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International Journal of Scientific Research in _______________________________ Research Paper . Biological Sciences Vol.6, Issue.4, pp.33-42, August (2019) E-ISSN: 2347-7520 DOI: https://doi.org/10.26438/ijsrbs/v6i4.3342 Influence of Organic Derivatives on Direct Regeneration of finger millet genotype CO 9 G. Atul Babu1, R. Ravindhran2* 1,2 T.A.L. Samy Unit for Plant Tissue Culture and Molecular Biology, Department of Plant Biology and Biotechnology, Loyola College (Autonomous), Affiliated To University of Madras, Chennai 600 034, Tamil Nadu, INDIA Corresponding author:

Available online at: www.isroset.org Received: 22/Jul/2019, Accepted: 18/Aug/2019, Online: 31/Aug/2019 Abstract- Finger millet (Eleusine coracana (L.) Gaertn.) is a commercially important food crop extensively cultivated in the arid and semi-arid tropical regions of India and East Africa. The current study was designed to develop an efficient protocol for micropropagation by the influence of additives to enhance the number of shoots per explant from Shoot Apical Meristems (SAMs) of finger millet genotype CO 9. The highest shoot regeneration frequency (95.89%) with an average of 46.52 shoots per explant and 10.86 cm shoot length per explant was achieved when SAMs were cultured in Shoot Regeneration Medium (SRM) containing Murashige and Skoog’s (MS) medium supplemented with 3.0 mg/L 6-Benzyl Amino Purine (BAP), 2.0 mg/L Kinetin and 5% coconut water, 300 mg/L proline and 400 mg/L casein enzymatic hydrolysate, and 3 mg/L glycine. Sub- culturing the SAMs in SRM at 2 weeks interval for 8 weeks resulted in an increase in the number of shoots per explant. The highest rooting frequency (100%) with an average root length of 7.32 cm was obtained on full-strength MS medium supplemented with 0.25 mg/L IAA. and successfully acclimated in the field, subsequently developed into fertile plants. Thus, the procedure described is a rapid and consistent method useful for efficient large-scale propagation and genetic transformation in finger millet. Keywords- Eleusine coracana, shoot apical meristems, Shoot Regeneration Medium, Random amplified polymorphic DNA I. INTRODUCTION transformation have been reported in cereal crops (rice, wheat, maize, oats, and barley), very less attention given so Finger millet (Eleusine coracana [L.] Gaertn.), a small grain for improvement of millets [7]. There are various explants crop, cultivated in more than 4 million ha worldwide is the used for in vitro regeneration in finger millets like staple diet for underdeveloped populations of Africa [1]. mesocotyl, leaf base segments, root and immature Millets are coarse-grained hardy crops of the Poaceae inflorescence [8-11], mature seeds [12-16], shoot tips [17- (Gramineae) family that include 10 genera and 14 species. 18]. Regeneration of whole plants from callus is a time- Millets are the first domesticated crops of the tropics owing consuming process as redifferentiation of shoots requires to their rapid growth, minimal harvesting period, and multiple sub-culturing cycles. During the prolonged culture, tolerance to abiotic stress like drought and high temperatures most of the calli lose their regeneration potential [19-21]. In [2-3]. Compared to other cereal crops, Finger millet rich in addition, the use of callus tissue for transformation makes it amino acids (cysteine, tyrosine, methionine, and tryptophan) difficult to identify competent cells for plant regeneration and minerals (calcium, phosphorus, and iron) [4]. These [22-23]. From the literatures that there is no universal recipe granules are vastly used in the food industry for the for efficient culture and induction of direct shoot preparation of cakes and bread. There is a high demand for regeneration depends on the nature of the plant organ from nutrient-rich finger millet is on the