Somatic embryogenesis and regeneration using Gracilaria edulisand Padina boergesenii seaweed liquid extracts and genetic fidelityin finger millet (Eleusine coracana)

Lakkakula Satish1& Periyasamy Rathinapriya1 & Arokiam Sagina Rency1 &

Stanislaus Antony Ceasar2 & Subramani Pandian1& Ramakrishnan Rameshkumar1 &

Manikandan Ramesh1

Received: 17 March 2015 /Revised and accepted: 15 August 2015# Springer Science+Business Media Dordrecht 2015

Abstract The effect of seaweed liquid extracts (SLEs) fromGracilaria edulis and Padina boergesenii was studied onfinger millet bioassays. Finger millet seeds were germinatedon 0–100 % of SLEs. Genotype ‘PR-202’ showed superiorresponse with higher frequency of germination (99.6 %),fresh weight (1.1 mg seedling−1), shoot length (13.4 cm),root number (4.8), and root length (8.6 cm) with 60 % ofG. edulis extract. Influence of SLEs and various phytohor-mones on somatic embryogenesis and regeneration of fin-ger millet was studied. Shoot apical meristem was inocu-lated on Murashige and Skoog (MS) medium containing 3–5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4,5-trichlorophenoxyacetic acid individually or in combinationof 0.1–1.0 mg L−1 kinetin or 10–40 % each SLEs ofG. edulis or P. boergesenii was used for callus inductionand somatic embryogenesis. SLEs of each species wereused individually at 20–60 % and 25–75 % for somaticembryogenesis and regeneration, respectively. The optimumlevels of either SLE were found to be 20, 40, and 50 % forcallus induction, somatic embryogenesis, and regeneration

from somatic embryos, respectively. The MS medium con-taining 4.0 mg L−1 2,4-D and 0.5 mg L−1 kinetin produced53.2 % somatic embryogenesis in genotype ‘PR-202’where G. edulis extract improved the somatic embryogenesisto 66.6 %. Further, 40 % of SLEs emerged somatic embryo-genesis 89.8 %, regeneration of embryogenic callus 96.3 %,and rooting of elongated shoots 97.4 %, respectively. Well-grown plantlets were acclimatized in the greenhouse with94 % survival rate. RAPD profiles of in vitro regeneratedand mother plants were similar and no somoclonal variationwas detected. The current study confirmed that SLEs can beused for somatic embryogenesis and plant regeneration.

Keywords Finger millet . Phytohormones . Regeneration .

Seaweed liquid extracts . Somatic embryogenesis

Introduction

The phytohormone 2,4-dichlorophenoxyacetic acid (2,4-D) iscommonly known as synthetic auxin (a type of herbicide). It isextensively used in plant tissue culture experiments as a phy-tohormone for callus induction and somatic embryogenesis inseveral plant species including cereals and pulses (Rai 2007).In the USA, 2,4-D is a constrained use pesticide (RUP) whichmeans it may be acquired and used merely by approved users(Papaefthimiou et al. 2002). European community legislationon the discharge of dangerous substances into the aquaticenvironment restricted the releasing of 2,4-D into groundwa-ter (WHO 1987). 2,4-D possesses carcinogenic and teratogen-ic properties, and there are several reports on the effects ormode of action of 2,4-D and its metabolites on amphibians(Papaefthimiou et al. 2002), fish (Sarikaya and Yilmaz 2003),human health (Bukowska 2003), and also on environment and

Electronic supplementary material The online version of this article(doi:10.1007/s10811-015-0696-0) contains supplementary material,which is available to authorized users.

* Lakkakula Satishpandu.pine@gmail.com

* Manikandan Rameshmrbiotech.alu@gmail.com

1 Department of Biotechnology, Science Campus, Karaikudi,Tamil Nadu 630 004, India

2 Division of Plant Biotechnology, Entomology Research Institute,Loyola College, Chennai 600 034, India

J Appl PhycolDOI 10.1007/s10811-015-0696-0

plants (Teixeira et al. 2007). So, it is essential to find a goodand eco-friendly alternative for 2,4-D.

Seaweeds are multicellular, macroscopic, benthic marinealgae, and they are sustainable and economical assets alongcoastal areas (Hernandez-Herrera et al. 2014; Satish et al.2015b). Seaweeds contain various phytohormones, such ascytokinins (Stirk et al. 2003), auxins (Stirk et al. 2004), gib-berellins (Stirk et al. 2014), and abscisic acids (Vinoth et al.2014), which enhance shoot growth, root growth and matura-tion, nutrient uptake, chlorophyll content, and flower and fruitset in many crop plants (Satish et al. 2015b; Rengasamy et al.2015). In addition, seaweed extracts are natural sources andthey possess a number of bio-active compounds such as com-plex minerals, vitamins, macro-nutrients (Ca, K, and P) andmicro-nutrients (Cu, Zn, B, Mn, Co, and Mo), antioxidants,organic acids, and amino acids (Khan et al. 2011; Satish et al.2015b). Seaweeds also have been used to improve soil qualityand applied as an alternative for chemical fertilizers which aretoxic and costly when compared to natural extracts (Crouchand Van Staden 1993; Ali et al. 2015). The southern coast ofIndia has abundant seaweed resources with more than 200species being found in this area (Satish et al. 2015b).Previous studies on the effect of seaweed liquid extracts(SLEs) on in vitro somatic embryogenesis and regenerationsuggested that SLEs can be extracted simply and serve as asubstitute for synthetic phytohormones, but unfortunately theyare underutilized so far (Vinoth et al. 2014; Satish et al.2015b). Optimization of a somatic embryogenesis and regen-eration protocol for finger millet using SLEs may advanceorganic cultivation of this crop.

Finger millet (Eleusine coracana L. Gaertn.) is grownworldwide in more than four million hectares, and it is animportant staple food for millions in developing countries ofAsia and Africa (Ceasar and Ignacimuthu 2011; Satish et al.2015a). Finger millet is considered as a traditional food cropfor farmers in drought-prone regions, and its seed can bestored safely for many years without any harm by insects(Latha et al. 2005). It is a recommended nutritious food forchildren and diabetic patients; in addition, its grains may alsobe powdered and provided as a healthful food for infants as itdigests easily and is rich in calcium, proteins, iron, and phos-phorus (Ignacimuthu and Ceasar 2012). There are several re-generation and genetic transformation protocols available forcereal crop varieties, but finger millet has not received muchfocus when compared to other major cereals like wheat, rice,barley, maize, and oats (Ceasar and Ignacimuthu 2009; Satishet al. 2015a). In previous in vitro studies, different types ofphytohormones were used for callus induction, somatic em-bryogenesis, maturation of somatic embryos, and plant regen-eration (e.g., Rangan 1976; Mohanty et al. 1985; Gupta et al.2001; Ceasar and Ignacimuthu 2008, 2011; Ignacimuthu andCeasar 2012; Satish et al. 2015a). However, no detailed studyis available on seed germination, in vitro callus induction,

somatic embryogenesis, and regeneration from embryogeniccallus using SLEs in finger millet genotypes.

The present study was undertaken with the aim to test theinfluence of SLEs along with phytohormones on embryogeniccallus induction, somatic embryogenesis, and shoot regenera-tion from embryogenic callus in four genotypes of finger mil-let, viz. ‘CO(Ra)-14’, ‘Hosur-1’, ‘PR-202’, and ‘Try-1’, inorder to reduce the use of synthetic phytohormones. In addi-tion, globular, heart, and torpedo embryos formed in fingermillet genotype ‘PR-202’ were analyzed by scanning electronmicroscope (SEM) and light microscopy. Random amplifiedpolymorphic DNA (RAPD) is an efficient marker system thatmakes use of arbitrary primers to cover the entire regions ofthe genome and has been routinely used to identify intra- andinterspecies genetic variation in crops (Williams et al. 1990).RAPD analysis was performed in this study to check thegenetic fidelity of the in vitro grown plants. This studymay form the basis for the utilization of SLEs in this andother millets for sustainable and cost-effective in vitro andfield-related studies.

Materials and methods

Collection of seaweed samples Two different seaweeds,Gracilaria edulis (Rhodophyta) and Padina boergesenii(Phaeophyta), were used. G. edulis was collected fromKilakarai, Gulf of Mannar Biosphere (9.23°N, 78.78°E), inthe intertidal region whereas P. boergesenii was obtainedfrom Mandapam, Gulf of Mannar Biosphere (9.28°N79.12°E), in the intertidal region during April andMay 2013. Seaweed samples were cleaned with sea waterto remove debris, mud, and other pollutants, and thenpacked in polythene bags and transported to theDepartment of Biotechnology, Alagappa University,Karaikudi, Tamil Nadu for further studies.

