Callus induction and plant regeneration via leaf segments of three accessions of African rice (Oryza glaberrimaStued.)

A study conducted with the aim of developing a protocol for callus induction and plantlet regeneration in vitro from leaf segments of three accessions of African rice (O. glaberrimaSteud.) indigenous to Ghana. Leaf segments of the accessions namely, Guame, N/4 and SARI 1 were assessed for callus induction and plantlet regeneration ability on different concentrations of plant growth regulators, incorporated into Murashige and Skoog, (1962) (MS) basal medium. Frequency of callus induction which was achieved on MS medium supplemented with (0-10) mg/l 2,4-D differed significantly (p≤0.05) among the accessions, as well as among the levels of 2,4-dichlorophenoxyacetic acid (2,4-D) tested. Highest callus induction frequency was exhibited at a concentration of 6 mg/l 2,4-D for all three accessions.Sub-culturing of callus on regeneration medium, which consisted of MS supplemented with (1:0-5) mg/l NAA:BAP resulted in no plantlet regeneration in all tested accessions. Instead, prolific root formation was observed.


INTRODUCTION
Rice is the most important food crop in the world and feeds over half of the global population (Sasaki, 2005).Increased production of the commodity is necessary to meet the predicted demands of an ever increasing population. One option is to increase the area under rice cultivation, which is getting more difficult as more farmlands are being converted to residential areas in the developing world. The most viable option, therefore, is to increase productivity by advances in biotechnology (Bajaj and Mohanty, 2005). Biotechnological techniques have been used to improve the existing cultivars, for the synthesis of novel plants and early release of high-yielding plants and plants resistant to various diseases, pests, stresses and temperature. The successful application of technology for crop improvement requires suitable in vitro plant regeneration methods. Callus, which is an unorganised, proliferative mass of differentiated plant cells, is one of such means by which crop improvement can be undertaken. The ability to regenerate plants from callus is influenced by physiological factors as well as the genotype of the plant (Henry et al., 1994). The regeneration of plants of some cereal crops such as bread wheat (Redwayet al., 1990;Vasilet al., 1990), barley (Luhrs and Lorz, 1987),rice (Yamada et al., 1986) and maize (Duncan et al., 1985), from callus have been documented. In rice, there are reports on successful plant regeneration from explants such as coleoptile (Oinam and Kothari, 1995), root tips (Sticklen, 1991), immature embryos (Koetjeet al., 1989), leaf blades (Yan and Zhao, 1982), and other parts of O. sativa. However, protocol for callus induction and plant regeneration for O. glaberrima accessions have not been achieved. The objective of this study was to induce callus and regenerate plantlets in vitroin three different accessions of O. glaberrima by determining the hormone types and their concentrations suitable for inducing callus from their leaf segments as well as determining the hormone types and hormone concentrations /combinations for regenerating plants from leaf-derived calli of three accessions of O. glaberrima.

II. MATERIALS AND METHODS 2.1 Callus induction from leave segments of O. glaberrima.
Seeds of three Oryza glaberrima accessions namely N/4, Guame and SARI 1 were manually dehusked and were surface sterilized by immersing in 0.1% mercuric chloride (HgCl2) andvigorously agitated for 2 minutes under the laminar flow hood and thereafter rinsed with threechanges of sterile distilled water. The sterilized seeds were inoculated in test tubes containing 1.15ml of hormone-free Murashige and Skoog (MS) (1962) basal medium prepared from stock, supplemented with 30 g/l sucrose and 100 mg/l myo-inositol with pH 5.8 adjusted using 1M KOH prior to addition of 3.5 g/l phytagel and autoclaving at 121°C for 15 minutes at 15 psi. The cultures were kept in a growth room at a temperature of 21°C under a 16/8-hr (light/dark) photoperiod with light provided by white fluorescent tubes (T 5 fluorescent fitting, UK) at an intensity of 3000 lux. Seeds were allowed to germinate under these conditions to produce seedlings. Leaves obtained from four-days old in vitro germinated O. glaberrima seedlings were excised from base and cut into three pieces (referred to as segments 1-3, with 1 being the leaf base segment closest to the seed and 3 being the segment closest to the tip of the leaf) and inoculated in culture jars containing MS medium supplemented with different concentrations of picloram (0-10 mg/l) or 2,4-dichlophenoxyacetic acid (2,4-D) (0-10 mg/l) together with30 g/l sucrose and 100 mg/l myoinositol. The pH was adjusted to 5.8 and 3.5 g/l phytageladded to the medium before autoclaving at 121°C for 15 minutes at 15 psi. The cultures were subsequently incubated in total darkness at 21°C for 12 weeks. The experiment was set up as a completely randomized factorial design. The factors tested were three accessions of O. glaberrima by six concentrations of 2,4-D by six concentrations of picloram. Data were subjected to analysis of variance (ANOVA) based on 5 replications for percentage callus formed. The means were separated, where appropriate, at the 5% significance level using the least significant difference (LSD). Data were analyzed using Genstat statistical package.

