis via embryogenic or organogenic plant regeneration. Somatic embryo induction can be stimulated in callus culture using the right type and concentration of growth regulators (Titov et al., 2006). 2,4-D alone or in combination with cytokinins, is mostly preferred for the purpose (Castillo et al., 1998).

Embryogenic cell mass is produced on the surface of the calli and these grow faster and gives rise to plants through somatic embryogenesis (Vasil, 1987). 2,4-D is in vogue has role for increased rate of cell division and profuse callusing. In the present investigation, 2,4-D at 2.5 mg/l alone had shown better callusing response (70.2%) and that with 0.5 mg/l Kn further increased callus induction frequency (76.0%) (Table 2, Figure 1) as well as excellent callus growth. Higher proportion of the calli induced was creamy, embryogenic and nodular with minimum necrosis. The calli formed were friable having easily dissociating clusters with a few cells. Such calli are in vogue amenable for genetic transformation. In contrast, 2,4-D with BAP induced non-embryoenic soft calli unsuitable for plant regeneration. Revathi and Pillai (2011) proved 2 mg/l 2,4-D alone to be the most favourable for callus induction and callus proliferation. Zafar et al. (1992) achieved somatic embryogenesis on MS and N6 medium with 2,4-D and Kn. Similarly, Karthikeyan et al. (2009) obtained friable, nodular and creamish-white embryogenic callus cultures from mature rice seeds on LS medium with 2.5 mg/l 2,4-D and 1.0 mg/l Thiamine-HCl.

At low concentration (1.0 mg/l), 2,4-D reduced the incidence of necrosis, but it could have a negative influence on the callus induction frequency and follow up differentiation in the regeneration medium. Tariq et al. (2008) achieved higher callusing response in N6 medium than MS medium each at 2.5-3.0 mg/l 2,4-D. But Vikrant et al. (2012) reported highest organogenic and somatic embryogenic plant regeneration response using calli derived from MS + 5 mg/l 2,4-D. In contrast, Jubair et al. (2008) realized 100% callusing response in a Bangladeshi rice land race ‘Topa’ in MS with 2 mg/l 2,4-D. Therefore, 2,4-D concentration in the callus induction medium should be at least not less than 2.0 mg/l. Somatic embryos once developed in the callus, the callus can be maintained to continuously produce somatic embryos. In the present investigation, organogenic calli were suitably maintained in MS + 2,4-D (2.5 mg/l) + 0.5 mg/l Kn for few subcultures, but still reduced concentration of 2,4-D (2 mg/l) with 0.5 mg/l Kn was needed for continuity in somatic embryo production (Figure 2).

1.3 Hormonal recipe for plant regeneration

Low auxin coupled with high cytokinin concentration is in vogue results shoot morphogenesis (Evans et al., 1981). Shoot buds emerge from the totipotent organized cell clumps and develop into shoots which thereafter need transfer to rooting medium for rhizogenesis. While, somatic embryos once develop in the callus, such calli on transfer to regeneration medium undergo a process of maturation of somatic embryos and finally develop into whole plantlets with defined shoot and roots. Hence, somatic embryogenic plant regeneration is preferred over organogenic pathway.

Sequential or complete withdrawal of 2,4-D is necessary for plant regeneration from organogenic and somatic embryogenic callus. Evans et al., (1981) obtained shoot regeneration (51.1%) at low concentration of NAA relative to BAP. Azria and Bhall (2000) stressed the importance of either BAP or TDZ in combination with NAA for shoot initiation. In the present investigation, small green projections appeared on the callus, which later developed into shoot buds. These shoot buds developed into plantlets, when the callus along with the shoot buds, was sub-cultured on a medium containing the same hormonal combination. MS basal (without glycine) + 2.0 mg/l BAP + 1.0 mg/l NAA elicited better organogenic plant regeneration (60.2%) (Table 3) within 2 weeks of culture but, it results more number of albino plantlets. However, MS at 2.0 mg/l BAP + 0.5 mg/l NAA added with 0.5 g/l ADS (Figure 3) revealed equivalent organogenic response (58.4%) with profuse green shoots/calli (10.2) and higher frequency of plant survival (80.2%). Kn or combination of Kn and BAP with NAA did not reveal good response over the above BAP + NAA combination for regeneration response. However, Jubair et al. (2008) realized 80% regeneration response with an average of 3 shoots per explant in MS with 3.0 mg/l BAP + 0.5 mg/l NAA + 0.5 mg/l Kn.

