Introduction

Establishment of efficient embryogenic cultures has become an integral part of plant biotechnology, as regeneration of transgenic plants in most of the important crops is dependent on the formation of somatic embryos (SEs) (Vasil 2008). Large-scale production of SEs can be applied to the production of artificial seeds (Thorpe 1995). A somatic embryogenesis pathway for plant regeneration has also been considered desirable for Agrobacterium-mediated transformation because it is assumed that SEs originate from single cells; thus, the derived transgenic plants are unlikely to be chimeric and will originate from low numbers of transgene integration events (Zhang et al. 2008). Many reports are available for somatic embryogenesis of millets (reviewed by Kothari et al. 2005). Recently, we have also reported a highly efficient somatic embryogenesis and plant regeneration system for finger millet (Ceasar and Ignacimuthu 2008).

Millets are capable of surviving in some of the most inhospitable ecosystems of the world providing food and fodder to millions where other quality cereals fail to grow. The projected food demand for 2025 will require the yield of cereals including millets to rise from 2.5 to 4.5 t ha−1 (Borlaug 2002). Production of millets is also constrained by biotic and abiotic stresses; development of rapid in vitro propagation system is more helpful in the improvement of millets. Establishment of an efficient somatic embryogenesis and regeneration system is also a vital prerequisite for the successful transformation and recovery of transgenic millet crops (reviewed by Ceasar and Ignacimuthu 2009). Any further improvement in the system for somatic embryogenesis and plant regeneration is essential for transformation and molecular studies of kodo millet in the future.

Several reports are available for in vitro studies of kodo millet: somatic embryogenesis was obtained from explants such as immature inflorescences (Nayak and Sen 1989; Vikrant and Rashid 2001; Kaur and Kothari 2004), immature and mature zygotic embryos (Vikrant and Rashid 2002a), mature caryopses (Vikrant and Rashid 2002b), root culture (Kaur and Kothari 2003) and mesocotyl and leaf segments (Vikrant and Rashid 2003). Direct organogenesis from immature inflorescence was also reported in another study (Kavi Kishor et al. 1992). We have already developed a regeneration protocol for kodo millet from shoot apex explants (Arockiasamy et al. 2001). Recently, a highly efficient plant regeneration system through embryogenesis was also developed from immature inflorescences by micronutrient modification (Kothari-Chajer et al. 2008). In this study, we report a highly efficient somatic embryogenesis and plant regeneration system from shoot apex explants of kodo millet. We developed an innovative two-step culture procedure using two different (induction and maturation) media for induction and maturation of SEs and from shoot apex explants. In this study, embryogenic callus (EC) was obtained on an induction medium containing auxin and cytokinin; this EC was then transferred to a maturation (second) medium for SE maturation and SE conversion to plantlets. The effects of amino acids, carbohydrates and cytokinins were tested in the maturation medium adopting a two-step culture method which has not been investigated so far in any millet. The two-step culture method was applied in this study with an attempt to develop an efficient system for SE maturation and plantlet formation in kodo millet.

Materials and methods

Plant material

The kodo millet cultivar used in this study was RK98; seeds of this cultivar were procured from the University of Agricultural Sciences, GKVK campus, Bangalore, India. Seeds were immersed in 0.1% (v/v) detergent solution of Tween-20 in distilled water for 15 min and rinsed in 70% (v/v) alcohol for 30 s and washed with sterile distilled water. They were then immersed in 0.1% HgCl2 for 5 min for surface sterilization and washed a further three times for 5 min each with distilled water before transferring to 100-ml Erlenmeyer flasks (30 seeds in each flask) containing Murashige and Skoog (1962; MS) basal medium supplemented with 3% (w/v) sucrose (Hi-media, Mumbai, India) and solidified with 0.6% (w/v) agar (bacteriological grade; Himedia) for germination. The flasks were incubated at 25 ± 2°C in the dark for 7 days. Shoot tips, consisting of the apex (4–6 mm in size), were excised inside a laminar airflow hood with the help of a scalpel blade from aseptically grown, 7-day-old seedlings. This shoot apex was used as the explant in this study. The pH of this and all other media used in the study was adjusted to 5.8 using 0.1 M NaOH before autoclaving for 15 min at 121°C and 1.3 kg cm−3 pressure.