rise in recent years. In which the explant was derived, and is highly dependent on order to meet the worldwide dietary demand, there is an plant genotype. Direct regeneration is an alternative immediate necessity to improve existing crops with new approach to establish whole plants using shoot apical approaches and technologies for abiotic and biotic stress meristems (SAMs) present at the apical dome. SAMs tolerance [5]. One of these methods involves the generation possess superior redevelopment ability and thus stimulate of transgenic crops with desired traits. In the case of millets, multiple shoots clumps within a short duration of time [23- little genetic improvement has been done so far, specifically 24]. Direct shoot regeneration is achieved by SAMs in using wide- or cross-hybridization among the closely related monocots like rice [23- 24] wheat [25] maize [26-27] species [6]. Advances in regeneration and genetic sorghum [28] pearl millet [29] oats, and barley [30-31]. © 2019, IJSRBS All Rights Reserved 33 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 Reports are also available on use of amino acids in media, explant and average shoot length, were recorded. Shoot plays an important role to improve callus induction, shoot explants were subcultured at the 2nd week in the same and root formation in various plants; viz: wheat [32-33] medium. The experiment was repeated thrice with each pearl millet [34] maize [35], sorghum [36], and naga chilli treatment using four replicates. [37-38]. Micropropagated plantlets are commonly subjected to genetic fidelity analysis using molecular markers to Effect of cytokinins and coconut water confirm their clonal identity to the mother plant [39]. Shoot apical meristem were cultured on MS medium Random amplified polymorphic DNA (RAPD) is an containing 3% (w/v) sucrose with 0.8% (w/v) agar and efficient marker system that makes use of arbitrary primers supplemented with 5% (w/v) coconut water and different to cover the entire regions of the genome and has been concentrations and combinations of plant growth regulators, routinely used to identify intra- and interspecies genetic including BAP 0.5-4.0 mg/L, KIN 0.5-4.0 mg/L and variation in crops [40]. In the Present study, we report an combination of BAP 3.0 mg/L and KIN 0.5-4.0 mg/L . efficient direct plant regeneration protocol for Indian finger millet genotypes CO 9 using amino acids and organic Effect of amino acid and organic derivative derivates. The genetic identity of the micropropagated The best PGR identified (3.0 mg/L BAP and 2.0mg/L KIN) plantlets from the genotype CO 9 was confirmed using from the above experiment were further examined to RAPD markers. maximize multiple shoot regeneration. The influence of some of the essential amino acids and organic derivatives II. MATERIALS AND METHODS such as proline (Pro; 50, 100, 200, 300, 400, 500 mg/L), glycine (Gly; 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/L) casein Plant Material and Surface Sterilization enzymatic hydrolysate (CEH; 200, 300, 400, 500, 750, or Seeds of finger millet genotype CO9, obtained from the 1000 mg/L), and 5% coconut water was systematically Tamil Nadu Agriculture University, Coimbatore, India. The observed to improve shoot induction and proliferation. The seeds were de-husked in running tap water for 5 min and cultures were incubated at 25±2°C for 16/8-h light/ dark surface-disinfected using 0.1% (w/v) mercuric chloride for 5 conditions. The percentages of responding explants, mean min followed by 70% (v/v) ethanol for 25 s. The seeds were number of shoots induced in each explant, and shoot lengths then rinsed five times with autoclaved, double-distilled were calculated after 8weeks incubation in light. water, blot dried on sterile filter paper. Rooting Medium Culture Conditions In vitro grown shoots (3 – 6 cm long, 8 weeks old) were Surface