Direct plant regeneration from in vitro-derived shoot apical meristems of finger millet (Eleusine...

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In Vitro Cellular & DevelopmentalBiology - Plant ISSN 1054-5476Volume 51Number 2 In Vitro Cell.Dev.Biol.-Plant (2015)51:192-200DOI 10.1007/s11627-015-9672-2

Direct plant regeneration from invitro-derived shoot apical meristemsof finger millet (Eleusine coracana (L.)Gaertn.)

Lakkakula Satish, Stanislaus AntonyCeasar, Jayabalan Shilpha, ArockiamSagina Rency, Periyasamy Rathinapriya& Manikandan Ramesh

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PLANT TISSUE CULTURE

Direct plant regeneration from in vitro-derived shoot apicalmeristems of finger millet (Eleusine coracana (L.) Gaertn.)

Lakkakula Satish & Stanislaus Antony Ceasar &

Jayabalan Shilpha & Arockiam Sagina Rency &

Periyasamy Rathinapriya & Manikandan Ramesh

Received: 18 September 2014 /Accepted: 16 January 2015 /Published online: 6 March 2015 / Editor: John Forster# The Society for In Vitro Biology 2015

Abstract A rapid and reproducible protocol for direct plantregeneration has been established using in vitro-derived, ac-tively growing shoot apical meristems (SAMs) of the geno-type 'CO(Ra)-14'. Shoot apical meristems from 3-day-oldseedlings of three finger millet genotypes viz, 'CO(Ra)-14','Try-1', and 'Paiyur-2' were assessed for the efficiency of directshoot induction. The regeneration efficiency of 'CO(Ra)-14'surpassed the other two genotypes and produced a mass ofgreen, multiple shoots within 12 d on Murashige and Skoogmedium containing 17.6 μM 6-benzylaminopurine (BA),0.9 μM 2,4-dichlorophenoxyacetic acid in combination with750 mg L−1 proline, 500 mg L−1 casein enzymatic hydroly-sate, and 2 mg L−1 glycine. 'CO(Ra)-14' showed a maximumfrequency of multiple shoot regeneration (96%) with an aver-age of 8.3 shoots per explant. The age and size of shoot apicalmeristems played a crucial role in triggering adventitiousshoot bud proliferation. The multiple shoot buds elongatedspontaneously upon reducing the concentration of BA (to11.0 μM) in the shoot induction medium. The in vitro prolif-erated shoots were rooted best in half-strength MS mediumcontaining 2.8 μM indole-3-acetic acid and successfully ac-climated in the field, subsequently developing into fertileplants. Thus, we report a short tenure (45 d) in vitro wholeplant regeneration system, which could be effectively utilizedfor the production of transgenic finger millet plants.

Keywords Direct regeneration . Finger millet . Shootproliferation . Apical meristem

Introduction

Finger millet (Eleusine coracana [L.] Gaertn.) is grownworldwide in more than 4 million ha and is the staple foodfor millions of people in less developed countries of Africaand Asia (Sastri 1989). Its high nutritional value and ability togrow under harsh, severe drought conditions and survive inpoor soils makes it an outstanding crop among other minormillets. The crop is considered as a traditional component offarmers’ risk avoidance strategies in drought-prone areas andits seed can be stored safely for many years without risk ofinsect damage. The nutritional qualities of finger millet aresuperior to that of other cereals as they are rich in calcium,phosphorus, iron, cysteine, tyrosine, tryptophan, and methio-nine (Latha et al. 2005).

An efficient plant regeneration system is a prerequisite forgenetic transformation of crops (Sharma et al. 2011).Although several regeneration and genetic transformation pro-tocols are available for cereal crop varieties, finger millet hasreceived little attention compared to rice, wheat, maize, oats,and barley (Ceasar and Ignacimuthu 2009). In earlier regener-ation studies of finger millet, different types of explants likeembryogenic seed (Kothari et al. 2004; Latha et al. 2005;Sharma et al. 2011; Babu et al. 2012; Jagga-Chugh et al.2012), shoot tips (Eapen and George 1990; Ceasar andIgnacimuthu 2008, 2011), somatic tissue (Rangan 1976), leafsheath segments (George and Eapen 1990; Gupta et al. 2001),mature embryos (Kumar et al. 2001), immature embryos (Piuset al. 1994; Kumar et al. 2001; Yemets et al. 2003), immatureinflorescences (George and Eapen 1990; Kumar et al. 2001),

L. Satish : J. Shilpha :A. S. Rency : P. Rathinapriya :M. Ramesh (*)Department of Biotechnology, Science campus, AlagappaUniversity, Tamil Nadu 630004, Indiae-mail: [email protected]