Preparation of seaweed liquid extracts Seaweed liquidextracts (SLEs) were prepared according to Satish et al.(2015b). In brief, the seaweeds were washed with fresh waterfor 5–10 min to remove impurities like macroscopic epiphytes,surface salt, sand particles, shells, and debris. Cleaned sea-weeds were shade dried for 7 days and made into a coarsepowder with the help of an electric mixer grinder. Powderedseaweed (0.5 kg) was added in 1.0 L sterile double distilledwater (1:2 w/v), stirred for 1 h, and sterilized at 121 °C for30 min. Autoclaved extract was filtered by gravity throughmuslin cloth (pore size 150 μm), allowed to cool at room tem-perature, and then filtered through Whatman no. 1 filter paper,and the filtrate was used for further studies. From 1 L of prep-aration, 840 and 760 mL of filtrate was obtained fromG. edulisand P. boergesenii, respectively. The SLEs were stored at−20 °C for further experiments. Different concentrations of

J Appl Phycol

SLE (10–100 %) were prepared by diluting this filtrate withsterile double distilled water.

Plant material and explant preparation Seeds of finger mil-let genotypes ‘CO(Ra)-14’, ‘Hosur-1’, ‘PR-202’, and ‘Try-1’were obtained from Tamil Nadu Agricultural University,Coimbatore, India. Seeds were surface sterilized according toSatish et al. (2015a). In brief, seeds were first treated withsterile water for 20 min in order to eliminate the husk.Dehusked seeds were surface disinfected with 70 % ethanolfor 1 min followed by two washes with sterile distilled water.Seeds were subsequently rinsed with 0.1 % HgCl2 for 5 minand further washed with sterile distilled water three times eachfor 1 min and used for further experiments.

Bioassay of seedlings germinated in SLEs Bioassay of fin-ger millet seedlings was done according to Satish et al.(2015b). In brief, surface sterilized seeds of the four fingermillet genotypes were grown with various concentrationsof SLEs (20, 40, 60, 80, and 100 %) diluted with steriledouble distilled water (optimized pH 5.4) on tissue towelsin polycarbonate jars. Seeds germinated in Murashige andSkoog (MS; Murashige and Skoog 1962) liquid mediumalone without SLEs were used as control. Cultures wereincubated at 27±1 °C for 3 days in complete dark andshifted to light by providing 50 μmol photons m−2 s−1

with white fluorescent tubes (Philips, India) for furthergrowth. After 7 days incubation, the percentage of germi-nation and fresh weight of the seedlings were measured.Other growth parameters like shoot length, root length,and root number were calculated 15 days after seed ger-mination in the light.

Effect of phytohormones and SLEs on embryogenic callusinduction Surface sterilized seeds were germinated in com-plete dark for 3 days on MS basal medium (pH 5.75) contain-ing 3 % sucrose, 0.8 % (w/v) agar-agar type 1, and 0.2 % (w/v)CleriGel (Hi-Media, India) for better solidification. After3 days incubation, the shoot apical meristems (SAMs) (1–2 mm in size) were aseptically detached and cultured in hor-izontal position for callus induction on MS medium contain-ing various concentrations and combinations of two phytohor-mones and two SLEs, individually.

In the first experiment, SAMs of ‘CO(Ra)-14’, ‘Hosur-1’,‘PR-202’, and ‘Try-1’ were randomly cultured for callusinduction on MS medium containing 3 % sucrose, 0.8 %(w/v) agar-agar type 1, and 0.2 % (w/v) CleriGel alongwith two different auxins, 2,4-dichlorophenoxyacetic acid(2,4-D) (3.0, 3.5, 4.0, 4.5, or 5.0 mg L−1) or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (3.0, 3.5, 4.0, 4.5,or 5.0 mg L−1), separately. SAMs cultured on MS mediumdevoid of phytohormones were used as a control. The pH ofthe medium was adjusted to 5.75 before adding solidifying

agents and autoclaved for 20 min at 121 °C. For callus induc-tion, all cultures were incubated at 27±1 °C for 4 weeks incomplete dark. After 4 weeks incubation, callus induced onMS medium containing 4.0 mg L−1 2,4-D or 2,4,5-T wassub-cultured for embryogenic callus formation on the samemedium along with various concentrations of kinetin (Kin)(0.10, 0.25, 0.50, 0.75, or 1.0 mg L−1) and incubated inthe dark for four more weeks at 27±1 °C.

In another experiment, SLEs of G. edulis (10, 20, 30, or40 %) or P. boergesenii (10, 20, 30, or 40 %) devoid of phy-tohormones were used along with 3 % sucrose, 0.8 % (w/v)agar-agar type 1, and 0.2 % (w/v) CleriGel for callus induc-tion. MS medium devoid of SLEs was used as a control. ThepH of the medium was adjusted to 5.75 after adding solidify-ing agents and sterilized at 121 °C for 20 min. Cultures wereincubated in the dark for 6 weeks at 27±1 °C. After 6 weeksdark incubation, callus induced on optimized MS mediumcontaining 20 % SLEs of G. edulis or P. boergesenii weresub-cultured on MS medium containing G. edulis (10, 20,30, or 40 %) or P. boergesenii (10, 20, 30, or 40 %) for em-bryogenic callus formation. All cultures were maintained at27±1 °C for 6 weeks in complete dark.

Effect of SLEs on somatic embryogenesis For somatic em-bryogenesis, 12-week-old yellowish white friable calli in-duced on MS medium containing 20 % of G. edulis orP. boergesenii SLEs were used. For proliferation of somaticembryos, white and friable calli induced on optimized MSmedium containing 20 % of G. edulis or P. boergeseniiSLEs were inoculated on MS medium amended with in-creased concentrations of SLEs of G. edulis (20, 40, or60 %) or P. boergesenii (20, 40, or 60 %) for somatic embryo-genesis. Callus cultured on MS medium devoid of SLEs wasused as a control to analyze the percentage of somatic embryo-genic callus formation in all four genotypes of finger millet.Cultures were maintained in the dark at 27±1 °C for 8 weeks.After 4 weeks incubation, somatic embryos proliferatedon MS medium containing both SLEs and were selectively(whitish-yellow friable callus) transferred on the samemedium for promoting somatic embryogenesis, and fur-ther cultures were incubated at 27±1 °C for four moreweeks in the dark. The percentages of somatic embryo-genesis and mean number of globular shape embryosformed on MS medium containing 20–60 % of SLEswere calculated after 8 weeks of incubation in the dark. Foranalysis of percentage of somatic embryogenesis and meannumber of somatic embryos formed, embryogenic calli wereused from all treatments. Influence of SLEs on the formationof globular, heart, and torpedo shape embryos was analyzedafter 8 weeks of incubation in genotype ‘PR-202’. Then thestages of embryo development, viz. globular, heart, and tor-pedo, were analyzed under a light microscope at ×4 and ×10magnifications and photographed.

J Appl Phycol

Influence of sucrose on maturation of globular somaticembryos The influence of sucrose concentrations at 10, 20,30, 40, or 50 g L−1 was studied on somatic embryogenesis infour genotypes of finger millet. Effect of sucrose was tested inMS medium supplemented with optimized concentration of40 %G. edulis SLE. MS medium devoid of sucrose wasused as a control. The cultures were incubated in the darkat 27±1 °C for 8 weeks. Percentage of somatic embryo-genesis and mean number of somatic embryos formed perembryogenic callus were calculated. Percentage of globu-lar, heart, and torpedo shape embryos formed on MS me-dium containing 10–50 g L−1 of sucrose was analyzedafter 8 weeks of incubation for genotype ‘PR-202’.

Scanning electron microscopy analysis Eight-week-old so-matic embryos obtained from the MS medium containing40 % of G. edulis or P. boergesenii SLEs were analyzed bySEM. Somatic embryogenic calli were fixed in 2 % (w/v)glutaraldehyde (pH 7.0 using 0.1 M phosphate buffer) for24 h at 4 °C (Dawns 1971). After 24 h, tissues were washedfor 10 min with phosphate buffer (0.1 M, pH 7.0) and thenpostfixed for 2 h in 1 % (w/v) osmium tetroxide. Then tissueswere dehydrated in a graded ethanol series and finally theexplants were layered with gold palladium. The preparedsamples were examined and photographed under SEM(Quanta 250 FEG, Japan) operating at 10 kV between×100 and ×400 magnification.

Histochemical analysis Histological observations were car-ried out on the calli with developing somatic embryos. Thesecalli were fixed in formaldehyde/glacial acetic acid/alcohol(FAA) (5:5:90, v/v) for 48 h at 28 °C. Then explants weredehydrated using graded concentrations of tertiary butyl alco-hol and embedded in paraffin wax. Serial sections of 10 μmthickness were cut and stained with 0.1 % Toluidine blue-Ofor 10 min at room temperature.