Induction of EC

Shoot apex explants were inoculated in the induction medium. This medium consisted of MS basal salts supplemented with 3% (w/v) sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 4.5, 9.0 or 13.5 μM. To determine the combined effect of auxin and cytokinin on EC induction, MS medium containing 9.0 μM 2,4-D or 2,4,5-T was further supplemented with 2.25 μM Kn or zeatin (Zn). These cultures were incubated at 25 ± 2°C in the dark. The resulting callus was subcultured routinely every 2 weeks onto fresh medium with the same composition, and the callus was observed closely at 2-day intervals for any change in the morphology. Only EC with yellow color, nodular structure, and embryogenic in nature were considered in this study. The average percentage of shoot tips which produced EC was counted after 5 weeks of incubation and the number of globular stage SEs formed in each EC was determined after 7 weeks of incubation in the dark at 25 ± 2°C.

SE maturation and plantlet formation

Five-week-old EC with nodular structures, obtained from induction medium, was transferred to 250-ml Erlenmeyer flasks containing maturation medium for SE maturation and plantlet formation. EC obtained from two different induction media (9.0 μM 2,4-D and 9.0 μM 2,4-D + 2.25 μM Kn) were transferred separately into the maturation medium. This was performed to study the role of induction medium on SE maturation and plantlet formation. Only SEs with globular stage were considered in this investigation. The effects of cytokinins, carbohydrates and amino acids were tested in the maturation medium for the efficient SE maturation and plantlet formation. One set of EC was also subcultured in the same induction medium to serve as control.

In the first maturation medium, the effect of cytokinin was tested on SE maturation and plantlet formation by adding Kn, benzyladenine (BA) or thidiazuron (TDZ) at 2.25 and 4.50 μM separately to MS medium supplemented with 3% (w/v) sucrose. This was performed to find out the optimum concentration and type of cytokinin for SE maturation and conversion to plantlets. MS basal medium containing 3% (w/v) sucrose and devoid of plant growth regulators (PGRs) was also included in this experiment as one of the maturation media to evaluate the role of MS basal medium on SE maturation and plantlet formation.

The effect of carbon source was examined in a separate experiment on SE maturation and plantlet formation; three different concentrations each of glucose (170, 220 and 275 mM), sucrose (87.7, 120 and 150 mM) and maltose (90, 120 and 150 mM) were added separately to MS basal medium supplemented with 4.50 μM TDZ but devoid of sucrose. TDZ was added in this maturation medium based on the response obtained in the previous experiment with the addition of different cytokinins; the medium containing 4.50 μM TDZ gave the superior response. This experiment was carried out in order to pick out the optimum concentration and type of carbon source for efficient SE maturation and plantlet formation.

The influence of amino acids on SE maturation and plantlet formation was also tested in another experiment; three amino acids (l-serine, l-proline and l-glutamine) were added separately each at 100, 200 and 300 μM to MS basal medium supplemented with 4.50 μM TDZ and 120 mM maltose to investigate the role of amino acids on SE maturation and plantlet formation. TDZ (4.50 μM) and maltose (120 mM) were added based on the optimum response observed in previous two experiments, and they were added in this assessment along with different amino acids to enhance the SE maturation and conversion to plantlet.

In all the above experiments, flasks containing maturation media inoculated with EC cultures were initially incubated in the dark for 2 weeks for SE maturation and then kept in the light (16 h) with the light intensity of 50 μm−2 s−1 photosynthetic photon flux density (PPFD) at 25 ± 2°C for plantlet development. The mean number of SEs formed and plantlets produced in each EC were determined after 2 and 5 weeks of incubation, respectively.