S. A. CeasarDivision of Plant Biotechnology, Entomology Research Institute,Loyola College, Chennai 600034, India

In Vitro Cell.Dev.Biol.—Plant (2015) 51:192–200DOI 10.1007/s11627-015-9672-2

Author's personal copy

mesocotyl (Rangan 1976; Mohanty et al. 1985; Eapen andGeorge 1990), root (Mohanty et al. 1985), and leaf base seg-ments (Rangan 1976; Mohanty et al. 1985) were used. Allthese reports have described indirect plant regeneration proto-cols, predominantly involving a callus phase from varioustypes of explants, and have not emphasized methods of directregeneration without an intervening callus step. The simpleprocess of in vitro shoot regeneration involves culturing shootapical meristems (SAMs), present at the primary apex or ax-illary buds. However, SAMs have been successfullyemployed for direct multiple shoot regeneration in other cropslike rice (Nhut et al. 2000; Sticklen and Oraby 2005;Arockiasamy and Ignacimuthu 2007; Dey et al. 2012), wheat(Ahmad et al. 2002), maize (Zhong et al. 1992, 1996), sor-ghum (Zhong et al. 1998), pearl millet (Devi et al. 2000), oats,and barley (Zhang et al. 1996, 1999), with maximum regen-eration and transformation efficiency achieved relatively inde-pendent of genotype. To our knowledge, no detailed study isavailable regarding direct multiple shoot regeneration in fin-ger millet using SAMs.

Direct plant regeneration is an effective approach to mini-mize somaclonal variation significantly, as it reduces the cul-ture duration (Karp 1991; Burner and Grisham 1995). Also, itresults in minimal or substantial reduction of callus formationand subculturing cycles (Arockiasamy and Ignacimuthu2007). To prevent the rapid loss of regeneration potential infinger millet, calli requires a multi-stage subculturing proce-dure, which is a long process and labor intensive for the de-velopment of the whole plant propagation system (Nhut et al.2000; Lakshmanan et al. 2006; Dey et al. 2012). As such, theidentification and selective use of a suitable type of callus fortransformation and regeneration studies has been difficult(Labra et al. 2001; Arockiasamy and Ignacimuthu 2007).Here, we report a rapid and efficient direct plant regenerationsystem for the Indian finger millet genotype 'CO(Ra)-14'using in vitro-derived SAMs as explants, without an interme-diate callus phase. For multiple shoot induction and regener-ation, we optimized the age of the SAMs, explant size, and theconcentration and combination of plant growth regulators(PGR). This is the first report on direct regeneration fromSAMs of finger millet and can effectively be utilized for ge-netic transformation studies of this species in the future.

Materials and Methods

Plant material and explant preparation. Seeds of the Indianfinger millet, Eleusine coracana (L.) Gaertn, cultivars'CO(Ra)-14', 'Piyur-2', and 'Try-1' were obtained from TamilNadu Agricultural University, Coimbatore, Tamil Nadu,India. Seeds were pretreated with sterile water for 30 min inorder to remove the husk. Dehusked seeds were subjected tosurface disinfestations with 70% (v/v) ethanol for 1 min

followed by two washes with sterile distilled water. Seedswere subsequently rinsed with 0.1% (w/v) mercuric chloride(HgCl2) for 5 min and further rinsed three times, each 1 min,with sterile distilled water. Surface-sterilized seeds were inoc-ulated on Murashige and Skoog (MS; Murashige and Skoog1962) basal medium (pH 5.8) containing 3% (w/v) sucrose,0.8% (w/v) agar-agar type 1, and 0.2% (w/v) CleriGel™ (Hi-Media, Mumbai, India) and incubated in dark at 26±2°C for3 d. After 3 d of incubation, the shoot apical meristem wasexcised from the seedling and used as the explant source forthe multiple shoot induction in the subsequent experiments.Experiments were carried out to validate the effect of explantsize (2–8 mm in length), age of SAMs (1–5 d), and identifi-cation of the optimal PGR type and concentration on multipleshoot regeneration of finger millet genotypes.