Effect of SLEs on plant regeneration The embryogenic calli(12 weeks old) obtained from the MS medium containing20 % of G. edulis and P. boergesenii SLEs were used forefficient plant regeneration. The embryogenic calli were cul-tured on MS medium supplemented with various concentra-tions ofG. edulis (25, 50, or 75 %) and P. boergesenii (25, 50,or 75 %), individually. MS medium devoid of either SLE wasused as a control. Cultures were maintained at 27±1 °C in16:8 h light/dark cycles under cool white fluorescent lightsproviding 50 μmol photons m−2 s−1. After 6 weeks of incuba-tion, green embryogenic calli were sub-cultured onto the samemedium for multiple shoot induction. Percentage of shootregeneration from somatic embryogenic callus was calculatedafter 6 weeks of incubation, and mean length of regeneratedshoots was calculated after 12 weeks of incubation.

Rooting of plantlets Shoots induced on the medium contain-ing either 40% ofG. edulis orP. boergesenii extract were usedfor root induction. Regenerated shoots of 3–4 cm length weredetached from clumps of green shoots and transferred ontohalf-strength MS medium supplemented with various con-centrations of G. edulis (5, 10, 15, or 20 %) orP. boergesenii (5, 10, 15, or 20 %) extract for root induc-tion. Half-strength MS medium devoid of SLEs was usedas a control. All the cultures were maintained at 27±1 °Cin 16:8 h light/dark cycles under cool white fluorescentlights providing 50 μmol photons m−2 s−1 for 6 weeks.The percentage of root induction and mean length of rootswere calculated after 6 weeks of incubation.

Hardening and acclimatization Fully grown plantlets(6–7 cm long) were carefully taken out from culture tubesand rinsed carefully with deionized water to remove the me-dium bound on surface of the roots. Then the plantlets weretransferred into plastic cups containing SoilriteTM (Keltech,Bengaluru, India), vermiculite, and red soil (1:1:1v/v/v ratio).Plastic cups containing plantlets were maintained in a growthchamber for a week to maintain 60 % relative humidity andmoved to a greenhouse. Plants were watered every 2 days andgrown to maturity along with seedling-derived field-growncontrol plants in the greenhouse. Survival rate of the plantletswas calculated and phenotypic similarities were comparedwith field-grown plants on the 30th day after transfer to thegreenhouse.

RAPD analysis Random amplified polymorphic DNA(RAPD) analysis was performed to check the genetic fidelityby leaves of in vitro regenerated plants through somatic em-bryogenesis. Two regenerated plants grown on MS mediumcontaining SLEs and phytohormones were obtained from eachgenotype and analyzedwith control plants (seedlings grown ingreenhouse) using custom synthesized 07 RAPD primers(Sigma, USA) (Supplementary Table 1). Genomic DNAwasisolated from leaves of in vitro regenerated plants and controlplants by CTABmethod (Doyle and Doyle 1990). Polymerasechain reaction (PCR) was carried out in a volume of 12.5-μLreactions consisting of 30 ng of template DNA, 1.5 mMMgCl2, 200 μM dNTPs, 0.4 μM RAPD primers, and 1 U ofTaq DNA polymerase (MBI, Fermentas, Germany). The am-plification was carried out in a Thermal Cycler (AB AppliedBiosystems, USA). PCR was performed with an initial dena-turation at 94 °C for 5 min followed by 42 cycles of denatur-ation at 94 °C for 1 min, annealing at 55 °C for 1 min, andextension at 72 °C for 2 min. The last cycle was followed by afinal extension at 72 °C for 7 min. Amplification was repeatedthrice to confirm the reproducibility of the results. The ampli-fied samples were electrophoresed in 6 % (w/v) polyacryl-amide gels and stained with 2 μL ethidium bromide (from10 mg mL−1 stock), and fragments were scored using a gel

J Appl Phycol

documentation system (Molecular Imager Gel Doc XR+

System; Bio-Rad, USA).

Data collection and statistical analysis Each experiment wascarried out twice with three replicates. In the bioassay, meanpercentage of germination and mean fresh weight of the seed-ling were calculated 7 days after inoculation. The mean lengthof shoots, mean number of roots, and mean length of rootswere calculated 15 days after inoculation of seeds. In bioassay,50 seeds were used per replication for each finger millet ge-notype and concentration of SLE. Effect of phytohormones oncallus induction from SAMs was calculated after 4 weeks ofdark incubation and percentage of embryogenesis was calcu-lated after 8 weeks of incubation. Influence of SLEs on callusinduction was calculated after 6 weeks of dark incubation andembryogenic callus formation was calculated after 12 weeksof incubation. Effect of various concentrations and types ofSLEs on percentage of somatic embryogenesis and meannumber of somatic embryos formed were calculated after8 weeks of incubation in the dark. Influence of sucrose alongwith 40 % of G. edulis on somatic embryogenesis was calcu-lated after 8 weeks of culture. Percentage of regeneration andmean length of regenerated shoots produced at various con-centrations and types of SLEs were analyzed after 6 and12 weeks of incubation in the light, respectively. Influenceof half-strength MS medium with and without SLEs onrooting was analyzed after 6 weeks of incubation in the light.After 30 days, the survival rates of plantlets maintained in agreenhouse were determined. Mean values were estimated

and the significance of differences with means was carriedout by using Duncan’s multiple range test (one-way analysisof variance) with SPSS 17.0 (SPSS, IBM Statistics).

Results

Bioassay of seedlings germinated in SLEs

Seed germination was observed within 2 days after incubationat 27±1 °C in SLEs of both G. edulis and P. boergesenii at allconcentrations tested. SLEs of G. edulis 60 % showed maxi-mum percentage of germination. In the control experiment,maximum percentage of germination was found in genotype‘PR-202’ (Table 1). The germination percentage in all fourgenotypes of finger millet increased with 60 % of SLE andthere was a decline in percentage of seed germination above60 % of extracts. The lowest germination percentage was ob-served in ‘Hosur-1’ genotype treated with maximum percent-age (100 %) of P. boergesenii liquid extract. The extracts ofG. edulis and P. boergesenii each at 60 % (w/v) showed asignificant increase in fresh weight of the seedlings over thecontrol values. Higher concentrations of both SLEs testedwere adversely affected and also decreased the response ofpercentage seed germination in all four finger millet geno-types. Finger millet seeds of all four genotypes germinatedon MS liquid alone (control) devoid of SLEs showed 0.1–0.5 mg seedling−1 fresh weight after 7 days of incubation inthe light (Table 1).

Table 1 Effect of MS liquid medium and G. edulis and P. boergesenii seaweed liquid extracts on in vitro seed germination in four finger milletgenotypes after 7 days of incubation in the light

SLEs (%) (w/v) Percentage of germination (mean±SE) Fresh weight of the seedlings (mg/seedling) (mean±SE)

CO(Ra)-14 Hosur-1 PR-202 Try-1 CO(Ra)-14 Hosur-1 PR-202 Try-1

Control 74.7±0.9a 69.3±0.2a 77.3±0.5a 70.7±0.4a 0.3±0.2ab 0.1±0.1a 0.5±0.2a 0.2±0.1a

Gracilaria edulis

20 82.4±1.0c 81.6±0.3g 85.0±0.3d 80.7±0.8d 0.5±0.4ab 0.2±0.1a 0.5±0.3a 0.2±0.2ab

40 92.5±0.8e 88.5±0.3h 95.8±0.4g 91.3±0.4f 0.6±0.3ab 0.4±0.4a 0.8±0.4a 0.4±0.3ab

60 97.3±0.9f 94.4±0.3k 99.6±0.4i 96.8±0.6h 0.9±0.2b 0.5±0.3a 1.1±0.5a 0.7±0.6b

80 88.2±1.6d 76.6±0.3e 89.3±0.7e 77.1±0.6c 0.4±0.2ab 0.3±0.2a 0.6±0.6a 0.3±0.4ab

100 79.2±0.7b 71.5±0.5c 80.4±0.5c 72.5±0.2b 0.3±0.3ab 0.1±0.1a 0.4±0.4a 0.1±0.1a

Padina boergesenii

20 87.4±0.4d 80.3±0.2f 88.9±0.2e 81.2±0.1d 0.2±0.1a 0.1±0.1a 0.3±0.2a 0.2±0.1a

40 93.9±0.5e 89.4±0.1i 92.3±0.3f 89.5±0.4e 0.3±0.4ab 0.2±0.2a 0.5±0.4a 0.4±0.3ab

60 96.5±0.3f 93.1±0.3j 98.4±0.3h 95.2±0.4g 0.7±0.2ab 0.4±0.3a 0.9±0.7a 0.6±0.4ab

80 83.6±0.4c 75.1±0.3d 84.3±0.4d 76.4±0.2c 0.5±0.5ab 0.3±0.5a 0.7±0.6a 0.5±0.6ab

100 78.6±0.2b 70.6±0.4b 79.5±0.5b 72.3±0.6b 0.1±0.1a 0.2±0.3a 0.4±0.7a 0.3±0.5ab

Themediumwas composed of sterile distilled water and various concentrations (w/v) of SLEs.MS liquid mediumwithout any SLEs was used as control.For each concentration, 50 seeds were germinated on tissue wet towels with 10 mL of extract in polycarbonate jars. Percentage of germination and freshweight of the seedlings were determined after 7 days of germination.Means followed by the same letter are not significantly different at P ≤0.05 based onDuncan’s multiple range test