Data collection and statistical analysis

Each experiment was repeated three times and each experiment consisted of three replicates. In EC induction, 20 shoot apex explants were used per treatment; the mean percentage of EC induction (no. of explants forming EC/Total no. of explants × 100) and mean number of SEs produced by each EC were calculated after 5 and 7 weeks of incubation, respectively, in the dark. In SE maturation and plantlet formation, ten EC, each derived from one shoot tip explant, were used per treatment; the mean number of SE (with 0.2–0.5 mm in diameter) formed was calculated after 2 weeks of incubation in the dark and the mean number of shoots produced was calculated after a further 3 weeks of incubation in the light. For statistical analysis, data were analyzed using analysis of variance (ANOVA,) and Fisher’s least significant difference (LSD) was used to compare the means.

Results

Induction of EC

Mature seeds of kodo millet cultivar RK98 germinated and produced initial shoots after 7 days of culture on growth regulator-free MS medium. Shoot tips excised from these seedlings served as the initial explants for the induction of EC. Callus induction was observed on the cut ends and meristem regions of shoot apex explants after 2 weeks of incubation in the dark (Fig. 1a). Initially, all calli were white in color and non-regenerative in nature (Fig. 1b) with soft, friable and unorganized morphology and devoid of nodular structures. This callus was subcultured every 2 weeks onto fresh medium of the same composition after which most of the calli became yellow, nodular and embryogenic in nature after a total of 5 weeks (Fig. 1c). Only this latter type of tissue was investigated further and reported in this study.

Fig. 1
figure 1

Induction and maturation of somatic embryos in kodo millet cultivar RK98. a Initiation of callus from excised shoot apex explants after 1 week incubation in the dark on MS medium supplemented with 9.0 μM 2,4-D. b Enlargement of callus after 2 weeks of incubation in the dark on MS medium supplemented with 9.0 μM 2,4-D. c Formation of SEs after 7 weeks of incubation in the dark on MS medium supplemented with 9.0 μM 2,4-D and 2.25 μM Kn. d Maturation and germination of EC on MS medium containing 4.50 μM TDZ. e Shoot and root induction from SEs after 2 weeks of culture in the light on MS medium supplemented with 4.50 μM TDZ and 120 mM maltose. f Shoot elongation on MS medium supplemented with 4.50 μM TDZ, 120 mM maltose and 200 μM l-proline. g Plants maintained in paper cups covered with polythene bags for hardening. h Maturation and seed setting of soil grown plant

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The mean percentage of EC induction ranged from 23.5 to 87.6% based on the concentration and combination of PGRs (Table 1). MS medium supplemented with 2,4-D produced superior EC induction compared to MS medium with 2,4,5-T. The optimum response was observed at 9.0 μM for both auxins. Addition of cytokinin enhanced the induction of EC; Kn produced superior response over Zn when added with either auxin. The highest percentage of EC induction was obtained in the MS medium containing 9.0 μM 2,4-D and 2.25 μM Kn (87.6%). Nodular-shaped SEs emerged from the EC upon maturation, after 7 weeks of incubation in the dark (Fig. 1c). The mean number of SEs produced also varied based on the concentration and combination of PGRs (Table 1). Like the EC induction result, the MS medium containing 9.0 μM 2,4-D and 2.25 μM Kn produced the highest number of SEs (15.3).