Multiple shoot induction. Two different experiments wereused to evaluate the multiple shoot induction potential ofSAMs. In the first experiment, the regeneration efficiency ofthree genotypes was evaluated by culturing SAMs on MSbasal medium containing various concentrations of 6-benzylaminopurine (BA; 8.8, 13.2, 17.6, or 22.0 μM),thidiazuron (TDZ; 4.5, 9.0, 13.5, or 18.0 μM), and 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 6.7, 9.0, or11.3 μM) separately. The cultures were incubated in a growthchamber (Sanyo versatile environmental test chamber, Japan,model no MLR 351H) with 16/8-h light/dark and 75/50%relative humidity (RH), respectively, at 26±2°C for 12 d.The cultures were observed at 2 d intervals for any changein the morphology. The number of SAMs showing shoot in-duction and the total number of shoots regenerated from eachexplant were recorded after 2 wk of incubation. This experi-ment was carried out to identify the best PGR type and con-centration for inducing shoot buds directly.

In the second experiment, the best PGR and fingermillet genotype identified from the above experimentwere further examined to maximize multiple shoot re-generation. The influence of some of the essential ami-no acids such as casein enzymatic hydrolysate (CEH;200, 300, 400, 500, 750, or 1000 mg L−1), glycine(Gly; 0.5, 1.0, 1.5, 2.0, 3.0, or 4.0 mg L−1), and proline(Pro; 100, 250, 500, 750, 850, or 1000 mg L−1) alongwith 17.6 μM BA and 0.9 μM 2,4-D was systematicallyanalyzed to improve shoot induction and proliferation.The cultures were incubated at 26±2°C for 16/8-h light/dark conditions with 70/50% RH, respectively. The per-centages of responding explants, mean number of shootsinduced in each explant, and shoot lengths were calcu-lated after 12–14 d incubation in light.

Shoot elongation. The multiple shoots developed in the in-duction medium were transferred to elongation medium con-taining 6.6, 8.8, 11.0, 13.3, 15.5, or 17.7 μMBA, 0.9 μM2,4-

DIRECT PLANT REGENERATION FROM IN VITRO-DERIVED APICAL MERISTEMS 193


D, 500 mg L−1 CEH, 750 mg L−1 Pro, and 2 mg L−1 Gly.Cultures were maintained for 12 d at 26±2°C, 16/8-h light/dark and 75/50%RH, respectively. The percentage of explantswith elongated shoots, mean length of proliferated shoots andmean number of shoots per explant were calculated following2 wk incubation in light.

Root induction. Elongated shoots (5–6 cm long) were trans-ferred to half-strength MS medium containing indole-3-aceticacid (IAA; 1.4, 2.8, 5.6, 7.0, or 8.4 μM) with 1.5% (w/v)sucrose. CleriGel 4.5% (w/v) was used for solidifying rootingmedia. The cultures were grown at 26±2°C for 16/8-h light/dark for 1–2 wk. The percentage of shoots with roots, meannumber of roots, and average root lengths were recorded after2 wk of incubation.

Hardening and field transfer. Rooted plantlets were carefullyseparated from rooting media and washed with deionized wa-ter to remove excess media on the surface of the roots. Rootsinduced plantlets were transferred to the Yoshida solution(Yoshida et al. 1976) for 5–6 d at a temperature 26±2°C.Then, they were transferred to plastic cups containing sterileSoilrite (Keltech, Bengaluru, India) and peat (1:1, v/v) andmaintained in natural conditions for further development.

Data collection and statistical analysis. This study was car-ried out using a completely randomized design with threereplications of 75 explants per replication. Observations wererecorded on the percentage of explants with shoot induction,average number of shoots regenerated per explant, frequencyof shoot elongation, shoot length, frequency of rooting, rootnumber, and root length. Statistics on the number of rootedshoots were documented after 14 d of inoculation on rootingmedium. The survival percentage was documented after 30 dof transfer in plastic cups (11×15 cm). The data were analyzedusing one-way analysis of variance (ANOVA), the signifi-cance of variations within means was carried out at P=.05using Duncan’s multiple range test with SPSS Statistics 17.0(IBM SPSS Statistics).

Results

Induction of SAMs. Surface-sterilized mature seeds of thethree finger millet genotypes were inoculated on plant growthregulator-free MSmedia, and shoots were induced after 3 d ofinoculation. SAM segments excised from these seedlingswere used as preliminary explants for direct shootorganogenesis.