Plant regeneration from leaf-derived callus of O. glaberrima
Calli obtained were sub-cultured on MS medium supplemented with napthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) in a ratio of (1:0-5) mg/l NAA:BAP followed by addition of 30 g/l sucrose and 100 mg/l myo-inositol. The pH was adjusted to 5.8 and 3.5 g/l phytageladded to the medium before autoclaving at 121°C for 15 minutes at 15 psi. The cultures were subsequently kept in a growth room at a temperature of 21°C under a 16/8-hr (light/dark) photoperiod with light provided by white fluorescent tubes (T 5 fluorescent fitting, UK) at an intensity of 3000 lux. The experiment was set up as a completely randomized factorial design, (3x6). The factors tested were three accessions of O. glaberrima by six concentrationsof NAA:BAP.

Effect of concentration of 2,4-D or picloram on callus formation from leaf segments of O. glaberrima
Leaf segment explants of the three O.glaberrimaaccessions; N/4, Guame and SARI 1 cultured on MS medium supplemented with varying concentrations (0-10 mg/l) of picloram did not develop callus irrespective of the accession or concentration of picloram present in the medium. However there was development of callus from leaf segment explants cultured on the same MS medium but supplemented with varying concentrations of 2,4-D (0-10) mg/l , occurred after 12 weeks of culture. Amongst the three portions of leaf segments inoculated, only segment 1 (leaf base) was tissue culture responsive ( Fig.1(D)). Segments 2 (middle) and 3 (tip) did not form callus on any of the callus induction media tried. All calli obtained from this experiment were formed from the leaf bases (segment 1). Colour of callus ranged from cream ( Fig.1(E)) to pale yellow ( Fig.1(F)) to brown with callus becoming intense in colour as concentration of auxin in medium increased, eventually becoming necrotic at 8mg/l ( Fig.1(G)). Higher concentrations of 2,4-D caused necrosis of the initiated calli.

Fig.1 Callus induction from leaf segment explants of O. glaberrima seedling (A) Dehusked mature seed inoculated on hormone-free medium; (B) 4 days old in vitro germinated O. glaberrima seedling; (C) Leaf segments inoculated on callus induction medium; (D) Callus formed from leaf base (segment 1); [(E-G) Callus formed from the leaf base (segment 1) showing different colours; (E) Cream (F) Pale yellow (G) Necrotic calli]; (H) Calli showing only root formation
Callus formation was observed in all the three rice accessions following addition of4 mg/l, 6 mg/l and 8 mg/l 2,4-D to the culture medium (Fig.2). The highest percentage callus formation was recorded at 6.0 mg/l 2,4-D, recorded in all the three rice accessions, and which proved significantly higher compared to percentage callus formation at other concentrations of 2,4-D used, in each case. Percentage callus formation following an increase in concentration of 2,4-D from 6 mg/l to 8 mg/l was not statistically different (p≥0.05) from a decrease in 2,4-D concentration from 6 mg/l to 4 mg/l (Fig.2).Callus formation however, depended on the concentration of the auxinin the culture medium. The percentage callus formation increased as the concentration of 2,4-D in the media increased for all the O. glaberrima accessions used.

Effect of accession of O. glaberrima on callus formation from leaf segments
Callus induction from O. glaberrima used for this study was found to be variable and accession dependent. Among the three accessions, N/4 recorded the highest number of explants forming callus (12.2%) from inoculated leaf segments, which was significantly (p≤0.05) higher than the other two accessions (Fig.3) Frequencies of callus induction from the leaf segments ofGuame and SARI 1 (6.7% and5.5% respectively) were notstatistically different from each other (Fig.3).The three accessions showed significant (p≤0.05) differences as regards frequency of callus formed.

Interaction effect of O. glaberrima accession and level of 2,4-D in culture medium on callus formation from leaf segments
Generally, callus formation was low even with 2,4-D in the culture medium. However, some accessions performed better than others. Amongst the three O. glaberrima accessions, leaf segment explants from N/4 developed the most calli on a medium with 6.0 mg/l 2, 4-D, (40%), with Guame and SARI 1 both recording 20.0% (Fig.4) at the same concentration of 2,4-D. In all the three rice accessions, percentage callus formation increased with increasing concentration of 2,4-D from 4 mg/l to a peak at 6 mg/l (Fig.4) before dropping.. However, the increase was at different rates depending on the accession. The interaction between accession and concentration of 2,4-D in callus induction medium was however, not statistically significant (P≥0.05).