Embryogenic calli usually exhibit high regeneration response (Seraj et al., 1997). Physiological pathways of development that determine organogenic response is different from somatic embryogenesis. Gandonou et al. (2005) observed a high positive correlation between the ability to produce embryogenic callus and the capacity for plant regeneration. Thus, embryogenic callus percentage can offer a good index to assess plant regeneration potential (Wang et al., 1987). Hormonal balance is an important factor influencing plant regeneration from embryos (Jiang et al., 1998). Evans et al. (1981) obtained plant regeneration (48.9%) with defined shoot and roots at low concentration of NAA relative to BAP in LS medium. Tariq et al. (2008) reported higher regeneration response in N6 medium than MS medium each at 2.5 mg/l BAP + 1.0 mg/l NAA within 27-30 days. In this context, MS with 2.0 mg/l BAP + 0.5 mg/l NAA revealed excellent somatic embryogenic regeneration response (70.5%) with 8.8 shoots per responsive callus as compared to any other combinations of BAP/Kn with NAA (Table 3). In contrast, Karthikeyan et al. (2009) achieved plant regeneration from 24 days old embryogenic callus on MS medium with 1.0 mg/l BAP and 1.5 mg/l NAA.

1.4 Effect of hormonal concentrations on rhizogenesis

Auxin alone or with very low concentrations of cytokinin is important for induction of root primordia (Evans et al., 1981). But it is not always true. Excised shoots when transferred to hormone-free MS (Azria and Bhall, 2000) or half-strength MS basal medium either liquid (Verma et al., 2011) or in solid form (Wani et al., 2010) also is reported to induced rooting.

In the present investigation, full strength R basal medium failed to develop roots (Table 4). But, half-strength R medium was shown to initiate healthy rooting with few laterals, although %-response of rooting from the excised shoots was poor (35.05+0.65%). This is because low salt levels and more specifically a lower nitrogen level is usually favourable for root initiation. Therefore, an attempt was taken to optimize the hormonal combination at varying concentrations in half-strength R basal medium. NAA at 1.0 mg/l with increased BAP up to 0.2 mg/l gave highest rhizogenetic response (84.6+1.05%) and the excised shoots developed profuse normal rooting within a week (Figure 4a). Further, increase in BAP (0.3 mg/l) at 1.0 mg/l NAA had shown delayed rooting with short fibrous roots, and even no rooting response at concentrations beyond 0.3 mg/l BAP. In contrast, Bano (2005) reported that 0.5 mg/l BAP with 0.3 mg/l IAA was sufficient for induction of roots in the regenerated plantlets.

1.5 Acclimatization and field establishment

The plantlets were transferred to pot mixture (peatmoss: perlite-2:1), and successfully acclimatized in glasshouse under partial shade (Figure 4b). The plants regenerated from first few callus cultures were phenotypically normal and fertile. The in vitro rapid plantlet regeneration protocol formulated in this study may be suitably used for successful Agrobacterium mediated genetic transformation.

2 Materials and Methods

mature healthy dehulled kernels of cv. Khandagiri was treated with 1% bavistin (w/v) mixed with a drop of Tween 20 for 15 min. followed by washing (5x) with sterilized distilled water with continuous shaking for 10 min. Further, the seeds were treated with 0.1% (w/v) HgCl₂ solution for 8 min followed by rinsing (5x) with sterile distilled water and blot dried on sterilized filter paper before inoculation on culture medium. The pH of the media was adjusted to 5.8 before autoclaving at 121°C and 15 psi for 15 min.

The test treatments included four callus induction media and various hormone recipes singly or in combination for optimization of in vitro culture of cv. Khandagiri. The calli were partially desiccated in between filter papers on petridish before transfer to fresh medium. Each step of in vitro culture was repeated, at least twice.

Data were recorded for callusing response, callus quality (color, texture and relative growth), morphogenetic potential and plant establishment; and analyzed statistically by the Duncan’s multiple range test (Duncan, 1955). Means followed by the same letter within columns were considered not significantly different at p≤0.05.

Authors’ contributions

SKT contributed for planning, execution of the experiment and editing of the MS; MM, SP and BS helped in carrying out the experiment; DBS, SKB and BC contributed towards review collection, data analysis, tabulation and type setting. All authors read and approved the final manuscript.

Acknowledgements

The authors sincerely acknowledge and thank all researchers for their valuable contributions included in this pursuit as references.

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