Direct shoot organogenesis. The SAM explants of finger mil-let genotypes 'CO(Ra)-14', 'Try-1', and 'Paiyur-2' were

cultured in MS medium containing various concentrations ofBA, TDZ, or 2,4-D individually. Shoot initiation was ob-served at the cut ends of SAM explants within 1–2 d of incu-bation and most of the segments initiated green shoots at highfrequency without callus formation. In the beginning, explantsinduced shoots from all genotypes that were white in color andformed a single leaf-like structure (Fig. 1A); thereafter, theybecame green and formed multiple shoots after a total of 2 wk(Fig. 1B). The three genotypes exhibited remarkably differentregeneration responses depending on the growth regulatortype and concentration in the medium. Among variousPGRs tested, BA was found to be efficient for direct shootbud induction in all three genotypes analyzed (Table 1). Theshoot induction rate increased significantly when the BA con-centration in the medium was higher (17.6 μM) resulting in amaximum percentage of shoot induction in 'CO(Ra)-14'(88.9%), 'Try-1' (64.7%), and 'Paiyur-2' (79.8%). The role ofBA in the shoot induction medium appeared to be more pro-nounced at this particular concentration as it resulted in ≥6shoots per explant in 'CO(Ra)-14' (Table 1).

Next to BA, TDZ also showed induced shoot bud initiationresponses in which a higher rate of shoot induction ('CO(Ra)-14' (56.6%), 'Try-1' (38.3%), and 'Paiyur-2' (47.3%)) was ob-served when the MS medium was supplemented with13.5 μM TDZ. When 2,4-D was used alone at higher concen-trations, the shoot induction frequency decreased significantlydue to the formation of compact callus rather than directshoot induction. The addition of 2,4-D at lower concen-trations along with BA increased the efficiency of shootinduction (data not shown). Among the three genotypesstudied, 'CO(Ra)-14' showed superior responses forshoot induction over the other two genotypes with allthree PGRs tested.

Effect of genotype on multiple shoot induction. Marked differ-ences in shoot induction rates were observed among the threefinger millet genotypes. 'CO(Ra)-14' produced a superior re-sponse on both percent shoot induction and average numberof shoots, 'Paiyur-2' gave an intermediate response and 'Try-1'was the least responsive (Table 1). The genotype 'CO(Ra)-14'showed 88.9% of multiple shoot initiation with maximumnumber of shoots 6.2, while the genotype 'Paiyur-2' resultedmoderate multiple shoot induction 79.8% with 4.9 shoots and'Try-1' genotype gave less values in multiple shoot induction64.7% and average number of shoots 4.5, respectively.

Influence of amino acids on multiple shoot proliferation fromSAMs of 'CO(Ra)-14'. Although shoot buds were successfullyinduced in the initial experiments, they did not subsequentlyproliferate, elongate, or mature. The addition of supplementsin the media has been found to exhibit significant improve-ment in adventitious shoot bud induction, proliferation, andmaturation. Similarly, addition of cytokinins, carbohydrates,

194 SATISH ETAL.


and amino acids enhanced the maturation and regeneration ofembryogenic callus in kodo millet (Ceasar and Ignacimuthu2010). In this experiment, we evaluated additives such asCEH, Gly, and Pro for improved proliferation, elongation,and maturation. The SAMs of 'CO(Ra)-14' responded verywell to amino acids supplement in a dose-dependent manner.The frequency of shoot induction, proliferation, and elonga-tion increased rapidly when concentrations of amino acidswere increased systematically in the culture medium. A highermean shoot induction rate (96%), mean shoot number perexplant (8.3), and mean shoot length (5.8 cm) were obtainedfor 'CO(Ra)-14' genotype when the MS medium containing17.6 μM BA, 0.9 μM 2,4-D was supplemented with

750 mg L−1 Pro, 500 mg L−1 CEH, and 2 mg L−1 Gly(Table 2). The multiple shoots induced in response to thisunique combination of PGRs and amino acid supplementsremained green and continued to proliferate and elongate.When the concentrations of supplements were increased be-yond this, there was no further improvement in regeneration,but rather a decline in induction rate, shoot number, and shootlength.

Influence of age and size of SAMs on direct shootregeneration. The present study revealed the responsivenessof the age and size of the SAM to be optimal for shoot prolif-eration. SAMs from seedlings of various ages (1–5 d) were

Figure 1. Direct regenerationfrom shoot apical meristems ofE. coracana genotype 'CO(Ra)-14'. (A) Initiation of shoots fromshoot apical meristems (3–4 mm)inoculated on MS mediumcontaining 17.6 μM BA, 0.9 μM2,4-D, 750 mg L−1 Pro,500 mg L−1 CEH, and 2 mg L−1

Gly after 2 d incubation. (B)Induction of multiple shoots inMS medium containing 17.6 μMBA, 0.9 μM 2,4-D, 750 mg L−1

Pro, 500 mg L−1 CEH, and2 mg L−1 Gly after 12 dincubation. (C) Shoots induced onelongation medium containing11.0 μM BA, 0.9 μM 2,4-D,500 mg L−1 CEH, 750 mg L−1