Table 1 Effect of auxin and cytokinin on induction of embryogenic callus in kodo millet cultivar RK98
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Effect of cytokinins on SE maturation and plantlet formation

Five-week-old EC obtained from induction medium was transferred to the maturation medium containing MS basal devoid of PGR and MS basal medium supplemented with Kn, BA or TDZ. EC transferred to the maturation medium containing cytokinin was matured, producing SEs and later converted to plantlets (Fig. 1d, e). EC obtained from the induction medium containing auxin and cytokinin (9.0 μM 2,4-D + 2.25 μM Kn) gave larger numbers of SEs and plantlets than EC obtained from the medium containing auxin alone (9.0 μM 2,4-D) (Table 2). Maturation media containing cytokinins produced a superior response for SE maturation and plantlet formation compared to MS basal devoid of a cytokinin. The maximum number of SEs and plantlets were produced at 4.50 μM for all the three cytokinins tested (Table 2). TDZ at 4.50 μM produced a superior response both for the number of SE maturation (26.4) and plantlet formation (15.9). Conversion of SE to plantlet was also more rapid in the MS medium containing cytokinins compared to MS devoid of a cytokinin. SEs matured on MS medium containing cytokinin were also bigger in size.

Table 2 Effect of cytokinins on somatic embryo maturation and plantlet formation in kodo millet cultivar RK98
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Effect of carbohydrates on SE maturation and plantlet formation

The effect of three different carbon sources (glucose, sucrose and maltose) was tested on SE maturation and plantlet formation in MS basal medium containing 4.50 μM TDZ. EC obtained from the induction medium containing auxin and cytokinin (9.0 μM 2,4-D + 2.25 μM Kn) gave more SEs and plantlets than EC obtained from the medium containing auxin alone (9.0 μM 2,4-D) (Table 3). Maltose gave a superior response for SE production (31.3) and plantlet formation (21.0) at 120 mM compared to sucrose and glucose (Table 3). This was even higher than the MS basal medium containing 87.7 mM sucrose (MS level). Sucrose at 87.7 mM produced even fewer SEs (26.1) and plantlets (14.3). SEs formed on maturation medium containing maltose also gave rapid elongation of shoots on plantlet conversion. EC obtained from the auxin-containing medium (9.0 μM 2,4-D) gave an inferior response in different concentrations of carbohydrates. MS basal medium containing 170 mM glucose produced the least number of SEs (4.4) and medium containing 275 mM glucose produced the least number of plantlets (3.4) for EC obtained from auxin containing medium (Table 3).

Table 3 Effect of carbohydrates on somatic embryo maturation and plantlet formation in kodo millet cultivar RK98, in MS medium supplemented with 4.50 μM TDZ
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Effect of amino acids on SE maturation and plantlet formation

The influence of three different amino acids (l-serine, l-proline and l-glutamine) was tested in MS basal medium containing 4.4 μM TDZ and 120 mM maltose for SE maturation and plantlet formation. Addition of any of these three amino acids increased both the number of SEs and plantlets (Table 4). EC obtained from the induction medium containing 9.0 μM 2,4-D + 2.25 μM Kn gave more SEs and plantlets than EC obtained from the medium containing 9.0 μM 2,4-D alone upon transfer to the maturation medium containing different amino acids, TDZ and maltose (Table 4). MS medium supplemented with l-proline was found to be superior to that containing l-glutamine or l-serine on SE maturation and plantlet formation (Table 4). The optimum concentration of l-proline, glutamine and serine was 200 mM. The maximum number of SEs (39.4) and plantlets (31.3) were produced at 200 mM l-proline. MS medium containing l-glutamine produced moderate response and l-serine added medium produced the least response for SE maturation and plantlet formation (Table 4). The leaves produced in the medium containing l-proline were also longer and more greenish in nature.

Table 4 Effect of amino acids on somatic embryo maturation and plantlet formation in kodo millet cultivar RK98, in MS medium supplemented with 4.50 μM TDZ and 120 mM maltose
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Hardening and acclimatization

Plantlets were formed by the development of shoot and root apparatuses after 4 weeks of incubation in the light (Fig. 1f). The plants were carefully removed from the culture flasks, washed with sterile water to remove agar media, placed in paper cups (60 mm in diameter and 110 mm high) filled with sterilized vermicompost (Bharat organic fertilizers, Karnataka, India) and supplied with diluted (5 times) MS basal salts; the plants were covered with polythene bags (10 × 8 cm) to maintain high humidity (Fig. 1g). The plants were then removed from the cups after 3 weeks and transferred to the field outside the greenhouse with 100% survival rate. Regenerants grew well and did not show any variation in morphology and growth characteristics; flower, seed settings and yield of the grain were also similar to control plants (Fig. 1h).