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There were significant differences in seedling phenotypesat different concentrations of SLEs. The highest frequency ofshoot length was observed in genotype ‘PR-202’ in the pres-ence of 60 % of G. edulis liquid extracts. The extract ofG. edulis seaweed showed higher root length and root numberin genotype ‘PR-202’ over the control values after 15 days ofincubation in the light (Table 2). Higher concentrations ofSLEs (above 60 %) were interdictory and resulted in de-creased growth parameters in all four genotypes of finger mil-let. Extract of P. boergesenii produced highest shoot and rootlengths, with maximum root number over the control values ingenotype ‘PR-202’ (Table 2).

Effect of phytohormones on in vitro callus inductionand embryogenic callus formation

Surface sterilized finger millet seeds were germinated on MSbasal medium and the SAMs of seedlings (after 3 days) wereused as explant source (Fig. 1a). SAMs inoculated on MSmedium supplemented with 2,4-D or 2,4,5-T at 1.0–5.0 mg L−1 produced callus in all genotypes of finger millet(Fig. 1b). In this study, both types of auxin increased thedevelopment of callus in SAM explants within 3–4 days ofincubation in the dark at 27±1 °C. This callus was soft, friable,watery, white in color, grew slowly, and was non-regenerativein nature, with unorganized morphology and devoid of nodu-lar structures. However, after 4 weeks dark incubation, themaximum percentage of callus was produced in genotype‘PR202’ on MS medium supplemented with 4.0 mg L−1 2,4-D (Table 3). Callus induction percentage ranged from 3.5 to37.8 % on MS medium containing 2,4,5-T, and no callusinduction was observed on medium devoid of phytohor-mones. Maximum percentage of the callus developed intoembryogenic callus with nodular structures on MS mediumcontaining 4.0 mg L−1 2,4-D and 0.5 mg L−1 Kin after atotal of 8 weeks of incubation in the dark. The averagepercentage of embryogenic callus formed was 8.1–48.9 %on MS medium supplemented with 4.0 mg L−1 2,4-D and0.5 mg L−1 Kin. There was no callus induction on MSmedium devoid of phytohormones (Table 3).

Effect of SLEs on callus induction and embryogenic callusformation

Callus induction was observed at the cut ends of the SAMexplants after 10 days of incubation in the dark (Fig. 1c).Callus induced from SAMs in all four genotypes of fingermillet on MS medium containing 20 % of SLEs was soft,watery, friable, and non-regenerative when tested on MS re-generation medium (data not shown). Highest percentage ofcallus was induced in genotype ‘PR-202’ on MS mediumsupplemented with 20 % of G. edulis extract, after 6 weeksof incubation in the dark (Table 4). Lowest percentage of calluswas produced in genotype ‘Try-1’ at 40% ofG. edulis extracts.Callus induction percentage ranged from 5.8 to 41.1 % onMSmedium containing P. boergesenii extract after 6 weeks of darkincubation (Table 4; Fig. 1d). No callus was induced on MSmedium devoid of SLEs in the MS medium after 6 weeks ofincubation (Table 4).

Sub-culturing of whitish-yellow and friable callus on MSmedium containing 20 % of SLEs had a stimulatory effect onembryogenic callus formation after 6 weeks of incubation inthe dark. Both types of SLEs tested accomplished the embryo-genesis in all four finger millet genotypes used in this study.Maximum percentage of embryogenic callus was produced onMS medium containing 20 % of G. edulis extract in genotype

Table 2 Effect of MS liquid medium and G. edulis and P. boergeseniiseaweed liquid extracts on length of shoot, root and number of roots infour genotypes of finger millet after 15 days of incubation in the light

SLEs (%) (w/v) Finger millet genotypes

CO(Ra)-14 Hosur-1 PR-202 Try-1

Gracilaria edulis Mean length of shoot (cm) (mean±SE)

40 8.8±0.4c 5.4±0.5cd 11.1±0.8d 5.2±0.2ab

60 10.6±0.6d 7.5±0.6e 13.4±0.6e 9.5±0.9e

80 6.9±0.8b 6.3±0.5d 9.7±0.7c 7.7±0.5d

Padina boergesenii

40 7.0±0.2b 4.3±0.6ab 8.1±0.6b 6.4±0.3c

60 8.2±0.5c 5.8±0.8cd 11.7±0.2d 8.9±0.5e

80 6.3±0.5ab 5.1±0.5bc 6.5±0.5a 5.6±0.4bc

Control 5.6±0.3a 3.6±0.2a 6.3±0.2a 4.5±0.7a

Gracilaria edulis Mean number of roots (mean±SE)

40 2.6±0.4bc 0.6±0.4ab 2.4±0.5bc 0.9±0.6a

60 3.4±0.3c 1.9±0.3c 4.8±0.3d 2.6±0.5b

80 2.9±0.5bc 1.4±0.5bc 3.9±0.4d 1.5±0.8a

Padina boergesenii

40 1.3±0.4a 0.8±0.8ab 1.6±0.5ab 0.7±0.4a

60 2.3±0.5b 1.6±0.7bc 2.7±0.5c 1.8±0.8ab

80 0.5±0.3a 1.1±0.5abc 0.9±0.6a 1.2±0.7a

Control 1.1±0.7a 0.2±0.2a 2.1±0.7bc 0.5±0.5a

Gracilaria edulis Mean length of root (cm) (mean±SE)

40 5.9±0.6de 2.5±0.7b 6.8±0.8bc 3.4±0.5cd

60 6.7±0.4e 4.8±0.7d 8.6±0.9d 5.9±0.9e

80 4.8±0.7cd 3.9±0.5cd 7.5±0.6cd 2.9±0.6bcd

Padina boergesenii

40 4.1±0.3bc 0.5±0.4a 6.0±0.5bc 2.1±0.9abc

60 5.2±0.5cd 2.9±0.9bc 7.1±0.9cd 3.7±0.5d

80 2.0±0.9a 1.0±0.8a 5.3±0.5ab 1.6±0.7ab

Control 3.0±1.1ab 0.7±0.6a 4.1±1.6a 1.1±0.9a

The medium was composed of sterile distilled water and various concen-trations (w/v) of SLEs. MS basal medium without any SLEs was used ascontrol. For each concentration, 50 seeds were germinated on tissue wettowels with 10 mL of extract in polycarbonate jars. Means±SE followedby the same letter are not significantly different at P ≤0.05 based onDuncan’s multiple range test

J Appl Phycol

‘PR-202’ after 6 weeks of incubation in the dark (Table 4).Most of the calli turned light yellow and were regenerative innature, and nodular formation was also found when tested onMS medium containing 20 % of either SLEs after a total of4 weeks of incubation in the dark (Fig. 1d). There was nosignificant improvement in the embryogenic callus mass atabove 20% of SLEs, and no callus was grown onMSmediumwithout SLEs in all four genotypes of finger millet genotypes.

Effect of SLEs on in vitro somatic embryogenesis

Embryogenic callus cultures selectively transferred on MS me-dium containing 40 % of G. edulis extract with 3 % of sucroseproduced the maximum percentage of somatic embryogenesiswith the highest number of somatic embryos after 8 weeks ofincubation in the dark (Table 5). Embryogenic calli producedglobular, heart, and torpedo shape embryos on MS mediumcontaining 40 % of SLEs of both G. edulis and P. boergesenii(Fig. 1e–g). Both types of SLEs increased the proliferation ofsomatic embryos after 4 weeks of sub-culturing on the same

medium. The medium fortified with 40 % of G. edulis extractrecorded the highest percentage of globular-shaped embryos,followed by heart and torpedo embryos production (Table 5;Fig. 2a). MS medium amended with 40 % of P. boergeseniiextract produced a smaller percentage of somatic embryos whencompared toG. edulis extract after 8 weeks of incubation in thedark (Fig. 2a, b). In this experiment, it was found that increasingthe concentrations of SLEs up to 40% augmented the frequencyof somatic embryogenesis with the formation of a maximumnumber of somatic embryos in all four genotypes of fingermilletwhen incubated in the dark at 27±1 °C (Table 5; Fig. 2a). Incontrast, an increase in the SLE concentration beyond 40 %resulted in decreased somatic embryogenesis and increased ne-crosis of somatic embryogenic callus. MS medium devoid ofeither SLE showed no callus growth after 8 weeks of incubation.