Pro, and 2 mg L−1 Gly after 2 wkof incubation in light. (D) Rootdevelopment in half-strength MSmedium containing 2.8 μM IAAafter 2 wk of incubation. (E)Acclimatized plantlets in plasticcups containing 1:1 ratio of sterilesoil rite and peat. (F) Seed set ofin vitro regenerated plant grownin the field.



assessed for their regeneration ability. Among which, 3-d-oldSAM explants were found to possess the greatest regenerationcapacity, which was evident from the high frequency of shootsproliferated (95.8%) with an optimal height of 8 cm, andhigher shoot fresh weight (6.5 mg) on shoot induction medi-um containing 17.6 μM BA, 0.9 μM 2,4-D, 750 mg L−1 Pro,500 mg L−1 CEH, and 2 mg L−1 Gly (Table 3). Similarly, thesize of the SAM segments played a key role in the shootproliferation rate. The SAMs of various lengths (2–6) in mil-limeter were tested for their efficiency for multiple shoot re-generation. An optimum segment length of 4 mm favored the

profuse shoot proliferation rate of 98.9%, and the fresh weightof shoots regenerated from 4 mm SAM segment was found tobe the highest at 5.8 mg (Table 3).

Shoot elongation and maturation in genotype 'CO(Ra)-14'. Inorder to achieve maximum shoot elongation, reduced concen-trations of BA were tested. Interestingly, the superior shootelongation rate (96.4%) was prominent when the concentrationof BAwas reduced to 11.0 μM (Table 4). The shoots attained amaximum average length of 13.6 cm, and the mean number offully elongated shoots obtained at this concentration was 13.6

Table 1 Effect of different plant growth regulators on direct shoot regeneration from SAMs of three finger millet genotypes after 4 wk of incubation inlight.

Plant growth regulator (μM) Percent of multiple shoot initiation Average no. of shoots

Finger millet genotype

CO(Ra)14 Try-1 Paiyur-2 CO(Ra)14 Try-1 Paiyur-2

BA

8.8 61.9±0.5 i 49.3±0.3 i 69.4±0.2 i 2.5±0.4 c 1.5±0.3 c 2.6±0.1 e

13.2 81.3±0.8 k 53.6±0.2 j 73.5±0.3 k 4.6±0.3 e 2.4±0.2 d 4.2±0.4 i

17.6 88.9±0.4 l 64.7±0.2 l 79.8±0.1 l 6.2±0.4 g 4.5±0.3 f 4.9±0.1 j

22.0 79.4±0.5 j 61.1±0.3 k 71.6±0.2 j 5.4±0.3 f 3.5±0.3 e 3.6±0.4 gh

2,4-D

4.5 11.8±0.3 d 3.7±0.2 d 5.5±0.3 d 1.2±0.2 b 0.8±0.2 b 0.9±0.3 c

6.7 9.6±0.3 c 2.1±0.1 c 3.6±0.2 c 1.3±0.2 b 0.6±0.1 b 0.7±0.2 bc

9.0 5.2±0.3 b 1.6±0.1 b 1.5±0.3 b 1.2±0.2 b 0.5±0.2 ab 0.3±0.1 ab

11.3 3.1±0.1 a 0.8±0.2 a 0.6±0.1 a 0.5±0.1 a 0.2±0.1 a 0.1±0.1 a

TDZ

4.5 36.6±0.3 e 28.4±0.3 e 31.5±0.3 e 2.5±0.4 c 1.2±0.1 c 2.1±0.2 d

9.0 48.7±0.3 g 31.1±0.3 f 39.8±0.2 f 3.7±0.2 d 2.5±0.3 d 2.9±0.2 ef

13.5 56.6±0.3 h 38.3±0.2 h 47.3±0.2 h 4.1±0.3 e 3.1±0.2 e 3.9±0.1 hi

18.0 41.7±0.3 f 35.2±0.4 g 41.7±0.4 g 3.6±0.2 d 2.4±0.2 d 3.2±0.2 fg

For each plant growth regulator type and concentration, 75 SAM segments per each genotype were placed in Plantron vessels with 50 mL media withthree replicates. Means followed by the same letter in each column are not significantly different at P≤.05 based on Duncan’s multiple range test

Table 2 Effect of BA, 2,4-D, and amino acid supplements on multiple shoot proliferation in finger millet genotype 'CO(Ra)-14' after 12 d ofincubation in the light.