Discussion

In this study, we have used shoot apex as an initial explant to induce EC. Shoot apex explants have been used in millets for both regeneration (Arockiasamy et al. 2001; Ceasar and Ignacimuthu 2008) and transformation (Latha et al. 2005, 2006) studies. Shoot apical meristems have also been used effectively to develop regeneration systems across the other cereals, and the use of apical meristem explants have been successfully employed as starting material to recover stably transformed maize, wheat, rice, oat, barley, sorghum and millet (reviewed by Sticklen and Orabya 2005). Somatic embryogenesis and plant regeneration system developed in this study from shoot apex explants of kodo millet may be utilized in future for the in vitro culture and transformation experiments.

In order to induce EC, two auxins, 2 4-D and 2,4,5-T, were tested separately and in combination with Kn or Zn. 2,4-D gave the optimum response of somatic embryogenesis at 9.0 μM. This is in conformity with earlier publications on kodo millet (Vikrant and Rashid 2001, 2003). The induction of callus in cereals and millets is commonly achieved by 2,4-D (Mohanty et al. 1985; Chandra and Kothari 1995; Kothari-Chajer et al. 2008). However, in this study, addition of Kn or Zn to medium containing 2,4-D (9.0 μM) increased the percentage of EC induction from shoot apex explants with the optimal response achieved at 9.0 μM 2,4-D and 2.25 μM Kn. A similar response of superior somatic embryogenesis was observed by combining 2,4-D and Kn in finger millet (Ceasar and Ignacimuthu 2008). Recently, Kothari-Chajer et al. (2008) used 8.6 μM 2,4-D alone in the micronutrient-modified MS medium for somatic embryogenesis on the kodo millet genotype GPUK3.

Effects of cytokinin, carbohydrate and amino acid were tested in the maturation medium on maturation and of EC. The mineral composition of the media, type and concentration of carbohydrates and amino acids can play vital roles in somatic embryogenesis (Mohamed et al. 2004). Transfer of EC to the maturation medium containing cytokinin, modified carbohydrate and amino acid produced more SEs and plantlets compared to the control subcultured in the same induction medium; two-step culture was very effective for SE maturation and plantlet formation in this study. A two-step culture process has also been proved successful in other plant species for efficient somatic embryogenesis and regeneration (Tirajoh et al. 1998; Bellettre and Vasseur 1999; Gallego et al. 2001; Xie and Hong 2001; Seabrook and Douglass 2001).

In the first maturation medium, the role of cytokinin was examined on SE maturation and plantlet formation. Addition of cytokinin in the maturation medium promoted the SE maturation and conversion to plantlets. Cytokinin has been used most frequently in the medium for efficient regeneration of millets. Nayak and Sen (1989) used Kn and BA, while a combination of BA with naphthalene acetic acid (NAA) was also used in another study for kodo millet regeneration (Kaur and Kothari 2004). In a previous report, we have used BA separately and in combination with NAA for regeneration of kodo millet from shoot apex explants (Arockiasamy et al. 2001). Kn and BA have also been used for efficient shoot induction and plant regeneration in pearl millet (Mythili et al. 2001; Srivastav and Kothari 2002), finger millet (Ceasar and Ignacimuthu 2008) and foxtail millet (Xu et al. 1984). Among the three cytokinins tested, TDZ was superior to BA and Kn on SE maturation and plantlet formation; TDZ has been used most frequently in recent times for the tissue culture studies of many plants. It showed high cytokinin activity in promoting growth of cytokinin-dependent cultures (Mok et al. 2005). TDZ stimulated conversion of cytokinin nucleotides to more biologically active nucleotides (Capelle et al. 1983) and stimulated accumulation of endogenous purine cytokinins (Thomas and Katterman 1986). So far, TDZ has not been used most frequently in millet tissue culture studies. This work provides the proof for the proficient role of TDZ in kodo millet regeneration, so it can be used in future for the efficient regeneration of kodo and other millets.