sterilized seeds were blot dried using autoclaved isolated individually from the multiple shoot clumps. The filter paper in a dry petri dish. The seeds were transferred to separated shoots were transferred to MS medium amended Petri plate containing MS medium [41] amended with with different concentration of (0.1, 0.25, 0.5 and 1.0 mg/L) sucrose 3% (w/v) and solidified with 0.8% agar. The pH of Indole Acetic Acid (IAA), (0.1, 0.25, 0.5 and 1.0 mg/L) the medium was changed to 5.8 ± 0.02 using 1N NaOH or Indole Butyric acid (IBA) and (0.1, 0.25, 0.5 and 1.0 mg/L) HCl, before the inclusion of agar and autoclaved at 121°c 1-Naphthalene Acetic Acid (NAA) for rooting. /15 lbs pressure for 20 mins. The seeds were cultured at 25 ± 2°c with a photoperiod of 16 hours light and 8 hours dark for Acclimatization 8 days. Two weeks old, rooted shoots were detached from agar medium and they were thoroughly washed under running tap Effect of cytokinins water and transferred to the soil mixture. Root induction Three days old in vitro germinated seedlings with Shoot frequency (number of shoots with root / total number of Apical Meristem (SAM) 4 – 6 mm in size were used as shoots × 100) and mean root length (average length of three explants. SAM explants were then transferred to Shoot longest roots) were measured and recorded. This experiment Growth Medium (SGM) containing MS basal salts and was repeated thrice to evaluate the efficacy. Plantlets (6 -7 vitamins supplemented with 6 – Benzylaminopurine (BAP) cm in length) were then acclimatized to a plastic pot ,Kinetin (KIN) (0.5, 1.0, 2.0, 3.0 and 4.0 mg/L ) individually containing sterilized cow dung manure and red soil (1:1 and combination of BAP 3.0 mg/l and KIN ( 0.5, 1.0, 2.0, ratio, v/v) and they were watered at regular intervals. 3.0 and 4.0 mg/L) concentrations for in vitro propagation. Cultures were nurtured in glass bottles (15 cm height × 10 RAPD Analysis cm diameter) and incubated at 25 ± 2°c in 16/8h (light/dark) Genomic DNA of the haphazardly selected mother plant and with a photosynthetic photon flux density of 50 µ mol m-2 S- micropropagated plantlets were isolated from fresh leaves 1 supplied with cool white fluorescent lamps (Philips, using CTAB method [42]. RAPD reactions were performed Chennai, India). The shoot regeneration frequency was as described by [43]. RAPD study of both mother plant and observed (number of explants with shoots/shoots per micro propagated plantlets was performed using RAPD explants × 100) after 8 weeks, the total number of shoots per primers (Eurofins Genomics, Bangalore, India). PCR © 2019, IJSRBS All Rights Reserved 34 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 amplification was done using proflex thermocycler (Applied 37.5 ±0.2 shoots per explant (fig 2). A similar result was Biosystems, Singapore). The PCR amplified products were reported by [45] in Sorghum bicolor. However, the increase electrophoresed on 1.2% agarose gel in 1X TAE buffer at in the concentration of BAP or KIN in the medium resulted 75V for 1h. The results were photographed using a gel in decrease in shoot number and shoot length. The shoot documentation system (GELSTAN 1012 Economy Imaging length varied significantly in all combination of shoot System, Chennai, India). Lab Image ID Software, version regeneration medium (SRM) (fig 2). 