PGRs (μM) Amino acids (mg L−1) Explants with multipleshoot induction (%)

Mean no. of shootsinduced per explant

Mean length ofshoots (cm)

BA 2,4-D Pro CEH Gly

17.6 0.9 100 200 0.5 44.3±0.2 a 4.7±5.1 a 2.8±0.4 a

17.6 0.9 250 300 1.0 62.8±0.3 b 6.5±5.5 c 3.3±0.3 b

17.6 0.9 500 400 1.5 71.6±0.3 c 7.1±8.7 d 4.6±0.3 c

17.6 0.9 750 500 2.0 96±0.1 f 8.3±0.4 e 5.8±0.4 d

17.6 0.9 850 750 3.0 77.6±0.2 e 5.6±3.9 a 4.9±0.3 c

17.6 0.9 1000 1000 4.0 72.3±0.1 d 4.3±0.9 a 3.5±0.1 b

For all treatments, 75 explants from 3-d-old seedlings were studied through three independent experiments. Average values of all the experimentsrepresent the means±SE and all the data were analyzed by SPSS 17.0.Means followed by the same letter in each column are not significantly different atP≤.05 based on Duncan’s multiple range test

196 SATISH ETAL.


(Fig. 1C). A rapid elongation response was noticed when theBA concentration was reduced to 11.0 μM, and the shootsattained complete elongation and maturity after 2 wk. Theshoots remained green, thick, and continued growth duringelongation.

Rooting of shoots in genotype 'CO(Ra)-14'. Rooting occurredimmediately when the elongated shoots were transferred tohalf-strength MS medium with or without IAA. Half-

strength MS medium devoid of IAA induced very thin andshort roots. The inclusion of IAA at various concentrations(1.4–8.4 μM) significantly increased the rooting frequencyand root length. The best root growth response in terms offrequency of root induction (88.2%), mean number of rootsper shoot (5.4), and mean root length (6.2 cm) was observedwhen 2.8 μM IAA was incorporated into half-strength MSmedium (Table 5; Fig. 1D).

Acclimatization and field transfer. The rooted shoots weretransferred to the Yoshida solution (Yoshida et al. 1976) andmaintained in a growth chamber for a week before beingtransferred to greenhouse conditions for 5–6 d and finallytransplanted to plastic cups with 100% survival rate(Fig. 1E). All in vitro regenerated plants grew well and exhib-ited phenotypic homogeneity such as flowering, seed setting,and grain yield when compared to in vivo plants grown fromseeds (Fig. 1F).

Discussion

In cereals, shoot apex explants are an outstanding resource forbiotechnological uses due to considerate storage and availabil-ity. SAMs consist of a group of stem cells that divide to pro-duce viable cells to be employed in the continuous arrange-ment of new organs, such as leaves, stems, axillary meristems,and adventitious roots (Zhong et al. 1996; Bowman andEshed 2000; Itoh et al. 2006). The most important benefit witha shoot apex is its quick development, which allows rapid anddirect regeneration of plants (Sticklen and Oraby 2005; Ceasarand Ignacimuthu 2008; Dey et al. 2012). Shoot tips have beendesirable at all the times for in vitro culturing because they can

Table 4 Effect of BA on elongation and maturation of shoots inducedfrom SAMs in finger millet genotype 'CO(Ra)-14' after 2 wk ofincubation in light.

BA 2,4-D Pro CEH Gly Shootelongation(%)

Mean lengthof shoots(cm)

Mean no. ofshoots perexplant

6.6 0.9 750 500 2.0 61.7±0.4 a 8.2±0.2 a 8.6±3.8 b

8.8 0.9 750 500 2.0 87.4±0.3 d 9.8±0.2 b 10.9±9.1 d

11.0 0.9 750 500 2.0 96.4±0.1 f 13.6±0.4 e 13.6±0.8 f

13.3 0.9 750 500 2.0 90±0.2 e 12.1±0.3 d 11.5±7.1 e

15.5 0.9 750 500 2.0 81.5±0.3 c 11.2±0.3 c 10.4±3.8 c

17.7 0.9 750 500 2.0 72.5±0.3 b 9.3±0.4 b 7.7±9.2 a

SAM explants were cultured for 12-d and transferred to MS media with11.0 μMBA, 0.9 μM 2,4-D, 750 mg/L Pro, 500 mg/L CEH, and 2 mg/LGly for elongation andmaturation. Values represent the means±SE (n=3;analyzed by SPSS version 17.0). Means followed by the same letter ineach column are not significantly different at P≤.05 based on Duncan’smultiple range test

Pro proline, CEH casein enzymatic hydrolyzate, Gly glycine

Table 5 Effect of IAA on root induction from regenerated shoots offinger millet genotype 'CO(Ra)-14' on half-strength MS medium, after 2wk of incubation in light.