Influence of sucrose on somatic embryogenesis

MS medium supplemented with 40 % of G. edulis extractalong with 30 g L−1 sucrose produced a varied response of

Fig. 1 Effect of seaweed liquid extracts on callus induction, somaticembryogenesis, and somatic embryogenic callus formation of fingermillet genotypes using shoot apical meristems. a Germination of fingermillet ‘CO(Ra)-14’ genotype seeds on MS basal medium (bar=1.0 mm)after 3 days of incubation. b Shoot apical meristems from ‘PR-202’genotype inoculated on MS medium containing seaweed liquid extract(bar=1.8 mm). c Initiation of callus at the end regions of shoot apicalmeristems on MS medium containing 20 % of G. edulis after 2 weeks ofincubation in genotype ‘Hosur-1’ (bar=1.0 mm). d Friable and non-regenerative callus formed in genotype ‘Try-1’ on MS medium

containing 20 % P. boergesenii after 6 weeks of incubation in the dark(bar=1.0 mm). e Embryogenic callus with globular embryos in genotype‘PR-202’ after 6 weeks of incubation in the dark on MS mediumsupplemented with 20 % of G. edulis extract (bar=1.4 mm) (arrowsindicate globular embryos). f Beginning of conversion from globular toheart shape embryo stage in genotype ‘CO(Ra)-14’ on MS mediumamended with 40 % of G. edulis after 8 weeks of incubation in the dark(bar=1.0 mm) (heart-shaped somatic embryos are shown with arrow). gSomatic embryogenesis produced torpedo shape embryos in genotype‘PR-202’ (bar=0.5 mm) (arrows indicate torpedo shape embryos)

J Appl Phycol

somatic embryogenesis in all four genotypes after 8 weeks ofincubation in the dark (Table 6; Fig. 3). Higher concentrationsof sucrose (40 and 50 g L−1) advanced the development ofnormal embryos, but somatic embryo growth was less at theseconcentrations. MS medium containing 30 g L−1 sucrosehelped to produce somatic embryos when compared to lower(10 and 20 g L−1) or higher (40 and 50 g L−1) concentrationsof sucrose, after 8 weeks of culture in the dark (Table 6). Forsomatic embryogenesis, MS medium devoid of sucroseshowed no callus growth after 8 weeks of incubation. Thisconcentration is optimum for obtaining the late globular toearly heart stage of embryos and afterwards the torpedo shapeembryos in clumps of diameters. MS medium containing40 % of G. edulis extract containing 30 g L−1 sucrose wasfound to be optimum for somatic embryogenesis in genotype‘PR-202’. The globular, heart, and torpedo shape embryos

were produced after 8 weeks of incubation in the darkin this genotype (Fig. 3). In this experiment, globular-shaped embryos were produced at the highest percentagewhen compared to heart and torpedo embryos (Fig. 3).

Histology

Microscopic observations of somatic embryos revealed thepresence of globular, heart, and torpedo stages of embryos(Fig. 4). The embryos grown were freely attached on the cal-lus. Somatic embryos contained different layers of cells withwell-developed suspensor cells (Fig. 4a). Histological exami-nation of embryogenic torpedo shape of tissues confirmed thatthey were proliferated to form mass of cells, which is identi-fied as embryogenic mass. The central portion composed ofcompact tissue was equivalent to the nodular callus and the

Table 3 Effect ofphytohormones on callusinduction and somaticembryogenesis from shoot apicalmeristem of four genotypes offinger millet

Phytohormone concentration(mg L−1)

Finger millet genotypes

CO(Ra)-14 Hosur-1 PR-202 Try-1

2,4-D Percentage of callus formation (mean±SE)

3.0 28.1±0.8h 9.9±0.7de 29.3±1.5d 10.1±0.8d

3.5 31.3±0.7i 15.5±0.8g 39.0±0.9f 24.4±1.2h

4.0 35.8±1.3j 21.8±0.6h 43.4±0.6g 28.7±0.8i

4.5 19.4±0.5e 6.7±0.7b 35.1±0.8e 15.6±0.7f

5.0 11.6±0.6c 4.6±0.6a 25.1±1.2c 8.5±0.8c

2,4,5-T

3.0 16.5±0.4d 3.5±0.7a 20.3±0.6b 7.1±0.3bc

3.5 21.2±0.8f 7.7±1.4bc 27.9±0.8d 13.9±0.8e

4.0 25.5±0.5g 12.6±0.5f 37.8±0.6f 18.1±0.6g

4.5 8.3±0.2b 10.4±0.4e 19.5±1.2b 5.9±0.9b

5.0 5.6±0.5a 8.9±0.3cd 13.5±0.7a 4.2±0.7a

Control 0.0 0.0 0.0 0.0

2,4-D+Kin Percentage of embryogenic callus formation (mean±SE)

4.0+0.10 14.7±0.6d 18.0±0.3f 40.6±0.6g 19.9±1.2e

4.0+0.25 37.9±0.9h 24.9±0.5g 48.9±1.1i 30.6±0.6i

4.0+0.50 42.4±1.0j 27.8±1.2h 53.2±0.5j 34.9±0.4j

4.0+0.75 23.8±0.6f 11.2±0.6d 31.8±0.4e 26.8±1.1h

4.0+1.0 9.9±0.8b 8.1±0.6c 23.7±0.6c 20.3±1.0fg

2,4,5-T+Kin

4.0+0.10 18.1±0.9e 9.2±0.7c 26.1±1.1d 11.5±0.5c

4.0+0.25 33.8±1.1g 13.6±0.6e 36.4±0.5f 16.4±0.8d

4.0+0.50 39.7±0.6i 16.9±0.9f 46.5±0.9h 21.8±1.3g

4.0+0.75 12.5±0.5c 5.6±1.2b 16.8±0.8b 6.8±0.9b

4.0+1.0 7.6±0.7a 2.4±0.7a 11.3±0.6a 3.5±0.7a

Control 0.0 0.0 0.0 0.0

For each phytohormone type and concentration, 50 explants were inoculated from each genotype with threereplicates. MS medium devoid of phytohormones had been used as control. Percentage of callus induction wascalculated after 4 weeks of incubation and embryogenic callus formation was analyzed 4 weeks after sub-culturein both control and treated experiments. Means±SEwith the same letter within column is significantly different atP ≤0.05 according to Duncan’s multiple range test

J Appl Phycol

outward portion was composed of loose tissues correspondingto the friable callus. Morphology of the surrounding embry-onic callus is partially dedifferentiated and cells at the tips ofthe masses could be seen (Fig. 4a). It is also assumed that theembryogenic calli grown for 8 weeks on MS mediumcontaining 40 % of G. edulis liquid extract along with30 g L−1 sucrose gave rise to globular or early heart shape ofembryos (Fig. 4b). In cross-section, the cells of the masseswere compact with dense cytoplasm characteristic and em-bryo development was asynchronous. Embryos at variousstages (globular, heart, and torpedo) of growth could be no-ticed in the histological observations (Fig. 4a–d). In cross-section of heart-shaped embryogenic callus, the base is com-posed of more differentiated cells corresponding to the necrot-ic portion of the callus (Fig. 4b–d).