3.3.0 (Kapelan Bio-Imaging solutions, Leipzig, Germany) was used for the determination of amplicon size by visual Influence of amino acids and organic derivatives on shoot comparison with 1 Kb ladder (250 – 10000 bp). proliferation The addition of supplements in the media has been found to Statistical Analysis exhibit significant improvement in shoot tip initiation, All the above experiments were repeated thrice with four proliferation, and maturation. In this study, we evaluated independent replicates for each treatment. The study of additives such as CEH, 5 % coconut water, Gly, and Pro on variance appropriate for the design was carried out to spot improved proliferation, elongation, and maturation. The the implication of difference among the treatment means. SAMs of CO 9 responded very well to amino acids IBM SPSS Statistics version 20 Duncan’s multiple range test supplement in a dose-dependent manner. The induction, at P < 0.05 was used to compare the treatment means. proliferation, and elongation frequency of shooting improved quickly when amino acid levels were III. RESULT AND DISCUSSION systematically increased in the culture medium. A higher shoot frequency (95.89%), shoot number per explant Effect of cytokinins on shoot regenerations (46.52), and shoot length (10.8 cm) were obtained for CO 9 Shoot apical meristems (SAMs) excised from 3-d-old in genotype (Table 1), when the MS medium containing BAP vitro germinated seedlings of CO 9 (Fig. 3a) were evaluated 3.0 mg/L, KIN 2.0 mg/L was supplemented with 300 mg /L for multiple shoot induction in Shoot regeneration medium Pro, 400 mg/L CEH, 3.0 mg/L Gly and 5 % coconut water (SRM). The initial response of shoot regeneration was (fig 3). In responds to this unique combination of PGRs and observed within 3 d and multiple shoot buds emerged from amino acid supplement’s, the multiple shoots remained the bottom of the explants after 7 d, without any basal callus green and continued to proliferate and elongate. When the growth (Fig. 3b). Multiple shoot induction increased concentration of supplements were increased beyond this, spontaneously after 2 wk. Among the PGRs tested, 3.0 mg/L there was no further improvement in regeneration, but rather BAP showed 88.03% shoot regeneration frequency, a decline in induction rate, shoot number, and shoot length. 25.5±0.33 of shoots per explant and 5.5±0.15 cm of shoot Pro, CEH and 5% coconut water have been successfully length (fig 1). KIN also showed a positive influence on shoot employed for improving regeneration in rice and sorghum regeneration frequency of 73.67% and 14.7±0.31 of shoots via high-frequency embryogenesis [47]. CEH is primarily per explant (fig 1) when MS medium supplemented with 2.0 act as an organic nitrogen source shows beneficially impact mg/L KIN which is less compare to BAP. However, MS on multiple shoots development [48]. Multiple shoot medium amended with the combination of 3.0 mg/L BAP proliferation and plant regeneration in cereals depends upon and 2.0 mg/L KIN show highest regeneration frequency on numerous factors such as age of the explant, explant size, (90.56%), multiple shoot tip per explant (32.5±0.26) and genotype and region are most important [49-51]. In our multiple shoot clumps (5.8±0.17cm) (fig 1). BAP and KIN study, SAMs from 3-d-old seedlings were found to be commonly induced multiple clumps in millets and cereals optimal, along with amino acid resulting in an excellent [44, 45, and 46]. In pearl millet and wheat, higher shoot regeneration response. proliferation was observed in medium containing BAP greater than 3.0 mg/L [25]. Rooting and Acclimatization Eight-week-old shoots from shoot regeneration medium Effect of cytokinins and coconut water on shoot were rooted in MS medium amended with IAA, IBA, and regenerations NAA. Roots initiated spontaneously after 1 wk in MS Synergistic effect of cytokinins and coconut water on medium devoid of PGRs. However, the inclusion of IAA multiple shoot induction were tested (fig 2). Medium (0.1 – 1.0 mg/L) positively influenced the rooting frequency supplemented with (0.5-4.0 mg/L) BAP, (0.5-4.0 mg/L) KIN and root length. The best root growth response in terms of individually and combination of (3.0 mg/L) BAP with (0.5- frequency of root induction (100%), mean number of roots 4 mg/L) KIN along with 5 % coconut water, were tried for per shoot (9.05±0.10) and mean root length (7.32 cm) was