Concentration ofIAA (μM)

Shoots withroots (%)

Mean no. of rootsper shoot

Mean rootlength (cm)

0.0 36.3±0.4 a 1.4±0.4 a 0.7±0.1 a

1.4 59.7±0.3 c 3.6±0.2 c 3.6±0.3 c

2.8 88.2±0.1 f 5.4±0.1 e 6.2±0.2 f

5.6 74.4±0.4 e 4.6±0.3 d 5.6±0.3 e

7.0 61.6±0.2 d 4.5±0.4 d 4.4±0.3 d

8.4 54.2±0.1 b 2.9±0.1 b 2.9±0.2 b

Fully elongated (5–6 cm) shoots of 'CO(Ra)-14' were rooted on half-strength MS medium containing various concentrations of IAA. For eachtreatment, the no. of roots produced by single shoot was calculated for14 d. Fifteen explants for each genotype with three replicates were used.The highest rooting percentage is shown in italics. Means followed by thesame letter in each column are not significantly different at P≤.05 basedon Duncan’s multiple range test

Table 3 Influence of explant age and size on regeneration of fingermillet genotype 'CO(Ra)-14' in MS medium containing BA, 2,4-D andamino acid supplements after 12 d of incubation in light.

Explantsage (d)

Explantsproducing shoots(%)

Average length ofthe shoot (cm)

Fresh weight ofthe shoot (mg)

1 48.1±0.2 a 2.9±0.2 a 2.2±0.3 a

2 74.7±0.3 c 5.6±0.2 c 4.7±0.1 b

3 95.8±0.3 e 8.0±0.1 e 6.5±0.3 d

4 80.2±0.3 d 6.1±0.2 d 5.2±0.3 c

5 56.1±0.2 b 4.6±0.3 b 4.3±0.2 b

Explants size (mm)

2 63.1±0.3 b 5.8±0.4 a 3.6±0.2 b

3 81.0±0.4 c 11.4±0.3 c 4.9±0.1 d

4 98.9±0.2 d 13.5±0.4 e 5.8±0.3 e

5 62.5±0.3 b 12.3±0.3 d 4.1±0.2 c

6 47.7±0.4 a 7.2±0.1 b 2.4±0.2 a

Different size of SAM segments (2–6 mm long) from 1 to 5-d-old seed-lings of the finger millet genotype 'CO(Ra)-14' were tested in MS medi-um containing 17.6 μM BA, 0.9 μM 2,4-D, 750 mg/L Pro, 500 mg/LCEH and 2 mg/L Gly. Values are mean±SE (3 replicates). Means follow-ed by the same letter in each column are not significantly different atP≤.05 based on Duncan’s multiple range test



be handled more easily compared to other explants, and theseare morphogenetically plastic and can be induced to producemultiple shoots (Arockiasamy and Ignacimuthu 2007).

Regeneration of multiple shoot clumps from shoot tips wasfirst illustrated in maize (Zhong et al. 1992). Sharma et al.(2004) reported multiple shoot regeneration from meristemat-ic shoot segments excised from mature embryo derived seed-lings of barley on MS medium containing picloram and TDZ.Devi et al. (2000) in pearl millet, Cho et al. (2003) in oats, andCho et al. (2004) in rice reported successful establishment ofshoot meristematic cultures. Also, shoot apex tissues havebeen effectively used as explant material in rice, maize, wheat,sorghum, oats, barley, and millet (Sticklen and Oraby 2005) torecover stable transformed plants (Ignacimuthu and Ceasar2012). Shoot apices and immature embryos are often used asa primary source of explants in finger millet for callus induc-tion (Eapen and George 1990; Pius et al. 1994; Yemets et al.2003; Ceasar and Ignacimuthu 2008) and showed a stronginfluence of genotype (Ceasar and Ignacimuthu 2008).However, this is the first report of using shoot apical meri-stems of the finger millet genotype 'CO(Ra)-14' for directshoot induction and subsequent plant regeneration.

Direct plant regeneration without the accompanyingcallus phase is the most preferred pathway for plantregeneration. SAM explants of finger millet were report-ed to respond significantly in direct organogenesis(Eapen and George 1990). The induction of shoots incereals and millets is commonly achieved by BA orTDZ (Srivastav and Kothari 2002; Ceasar andIgnacimuthu 2008). In order to achieve direct adventi-tious shoot regeneration in finger millet, we tested threePGRs namely BA, 2,4-D, and TDZ at different concen-trations. In our study, BA (17.6 μM) was found to bemore effective in direct shoot induction from SAM ex-plants of three finger millet genotypes. However, Deyet al. (2012) reported that same concentration of TDZ(18.0 μM) is required for efficient direct regeneration inrice from shoot apices. BA is one of the active PGRsfrequently used for tissue culture of monocotyledonousplants (Ramakrishnan et al. 2013). We found that BA incombination with an amino acid supplement resulted informing consistent multiple shoot clumps within 12 dthat continued further proliferation and maturation.