SEM of somatic embryogenic callus

The globular, heart, and torpedo somatic embryos formed onembryogenic callus were studied using SEM after 8 weeks ofincubation. SEM of non-embryogenic callus revealed thread-like structures in genotype ‘CO(Ra)-14’ (Fig. 5a) under ×100magnification whereas in embryogenic callus different typesof somatic embryos, viz. globular, heart, and torpedo, wereobserved under ×100 to ×400 magnifications (Fig. 5b–d).SEM of somatic embryos showed that MS medium supple-mented with either SLE (40 %) along with sucrose (30 g L−1)-derived embryogenic callus showed a higher number ofglobular structures in genotype ‘PR-202’ (Fig. 5b). Alongwith globular embryos, heart and torpedo shape somaticembryos were also observed in SEM where globular structure

Table 4 Effect of seaweed liquid extracts on callus induction andembryogenic callus formation in four genotypes of finger millet

SLEs (%) (w/v) Finger millet genotypes

CO(Ra)-14 Hosur-1 PR-202 Try-1

Gracilaria edulis Percentage of callus induction (mean±SE)

10 36.7±0.9f 26.6±1.2f 43.4±0.8g 25.2±0.5f

20 40.2±1.2h 28.7±0.6g 49.8±0.4h 30.8±1.1h

30 31.9±1.1e 16.3±0.8d 38.9±0.9e 19.4±0.6e

40 27.1±0.8c 13.3±0.9c 29.5±0.8c 11.8±0.8c

Padina boergesenii

10 29.8±1.0d 10.3±0.5b 35.4±0.9d 17.7±1.4d

20 38.6±1.2g 22.8±0.6e 41.1±1.2f 28.7±1.0g

30 20.5±0.7b 17.5±1.1d 24.0±0.8b 9.1±0.8b

40 16.2±0.8a 8.1±0.9a 18.2±0.7a 5.8±0.7a

Control 0.0 0.0 0.0 0.0

Gracilaria edulis Percentage of embryogenic callus formation (mean±SE)

10 44.3±0.5d 38.8±0.6d 49.4±0.2c 40.8±0.4d

20 60.0±0.4h 49.3±0.2g 66.6±0.3h 53.8±0.9h

30 54.7±0.7f 42.7±0.4e 57.7±0.8f 47.7±1.0f

40 39.8±0.7b 34.5±0.9b 51.9±0.8d 36.1±0.3b

Padina boergesenii

10 42.9±0.5c 35.7±0.8c 46.7±0.4b 38.6±0.9c

20 57.6±0.8g 46.0±0.7f 62.9±0.4g 50.6±0.4g

30 51.7±0.6e 41.7±0.6e 55.3±0.7e 45.7±0.6e

40 38.4±0.5a 30.4±0.4a 41.2±0.3a 33.8±0.8a

Control 0.0 0.0 0.0 0.0

For primary callus induction, 75 shoot apical meristems were studiedthrough three independent experiments. For embryogenic callus forma-tion 6 weeks old, 50 calluses from each seaweed concentration were sub-cultured. MS medium without SLEs had been used as control. Averagevalues of all the experiments represent the means±SE, and all the datawere analyzed by SPSS 17.0 software. Means followed by the same letterin each column are not significantly different at P ≤0.05 based onDuncan’s multiple range test

Table 5 Effect of various concentrations and types of seaweed liquidextracts on somatic embryogenesis in four genotypes of finger millet

SLEs (%) (w/v) Finger millet genotypes

CO(Ra)-14 Hosur-1 PR-202 Try-1

Gracilaria edulis Percentage of somatic embryogenic callus formation(mean±SE)

20 73.4±0.8d 49.7±0.5c 75.7±1.0c 62.8±1.1d

40 82.4±0.7f 63.9±0.9f 89.8±0.8e 71.6±0.5f

60 65.5±0.4c 53.3±1.1d 68.9±0.9a 50.3±0.9c

Padina boergesenii

20 52.4±0.7a 44.7±0.8b 72.9±0.8b 48.6±0.9b

40 77.8±0.8e 58.9±0.9e 81.8±0.9d 66.6±1.1e

60 61.5±0.5b 37.3±0.8a 67.7±0.9a 39.6±0.8a

Control 0.0 0.0 0.0 0.0

Gracilaria edulis Mean number of somatic embryos (mean±SE)

20 5.2±0.2c 3.1±0.3ab 5.4±0.4abc 3.5±1.2ab

40 5.7±0.5c 3.7±0.4b 6.1±1.3c 4.2±0.2b

60 3.9±0.5ab 2.6±0.5ab 4.3±0.6ab 2.8±0.7ab

Padina boergesenii

20 3.5±0.4a 2.0±1.0a 4.8±1.2abc 2.4±0.7ab

40 4.8±0.7bc 2.9±1.1ab 5.6±0.4bc 3.9±0.8ab

60 2.8±1.1a 1.8±0.7a 3.9±0.6a 1.9±1.2a

Control 0.0 0.0 0.0 0.0

The percentages of somatic embryogenesis and mean number of globularshape embryos formed were calculated after 8 weeks of incubation in thedark. For all treatments, 12-week-old yellowish white friable callusinduced on MS medium containing 20 % of G. edulis or P. boergeseniiSLEs each 75 was tested for somatic embryogenesis with three repli-cates. Calluses cultured on MS medium devoid of SLEs have beenused as controls. Data collected after 8 weeks of dark incubation inthe medium amended with various concentrations of SLEs and con-trols tested for percentage somatic embryogenesis and mean numberof globular shape embryo formation. Means followed by the sameletter in each column are not significantly different at P ≤0.05 basedon Duncan’s multiple range test

J Appl Phycol

embryos were produced regularly in high number for all fourfinger millet genotypes used in this study (Fig. 5c, d).

Plantlet regeneration

Inclusion ofG. edulis and P. boergesenii liquid extracts in MSmedium initiated the regeneration of embryogenic calli in allfour genotypes of finger millet when incubated in the light(Fig. 6a). Embryogenic callus obtained on MS medium con-taining 20 % of G. edulis extract gave more regenerated

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Fig. 2 Effect of seaweed liquid extracts on globular, heart, andtorpedo shape embryo formation in ‘PR-202’ genotype. a MSmedium supplemented with 20–60 % of G. edulis extract. b MSmedium supplemented with 20–60 % of P. boergesenii extract

Table 6 Effect of sucroseconcentrations on maturation ofglobular somatic embryos on MSmedium supplemented with 40 %of G. edulis liquid extract in fourgenotypes of finger millet

Sucrose (g L−1) (w/v) CO(Ra)-14 Hosur-1 PR-202 Try-1Percentage of somatic embryogenic callus formation (mean±SE)

Control 0.0 0.0 0.0 0.0

10 76.6±0.7c 51.8±1.1a 80.8±0.5c 65.7±0.7b

20 81.8±0.4d 67.7±0.7d 87.9±1.1d 74.7±0.5d

30 88.9±0.9e 72.8±0.8e 95.3±0.2e 81.6.±0.4e

40 73.8±0.7b 61.8±0.5c 78.2±0.7b 69.8±0.7c

50 66.6±0.9a 56.7±0.4b 71.7±0.3a 58.6±0.6a

Sucrose (g L−1) (w/v) Mean number of somatic embryos per embryogenic callus (mean±SE)

Control 0.0 0.0 0.0 0.0

10 4.1±0.3a 2.7±0.6a 7.1±0.8b 3.3±0.2a

20 6.1±0.5b 4.5±0.4c 7.7±0.5bc 5.6±0.4c

30 7.6±0.4c 5.3±0.2d 8.2±0.2c 6.1±0.7c

40 5.7±0.2b 3.9±0.1c 6.8±0.3b 4.5±0.3b

50 4.8±0.6a 3.2±0.3ab 5.2±0.3a 3.8±0.7ab

Data collected after 8 weeks of dark incubation in the medium supplemented with different concentrations ofsucrose tested for somatic embryogenesis. MS medium devoid of SLEs had been used as controls. Each exper-iment was repeated two times with three replicates. In control and treated experiments, the percentages of somaticembryogenesis and mean number of globular shape embryos formed were calculated after 8 weeks of incubationin the dark. Average values of all the experiments represent the means±SE and all the data were analyzed bySPSS 17.0 software. Means followed by the same letter in each column are not significantly different at P ≤0.05based on Duncan’s multiple range test

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Fig. 3 Effect of sucrose concentrations on various developmental stagesof somatic embryos in ‘PR-202’ genotype on MS medium containing40 % of G. edulis extract

J Appl Phycol

plantlets than embryogenic callus obtained on MSmedium supplemented with 20 % of P. boergeseniiextract upon transfer onto the medium. As the embryo-genic callus phases were asynchronous, regeneratedshoots obtained from the same culture medium were atvarious developmental stages (Fig. 6a–c). MS mediumsupplemented with 50 % of G. edulis extract was foundto be more effective on regeneration in all four geno-types of finger millet (Table 7; Fig. 6c). An increase inthe concentration of SLEs progressively reduced thefrequency of regeneration when compared to controlsin all four genotypes of finger millet after 12 weeksof incubation in the light. The maximum percentage ofregeneration from embryogenic callus and mean length ofregenerated shoots was with 50 % G. edulis extract in geno-type ‘PR-202’, after 12 weeks of incubation in the light.Shoots regenerated on this medium were longer and moregreenish (Fig. 6d).