Effect of NAA and BAP on shoot and root formation from leaf-derived callus of O. glaberrima
Calli induced from leaves of the three O. glaberrima accessions were further cultured in vitro for assessment of shoot and root regeneration ability on MS medium supplemented with a single concentration of NAA(1 mg/l) and varying concentrations (0-5 mg/l) of BAP. Of the six regeneration media evaluated with different combinations of NAA and BAP, none was able to regenerate plantlets (shoots with roots). Instead media containing low levels (0-2 mg/l) of BAP led to prolific root formation from calli while media containing high levels (3-5 mg/l) of BAP led to necrosis of calli. There were no signs of plantlet regeneration from calli sub-cultured onto any of the regeneration media for any of the three O. glaberrima accessions.

IV. DISCUSSION 4.1 Effect of hormone type and hormone concentration on callus induction from leaf segments of O. glaberrima
Results from the experiment indicate that 2,4-D but not picloramcan induce callus from leaf base segments of O. glaberrima. Also, 2,4-D at concentrations of 4mg/l, 6mg/l and 8mg/l yielded calli for all three accessions of O. glaberrimatested with the best result recorded at 6mg/l, suggesting that the hormone 2,4-D plays a crucial role in callus induction as earlier reported by Chen et al., (1991).This observation also concurs with previous studies by Ramesh et al., (2009), which showed that 2,4-D at 3.0 mg/l was effective in inducing callus from leaf base segments of O. sativa. It is also consistent with findings by Abe and Futsuhara, (1984)   Furthermore, this study revealed that different portions of the leaf responded differently to callus induction with the leaf base segment showing the highest percentage callus induction in all the accessions used for the study. This is consistent with an earlier report by Ramesh et al.,(2009) which stated that induction of embryogenic calli from rice leaf is restricted to only the leaf base.
Another interesting observation from this study was the colour of calli obtained. Calli induced from media that had lower levels of 2,4-D were creamish. However, the colour intensified as the concentration of 2,4-D in the induction medium increased. In a related study in chick pea

Effect of accession of O. glaberrima on callus inductionfrom leaf segments
This study proved that the ability to induce callus was greatly influenced by the genotypeof O. glaberrima used.

Effect of interaction of O. glaberrima accession and 2,4-D concentration on callus induction medium on callus formation from leaf segments
The present investigation revealed that all three O. glaberrima accessions, concentration of 2,4-D as well as their interaction largely affected callus induction. This observation is in agreement with findings by Pandeyet al., (1994), who reported that the success of in vitro culture largely depends on the nutritional media, growth regulators, genotype and on the interaction of genotype and medium. Similar reports were also made by earlier workers (Abe and Futsuhara, 1986; Guo and Cao, 1982).

Effect of NAA and BAP on plantlet regeneration
who reported on the stimulatory effect of BAP in combination with NAA in facilitating plantlet regeneration in rice callus cultures. Differences in response may be due to different concentrations of hormone used. The rationale behind combining NAA and BAP was to simultaneously regenerate shoots with roots so as to reduce duration of culture of plantlets. NAA is a synthetic auxin that stimulates cell division in the pericylce leading to the formation of lateral and adventitious roots (Taiz and Zeiger, 1998) and BAP is a first-generation synthetic cytokinin that elicits plant growth and development responses, by stimulating cell division (Raven et al., 1999). The

V. CONCLUSIONS
In vitro culture holds potential for improving overall yield of Oryza glaberrimaSteud., the traditional rice of African origin, with multiple adaptation to several biotic and abiotic stress factors. Based on the results this study, Murashige and Skoog (MS) basal medium supplemented with 30 g/l and 100 mg/l myo-inositol with pH adjusted to 5.8 and incorporated with the plant hormone (auxin) 2,4diclorophenoxyacetic acid (2,4-D) at an optimal level of 6 mg/l provided a viable medium for induction of callus in three accessions of O. glaberrima, namely N/4, Guame and SARI 1, using leaf base segments as explants. Whole plantlet (shoot with roots) regeneration which was not successful in this study may be achieved through subculture of induced calli onto same MS-supplemented medium incorporated with varying combinations of the cytokinin benzy-aminopurine (BAP) and the auxinnaphthaleneacetic acid (NAA) to stimulate shoot and root development respectively.