Pro and CEH have been successfully employed for improv-ing regeneration in rice via high-frequency embryogenesis(Ozawa and Komamine 1989). CEH is mainly purported asan organic nitrogen source to stimulate positive effects ondevelopment of multiple shoots (Zhang et al. 1996; Sharmaet al. 2004) and somatic embryos (Ramakrishnan et al. 2013;Sezgin and Dumanoglu 2014). MS medium along with 2,4-Dand CEH was effectively utilized for callus initiation through3- or 4-d-old leaf base segments, mesocotyl, and root tissuesin finger millet (Mohanty et al. 1985). Effective multiple shoot

induction and maturation is habitually the sign of an optimalbalance between PGRs and of the physiological state of theexplants (Mallikarjuna and Rajendrudu 2007).

It is well known that the multiple shoot proliferation andplant regeneration in cereals depends on various factors ofwhich genotype, age of the donor explant, and explant sizeor region are most important (Cheng et al. 2003; Ramesh et al.2009; Piqueras et al. 2010). In our study, SAMs from 3-d-oldseedlings that were 4 mm size were found to be optimal,resulting in an excellent regeneration response with increasedfresh weight of shoots compared to other SAM sizes tested.Previously, a large number of studies focused on embryogeniccalli-mediated plant regeneration in cereals inclusive of fingermillet (Poddar et al. 1997; Srivastav and Kothari 2002; Ceasarand Ignacimuthu 2008), pearl millet (Mythili et al. 1997; Deviet al. 2000), barley (Sharma et al. 2004), rice (Ozawa andKomamine 1989; Nhut et al. 2000; Arockiasamy andIgnacimuthu 2007; Karthikeyan et al. 2009; Ramesh et al.2009), and wheat (Barro et al. 1999; Ahmad et al. 2002).However, our results show that this more complex regenera-tion procedure may not be required for finger millet cv.'CO(Ra)-14'.

In vitro rooting of regenerated shoots was carried out inhalf-strength MS medium. Roots developed spontaneouslyafter 2 wk even without the addition of IAA. Our results wereconsistent with Ahmad et al. (2002) in wheat. However, theinclusion of IAA in medium resulted in significant improve-ment of rooting frequency, root number, and length. Optimalrooting was observed in half-strength MS medium containing2.8 μM IAA. Sharma et al. (2004) observed good rootingresponse in barley on MS medium containing 2.4/4.9 μMIBA.

In the present study, induction of multiple shoots occurreddirectly from shoot apical meristems devoid of an interveningcallus phase. Our study reveals that SAMs developed tinyshoots with unexpanded leaves within 1 wk, and those ex-plants can be lucratively used to develop mature plants bythe addition of amino acid supplements to the culture medium.In addition, these shoots can continue to be cultured withability to completely regenerate over 2 mo. Furthermore, ourcomparison of several concentrations and combinations of thegrowth regulators has shown that the potential for multipleshoot formation is regulated by a fine balance between partic-ular types of growth regulators.

Conclusion

A simple, reproducible, efficient, and high-frequency multipleshoot regeneration protocol has been established using active-ly growing SAMs of the finger millet cultivar 'CO(Ra)-14'.This is the first report and detailed method described for de-velopment of an efficient in vitro plant regeneration system for

198 SATISH ETAL.


finger millet within a short duration (45 d) by employing 3- or4-d-old seedlings. The use of SAMs for rapid plant regenera-tion is considered as a safe and convenient approach, thusmaking this system potentially useful for large-scalemicropropagation as well as genetic improvement studies.

Acknowledgments The author L. Satish sincerely thanked the Univer-sity Grants Commission, New Delhi, India, for financial support in theform of UGC-BSR fellowship. We thank the Department of Small Mil-lets, Millet Research Station, Tamil Nadu Agricultural University forproviding seed material used in the present study. Also, the authors grate-fully acknowledge the Bioinformatics Infrastructure Facility of AlagappaUniversity (funded by the Department of Biotechnology, Government ofIndia: Grant No. BT/BI/25/001/2006) for providing the computationalfacility.

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