Effect of SLEs on rooting of in vitro derived shoots

Root induction was observed after 15 days of incubationin the light with both types of SLEs. Half-strength MSmedium containing 60 % of G. edulis extract induced ahigh frequency of rooting with maximum root length after6 weeks of incubation in the light (Fig. 6d). Root

induction frequency for P. boergesenii was higher at60 % of extract in half-strength MS medium, and beyondthis the effect of rooting decreased in all genotypes offinger millet (Table 8). G. edulis SLE at all concentrationstested (20–80%) produced the lowest percentage of rooting inthe ‘Hosur-1’ genotype compared to the other genotypes.P. boergesenii SLE and control (half-strength MS mediumwithout any SLEs) showed the lower percentage of rootingin all genotypes after 6 weeks of incubation in the light(Table 8). Half-strength MS medium devoid of SLEs inducedvery thin and short roots, whereas inclusion of SLEs signifi-cantly increased the rooting frequency and root length in allfour genotypes of finger millet, after 6 weeks of incubation inthe light.

Acclimatization of in vitro grown plantlets

Plants with well-developed roots were carefully taken outfrom culture tubes and rinsed carefully with deionized waterto remove the medium bound on surface of the roots. Allacclimated plants were finally transferred to the field andgrew normally in the natural environmental condition. Theplants transferred from the growth chamber to a green-house grew well with a >94 % survival rate (Fig. 6e).In vitro regenerated plants were phenotypically similarwhen compared to field-grown seed-derived plants, and

Fig. 4 Histological analysis ofsomatic embryogenic callusformation in finger milletgenotypes using 40% ofG. edulisor P. boergesenii liquid extracts. aCross-section of torpedo stageembryo from genotype ‘PR-202’showing torpedo-shaped embryo(bar=50 μm). b Somaticembryos derived from friableembryogenic callus showingglobular or early heart-shapedtissues (bar=50 μm). c Cross-section of embryogenic calluswith compact tissue in the middlepart (bar=100 μm). d Cross-section of heart-shaped somaticembryo composed of moredifferentiated cells (bar=75 μm)

J Appl Phycol

they revealed phenotypic homogeneity such as flowering,seed setting, and grain yield (Fig. 6f).

RAPD analysis

RAPD profiles obtained from in vitro regenerated plants usingSLEs and phytohormones, and that of the seed-grown plants,were similar in all respects. All the primers produced mono-morphic bands confirming the genetic homogeneity of thein vitro regenerated plants (Supplementary Fig. 1). No varia-tion was seen between plants regenerated from embryogeniccallus and seed-derived plants.

Discussion

In this study, SAMs were used as starting material for callusinduction and subsequent somatic embryogenesis. In previousreports, various types of explants such as leaf base segments(Rangan 1976; Mohanty et al. 1985) and shoot tips or shootapex (Latha et al. 2005; Ceasar and Ignacimuthu 2008, 2010,2011) were successfully used for both regeneration and genet-ic transformation of millet. SAMs have also been used suc-cessfully to improve regeneration methods in other cereals(Ceasar and Ignacimuthu 2010), and SAMs were also usedas a starting explant to produce highly transformed rice,

wheat, maize, barley, oat, sorghum, and millets (Baskaranand Dasgupta 2012; Satish et al. 2015a). Embryogenic callusinduction, somatic embryogenesis, and regeneration of plant-lets developed in this report from 3-day-old seedling-derivedSAMs of finger millet genotypes may be employed in the nearfuture for in vitro culture and genetic transformation studies.

In order to induce callus and to form the embryogeniccallus, two different types of auxins (2,4-D and 2,4,5-T) weretested individually or in combination with Kin along with30 g L−1 sucrose. The optimum response of embryogeniccallus formation was found on MS medium containing4.0 mg L−1 2,4-D. This is in conformity with previous studiesin finger millet (Latha et al. 2005; Ceasar and Ignacimuthu2008) and kodo millet (Ceasar and Ignacimuthu 2010). Callusinduction in millets or other cereals is frequently accom-plished by supplementing 2,4-D inMSmedium. In a previousstudy, Kothari-Chajer et al. (2008) used only 2,4-D with mod-ified MS medium for somatic embryogenesis in kodo milletgenotype ‘GPUK3’. However, in this study, addition of0.5 mg L−1 Kin to MS medium containing 4.0 mg L−1 2,4-Dincreased the percentage of embryogenic callus formationusing SAMs. Latha et al. (2005) also used 2,4-D and Kin forembryogenesis in 20 genotypes of finger millet, while Ceasarand Ignacimuthu (2008) obtained somatic embryos in eightgenotypes of finger millet when combining Kin or zeatin inMS medium.

Fig. 5 Scanning electronmicrographs of the developingcallus of finger millet genotypes.a Non-embryogenic callusshowing thread-like structures ingenotype ‘CO(Ra)-14’ (bar=500 μm). bGlobular pro-embryo-like structures formed on MSmedium containing 40 % ofG. edulis extract and 20 g L−1

sucrose after 8 weeks ofincubation (bar=300 μm). cGlobular- and heart-shapedsomatic embryos in genotype‘PR-202’ (bar=100 μm). dTorpedo and globular embryossowing in genotype ‘Try-1’ after8 weeks of incubation (bar=300 μm)

J Appl Phycol

Seaweeds and their extracts are a source of macro- andmicroelements, various kinds of plant growth regulators, ami-no acids, and organic acids (Khan et al. 2011; Hernandez-Herrera et al. 2014; Vinoth et al. 2014; Satish et al. 2015b).In the present study, an increased percentage of SLEs fromG. edulis and P. boergesenii had a significant effect on seedgermination, maximum fresh weight of the seedlings, healthyshoot and root induction and their development, and highestfrequency of rooting with more number of roots, and it alsoenhanced plant height in all four genotypes of finger millet.Our results are consistent with previous reports in Hordeumvulgare (Steveni et al. 1992), Triticum aestivum (Kumar andSahoo 2011), Vigna radiata, and Zea mays (Rengasamy et al.2015). Lower concentration (20 and 40 %) of the SLEs gave a

better response in the bioassays, and beyond 60 % of SLEsshowed a significant harmful outcome on the germination ofseeds and growth of shoots and roots in all four genotypesof finger millet tested. Similar results were observed inLycopersicon esculentum (Kumari et al. 2011; Hernandez-Herrera et al. 2014) and in Solanum melongena (Satish et al.2015b) with treatments of seaweed extracts. Recently,Rengasamy et al. (2015) reported a high frequency of sem-inal rooting in maize seedlings with the application of eckol,a new plant growth stimulant isolated from the brown sea-weed, Ecklonia maxima.

The influence of genotypes on in vitro morphogenesis hasbeen described in cereals and millets (Ceasar and Ignacimuthu2008). It is important to screen various genotypes for their

Fig. 6 Plant regeneration androoting frommature embryogeniccallus of finger millet genotypes.a, b Green shoot bud initiation ofembryogenic callus on MSmedium amended with 50 % ofP. boergesenii extract after6 weeks of incubation in the lightin genotype ‘PR-202’(bar=1.8 mm). c Regeneratedshoots from cultures of genotype‘CO(Ra)-14’ after 12 weeks ofincubation in the light on MSmedium supplemented with 50 %P. boergesenii extract (bar=4.0 mm). d Rooting of elongatedshoots in genotype ‘Hosur-1’ onhalf-strength MS mediumamended with 60 %G. edulis extract after 6 weeksof incubation in the light(bar=14 mm). e Hardened andin vitro regenerated finger milletplants in soil cups (bar=14 mm).f Seed setting of in vitroregenerated genotype ‘CO(Ra)-14’ in the field (bar=5.0 mm)

J Appl Phycol

response in culture since it allows the identification of geno-types which are further helpful for biotechnological ap-proaches. In this report, four genotypes of finger millet werestudied for their ability to undergo in vitro embryogenesis andsubsequent plant regeneration. All genotypes studied wereproficient in undergoing embryogenic callus formation, so-matic embryogenesis, and plant regeneration under the cultureconditions established. However, the percentage of morpho-genic response was found to be genotype dependent and the‘PR-202’ genotype remained the most responsive in all theexperiments.

Previously, phytohormones have been identified inCaulerpa scalpelliformis seaweed extract that induced thein vitro embryogenesis from leaf explants of tomato (Vinothet al. 2014). In the present study, medium supplemented withliquid extracts ofG. edulis or P. boergesenii increased somaticembryogenesis in all four genotypes of finger millet. The low-er concentrations (≤10 %) of both SLEs induced friable callusin all finger millet genotypes tested. In another experiment,transfer of embryogenic callus on MS medium containing40 % of G. edulis extract in addition to modified sucrose

concentration (30 g L−1) produced the highest frequency ofsomatic embryogenesis with a higher number of heart andtorpedo shape embryos when compared to lower concentra-tions of sucrose (≤30 g L−1). Carbohydrate has been found tobe essential for somatic embryo induction and sugars alsofunction as both a carbon source and as an osmotic regulatorin many plant species (Ceasar and Ignacimuthu 2010).Kamada et al. (1989) previously reported that supplementa-tion of sucrose in the culture medium affects the developmentof somatic embryos in carrot.