The effects of three carbohydrates (glucose, maltose and sucrose) were also tested in another maturation medium containing 4.50 μM TDZ to discover the role of carbon source on SE maturation and plantlet formation. Maltose gave the best response of SE maturation and plantlet formation compared to glucose and sucrose. Medium containing 120 mM maltose produced more SEs and plantlets than medium containing 87.7 μM sucrose (MS level). The superiority of maltose over sucrose on somatic embryogenesis was also proved in other plants including rice (Asano et al. 1994), wheat (Karsai et al. 1994), barley (Scott and Lyne 1994) and rubber (Blanc et al. 1999). Maltose also enhanced somatic embryogenesis from shoot apices in pea (Loiseau et al. 1995) and immature embryos in sunflower (Jeannin et al. 1995). Sugars function as both a carbon source and as an osmotic regulator in culture media (Zhang et al. 2008). The proficient role of maltose over sucrose for the initiation of SEs in cereal has been explained either in terms of the metabolism of sucrose leading to the accumulation of potential toxic products (Scott and Lyne 1994) or that maltose is degraded at a much slower rate than sucrose (Navarro-Alvarez et al. 1994). From this study, it is apparent that replacing sucrose with maltose can improve the SE maturation and plantlet formation in kodo millet.

Addition of amino acids improved both the mean number of SEs and plantlets formed in the MS medium containing 4.50 μM TDZ and 120 mM maltose. l-proline was more prominent compared to two other amino acids (l-glutamine and l-serine) on SE maturation and plantlet formation. l-proline has been shown to possess a stimulatory effect during embryogenesis and to provide osmo-tolerance (Shimizu et al. 1997; Bela and Shetty 1999; Hita et al. 2003). In a previous study, l-proline was found to give optimum response of somatic embryogenesis in kodo millet when added with 2,4-D (Vikrant and Rashid 2002a); this was added in a single medium. In this study, l-proline was used in a two-step culture method and it was added in the second (maturation) medium containing TDZ (4.50 μM) and maltose (120 mM). From this study, we found that addition of l-proline in the maturation medium is beneficial for SE maturation and plantlet formation. Marchant et al. (1996) showed both primary and secondary embryogenesis in the presence of l-proline in rose. l-proline was considered to provide organic nitrogen supply and protection against possible stress associated with in vitro culture and embryogenesis (Claparols et al. 1993). Other reports have also stated the vital role of l-proline on SE maturation in callus culture of corn (Armstrong and Green 1985; Suprasanna et al. 1994; Pareddy and Petolino 1990), somatic embryogenesis in maize (Santos et al. 1993) and somatic embryogenesis in long-term callus cultures of barley (Rengel and Jelaska 1986). It has been suggested that the presence of amino acids in the culture medium is important because they partially replace the NH4 +. Amino acids can also increase the levels of reduced nitrogen which stimulates the development of SEs in several species (George 1993). The highest number of SEs and plantlets were obtained in the maturation medium containing 4.50 μM TDZ, 120 mM maltose and 200 μM l-proline. Addition of these compounds in the maturation medium offers a rapid and efficient multiplication of kodo millet via somatic embryogenesis.

In conclusion, our two-step culture offered a highly efficient system for induction and maturation of SEs in kodo millet. Addition of l-proline, maltose and TDZ in the maturation medium increased the frequency of SE maturation and plantlet formation. This system might offer some possibilities in terms of the molecular study of somatic embryogenesis or transformation of kodo millet in future. This two-step culture procedure may also be adopted in future for the efficient somatic embryogenesis and regeneration of the rest of the millets.