shoot induction. Addition of coconut water (5 %( w/v)) observed when 0.25 mg/l IAA was incorporated into MS increased the shoot number as well as shoot length (fig 2). medium (Table 2; Fig. 3f.g.h). Our results were consistent The most significantly shoot proliferation frequency was with [25] in wheat. attained in MS medium added with 3.0 mg/L BAP, 2.0 mg/L Kin along with 5 % coconut water produced 94.36 %. And © 2019, IJSRBS All Rights Reserved 35 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 RAPD analysis [6]. S.Plaza-Wuthrich, Z.Tadele, “Millet improvement through Finger millet plantlets propagated under in vitro conditions regeneration and transformation. Biotechnology and Molecular Biology Review”, Vol 7, pp.48–61, 2012. were subjected to clonal fidelity analysis using RAPD [7]. S.A. Ceasar, S. Ignacimuthu, “Genetic engineering of millets: markers. 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CONCLUSION direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn)”. Euphytica Vol.48, pp.269–274, 1990 The method demonstrates an effective and low costs [11]. S. Kumar, K. Agarwal, S.L. Kothari, “In vitro induction and protocol that helps in the rapid multiplication, large-scale enlargement of apical domes and formation of multiple shoots in production in finger millets. Influence of additives on en- finger millet, Eleusine coracana (L.) Gaertn and crowfoot grass, hanced shoot multiplication was established by the use shoot Eleusine indica (L.) Gaertn”. Current Science, Vol.81, pp.1482– tip explants. Shoot tip proliferation, without any intervening 1485, 2001 [12]. J.Pius, S. Eapen, L. George, P.S. Rao, and R.S. Raut, callus phase, supposedly leads to the production of “Performance of plants regenerated through somatic genetically stable, true-to-type plantlets. Reproducible bands embryogenesis in finger millet (Eleusine coracana Gaertn.),” were obtained from RAPD marker confirmed the genetic Tropical Agricultural Research and Extention,Vol 2, pp. 87–90, integrity of regenerated plantlets. Hence, this method could 1999. be used as a potential alternative en masse propagation and [13]. P. Gupta, S. Raghuvanshi, A.K. Tyagi, “Assessment of the further for genetic transformation. efficiency of various gene promoters via biolistics in leaf and regenerating seed callus of millets, Eleusine coracana and Echinochloa crusgalli”,. Plant Biotech, Vol.18, pp.275–282, ACKNOWLEDGMENTS 2001. [14]. N. Nethra, R. Gowda, P.H.R. Gowda, “Influence of culture This work is funded by Loyola College – Times of India, medium on callus proliferation and morphogenesis in finger Chennai, India (Project approval code: 4LCTOI14PBB001) millet. In: Tadele, Z. 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Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 Table 1 Effect of BAP, Organic Derivatives and Amino acid supplements on multiple shoot proliferation in finger millet genotype CO9 after 8 wks of incubation in the light Organic derivatives Mean no. of Mean length PGRs mg/L Amino acids mg/L Explants with shoots induced of shoots Multiple shoot per explant (cm) induction (%) %Coconut BAP KIN pro Gly CHE water 3 2 50 0.5 100 5 60.35 19.65 ±0.2f 6.82 ±0.1j 3 2 100 1 200 5 67.46 23.45 ±0.2e 7.24 ±0.4hi 3 2 200 2 300 5 81.61 30.91 ±0.3b 9.11 ±0.6g 3 2 300 3 400 5 95.89 46.52 ±0.3a 10.86 ±0.2g 3 2 400 4 500 5 79.74 29.31 ±0.5c 8.60 ±0.6h 3 2 500 5 600 5 64.52 24.48 ±0.4d 7.21 ±0.5hi Table 2 Effect of Auxins on Root formation of finger millet genotype CO9 Auxins mg/L Percentage of Rooting (%) No. of Roots per Shoot Root Length (cm) IAA 0.1 81.83 4.44 ± 0.13e 3.58 ± 0.08f 0.25 100 9.05 ± 0.10a 7.32 ± 0.06b 0.5 98.26 8.61 ± 0.03b 4.443± 0.7e 1.0 92.59 5.45 ± 0.06d 2.34 ± 0.07g IBA 0.1 60.96 6.56 ± 0.10c 3.90 ± 0.05f 0.25 66.61 8.22 ± 0.10b 4.27 ± 0.07e 0.5 76.19 6.37 ± 0.09c 3.20 ± 0.08f 1.0 69.43 4.71 ± 0.07e 3.00 ± 0.11f NAA 0.1 64.62 