SEM was used to evaluate the effects of G. edulis orP. boergesenii liquid extracts on the cellular origins of matureembryos during somatic embryogenesis in four genotypes offinger millet. Previously, SEM analyses has been used to il-lustrate the sequential changes of callus explants in japonicarice (Nakamura and Maeda 1989) and also to confirm early

Table 7 Effect of various concentrations and types of seaweed liquidextracts on the conversion of embryogenic callus into plantlets

SLEs (%) (w/v) Finger millet genotypes

CO(Ra)-14 Hosur-1 PR-202 Try-1

Gracilaria edulis Percentage of regeneration (mean±SE)

25 79.6±0.4d 66.8±0.5e 82.5±0.4e 76.3±0.4f

50 91.5±0.5g 78.2±0.3g 96.3±0.2g 84.1±0.2g

75 86.2±0.3f 57.7±0.2c 73.1±0.1c 68.5±0.2d

Padina boergesenii

25 71.3±0.2c 62.8±0.2d 79.1±0.3d 64.6±0.7c

50 80.6±0.3e 69.2±0.1f 88.6±0.2f 72.4±0.4e

75 63.6±0.5b 53.6±0.3b 68.7±0.4b 59.5±0.3b

Control 48.2±0.6a 31.1±0.3a 53.6±0.5a 37.7±0.4a

Gracilaria edulis Mean length of regenerated shoots (cm) (mean±SE)

25 11.4±0.4f 6.3±0.5e 15.7±0.3e 7.3±0.2d

50 13.2±0.2g 9.8±0.1f 20.7±0.4g 11.4±0.1f

75 8.5±0.2d 3.3±0.3c 14.0±0.5d 5.6±0.3c

Padina boergesenii

25 6.2±0.2b 2.5±0.2b 12.2±0.3c 3.2±0.2b

50 10.6±0.4e 4.7±0.3d 16.8±0.2f 8.5±0.4e

75 4.4±0.3a 1.7±0.4a 9.4±0.4b 2.3±0.2a

Control 6.7±0.2c 4.8±0.2d 8.2±0.3a 5.9±0.1c

Data collected after 12 weeks of dark incubation in the medium contain-ing various concentrations of SLEs studied for regeneration embryogeniccallus. MS medium devoid of SLEs have been used as control. Embryo-genic calluses were cultured for 6–12 weeks on MS medium amendedwith different SLEs along with control experiments. Values represent themeans±SE of three replicates (analyzed by SPSS version 17.0). Meansfollowed by the same letter in each column are significantly different at P≤0.05 based on Duncan’s multiple range test

Table 8 Effect of seaweed liquid extracts on rooting of elongatedshoots in four finger millet genotypes after 6 weeks of incubation in thelight

SLEs (%) (w/v) CO(Ra)-14 Hosur-1 PR-202 Try-1

Gracilaria edulis Percentage of root induction (mean±SE)

20 68.7±0.4f 53.7±0.3f 75.4±0.3f 55.7±0.4e

40 76.6±0.5g 61.4±0.4h 88.3±0.2h 68.5±0.5h

60 87.5±0.6i 69.8±0.3i 97.4±0.3i 76.2±0.6i

80 56.6±0.7c 47.5±0.8e 66.7±0.2d 51.7±0.3d

Padina boergesenii

20 64.4±0.3e 40.4±0.5c 62.6±0.4c 43.7±0.7b

40 61.5±0.5d 44.5±0.6d 70.2±0.2e 58.6±0.4f

60 78.6±0.7h 58.8±0.9g 84.3±0.3g 62.4±0.3g

80 52.3±0.3b 36.1±0.7b 57.4±0.1b 47.1±0.2c

Half-strengthMS alone

31.7±0.6a 21.4±1.1a 43.4±0.3a 29.3±0.6a

Gracilaria edulis Length of roots (cm) (mean±SE)

20 5.2±0.5c 3.7±0.4de 8.9±0.4e 4.3±0.2d

40 7.7±0.4f 4.2±0.1e 9.7±0.1fg 6.2±0.3f

60 9.0±0.4g 5.9±0.2f 11.8±0.2h 7.6±0.3g

80 6.3±0.1d 2.8±0.2c 7.5±0.3c 5.4±0.4e

Padina boergesenii

20 4.9±0.2c 2.4±0.3c 8.1±0.2d 2.8±0.3c

40 6.5±0.3de 3.1±0.2cd 9.3±0.3ef 3.7±0.6d

60 7.1±0.6ef 4.0±0.9e 10.1±0.2g 5.8±0.4ef

80 3.8±0.2b 1.6±0.5b 6.6±0.3b 1.9±0.5b

Half-strengthMS alone

1.9±0.2a 0.4±0.3a 2.6±0.2a 0.8±0.1a

Shoots regenerated (3–4 cm long) on MS medium containing SLEs wereinoculated onto half-strength MS medium with SLEs. For each experi-ment, the number of roots produced by a single shoot was determinedafter 6 weeks of incubation in the light. Twenty-five explants for eachcontrol and SLEs treated experiments in all genotypes with three repli-cates were used. Means followed by the same letter in each column aresignificantly different at P ≤0.05 according to Duncan’s multiple rangetest

J Appl Phycol

stages of somatic embryos, unorganized structures, andsmooth structures in switchgrass (Burris et al. 2009). SEManalysis of all four finger millet embryogenic callus showedglobular, heart, and torpedo somatic embryos.

Plant regeneration and rooting was achievedwhen embryo-genic callus was sub-cultured ontoMSmedium amended withboth types of SLEs individually. Vinoth et al. (2014) showedthat seaweed extracts containing 6-benzylaminopurine andindole-3-butyric acid induced plant growth, regeneration,and development of L. esculentum. Two different compounds,phloroglucinol and eckol, have been found in E. maximawhich stimulated the growth and development in mung beanand maize seedlings (Rengasamy et al. 2015). In the presentstudy, supplementation of SLEs of G. edulis or P. boergeseniiat lower concentrations in MS medium improved the matura-tion of embryogenic calli, regeneration frequency, androoting, similar to previous reports on vegetables (Vinothet al. 2014; Satish et al. 2015b). Initially, embryogenic calluscultured on MS medium containing 20 % of both SLEs initi-ated the formation of roots rather than shoots. This is consis-tent with Vinoth et al. (2014) where G. corticata extract alsoproduced roots rather than shoots. This is the first report andsubstantial method to make use of SLEs in the embryogenesisand in vitro plant regeneration in four genotypes of fingermillet. In summary, the use of SLEs for micropropagation offinger millet is believed as a safe method, easy to extract, andsuitable for obtaining quick and high-frequency embryogene-sis, mass propagation, and rooting. Also, application of SLEsimproved the percentage of seed germination, fresh weight ofseedlings, and shoot or root length and number, as found inother seaweed extracts (Finnie and Van Staden 1985; Vinothet al. 2014; Rengasamy et al. 2015). This positive outcome ofSLEs represents a suitable approach to decrease the use ofenvironmentally hazardous phytohormones such as 2,4-D ortheir derivatives in tissue culture protocols for callus induction.

In conclusion, simple and high-frequency embryogeniccallus induction, somatic embryogenesis, and regenerationof four finger millet genotypes using SLEs has been success-fully established. SLEs of G. edulis and P. boergesenii influ-enced the development of mature embryos and conversion ofplantlets in all four finger millet genotypes tested. High fre-quency of mature embryo regeneration, multiple shoot induc-tion, and rooting was optimized using these two differenttypes of SLEs. This study shows that a small quantity ofSLEs can be developed as an alternative to commerciallyavailable phytohormones to use in MS media to improvein vitro regeneration of finger millet or other millet crop spe-cies. Other than synthetic hormones, SLEs contain plantgrowth stimulants, which enhance the in vitro growth of theplantlets. The use of SLEs for rapid plant regeneration is con-sidered as a convenient, environment-friendly, and safe meth-od, thus making this system possibly helpful for in vitro re-generation as well as genetic improvement of millet species.

Acknowledgments The author L. Satish sincerely thanks the Univer-sity Grants Commission, New Delhi, India for financial support in theform of UGC-BSR SRF. We thank the Department of Small Millets,Millet Research Station, Tamil Nadu Agricultural University, Coimba-tore, India for providing seed material used in this study. The authors alsogratefully acknowledge the Bioinformatics Infrastructure Facility ofAlagappa University (funded by Department of Biotechnology, Govern-ment of India, grant no. BT/BI/25/001/2006) for providing the computa-tional facility. The first author gratefully thanks all the three reviewers fortheir valuable comments to improve this manuscript.

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