3.40 ± 0.07f 1.94 ± 0.07h 0.25 61.21 3.70 ± 0.11f 2.45 ± 0.01d 0.5 77.41 5.46 ± 0.08d 2.51 ± 0.10g 1.0 72.72 4.59 ± 0.07e 2.18 ± 0.04g Table 3 List of RAPD primer sequences with the number and size of amplified fragments generated in genotype CO 9 of finger millet mother plant and micropropagated plantlets. Genotype CO 9 Primer code Primer sequence (5′–3′) No. of scorable bands Range of band sizes (bp) OPA 1 CAGGCCCTTC 9 460-1500 OPA 2 TGCCGAGCTG 8 430-1450 OPA 3 AGTCAGCCAC 7 530-1600 OPA 4 AATCGGGCTG 7 640-1500 OPA 5 AGGGGTCTTG 8 300-1500 OPA 6 GGTCCCTGAC 8 490-1990 OPA 7 GAAACGGGTG 5 520-1900 OPA 8 GTGACGTAGG 8 250-1480 OPA 9 GGGTAACGCC 6 360-1490 OPA1 0 GTGATCGCAG 5 330-1350 71 250-1990 © 2019, IJSRBS All Rights Reserved 38 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 Figure 1 Effect of Cytokinins in Direct Regeneration of finger Millet genotype CO 9. Values represent mean ± SE of individual treatments obtained from three repeated experiments. Each treatment consisted of four replicates with ten explants per replicate © 2019, IJSRBS All Rights Reserved 39 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 No shoots per explant 45 40 35 30 25 20 15 10 5 0 0.5 1 2 3 4 BAP mg/L KIN mg/L BAP 3 mg/L + 0.5- 4 mg/L KIN Figure 2 Effect of Cytokinins along with 5% coconut water in Direct Regeneration of finger Millet genotype CO 9 Values represent mean ± SE of individual treatments obtained from three repeated experiments. Each treatment consisted of four replicates with ten explants per replicate © 2019, IJSRBS All Rights Reserved 40 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 a b c d e f g h i j Figure 3 Direct regeneration from shoot apical meristems of E. coracana. (a) Initiation of shoot apical meristems (3–4 mm) from mature seeds germinated on MS medium after 3 d; (b) Induction of multiple shoots in shoot regeneration medium(SRM) containing MS basal salts and vitamins supplemented with with 3 mg/L 6-Benzyl Amino Purine (BAP), 2 mg/L μM Kinetin and 5% coconut water in combination with 300 mg/L proline, 400 mg/L casein enzymatic hydrolysate, and 3 mg/L glycine.; (c) Proliferation of shoots in SRM after 4 wk; (d) Multiple shoot regeneration in SRM after 6 wk; (e) Elongated shoots of CO 9 in SRM after 8 wk; (f,g) Rooting in MS medium supplemented with 0.25 mg/l IAA after 1 wk; (h) Rooting in liquid MS for 1 wk (i) Acclimatized plantlets in grow bags containing sterile red soil and manure (1:1, v/v); (j) Seed set of micropropagated plants grown in the field. © 2019, IJSRBS All Rights Reserved 41 Int. J. Sci. Res. in Biological Sciences Vol. 6(4), Aug. 2019, ISSN: 2347-7520 Figure 4 Assessment of genetic fidelity of the mother plant and micropropagated plantlets of Eleusine coracana using RAPD markers. (a)RAPD primers 1 (left) and 2 (right); (b) RAPD primers 3 (left) and 4 (right); (c) RAPD primers 5 (left) and 6 (right) (d) RAPD primers 7 (left) and 8 (right); (e) RAPD primers 9 (left) and 10 (right) The same seven ex vitro plants are represented in the same order in each of the 10 gel halves. In each panel, lanes 1 and 9 are mother plants; lanes 2–8 and 10–16 are ex vitro plants; lane M is 1 Kb DNA marker. Author’s Profile Dr.R.Ravindhran is presently working as Associate professor and Head of the Department, Department of plant Biology and Biotechnology, Loyola College, Chennai-600034. He has 20 years of experience in teaching and research. To his credit 7 students have completed Ph.D. under his guidance. His research team involved in developing bio fortified millets through genetic engineering and genome editing using CRISPR/Cas 9. G.Atul babu is a research scholar in the Department of Plant Biology and Biotechnology, Loyola College, Chennai-600034. He has completed master’s degree in Biotechnology. His area of research is plant tissue culture and molecular Biology. © 2019, IJSRBS All Rights Reserved 42