Direct regeneration, microshoot recovery and assessment of genetic fidelity in Helicteres isora L., a medicinally important tree

Journal Pre-proof Direct regeneration, microshoot recovery and assessment of genetic fidelity in Helicteres isora L., a medicinally important tree Mariappan Muthukumar, Selvaraj Muthukrishnan, Thiruppathi Senthil Kumar, Mandali Venkateswara Rao PII:

S1878-8181(19)31050-3

DOI:

https://doi.org/10.1016/j.bcab.2019.101415

Reference:

BCAB 101415

To appear in:

Biocatalysis and Agricultural Biotechnology

Received Date: 21 August 2019 Revised Date:

25 October 2019

Accepted Date: 31 October 2019

Please cite this article as: Muthukumar, M., Muthukrishnan, S., Kumar, T.S., Rao, M.V., Direct regeneration, microshoot recovery and assessment of genetic fidelity in Helicteres isora L., a medicinally important tree, Biocatalysis and Agricultural Biotechnology (2019), doi: https://doi.org/10.1016/ j.bcab.2019.101415. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Direct regeneration, microshoot recovery and assessment of genetic fidelity in Helicteres isora L., a

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medicinally important tree

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Mariappan Muthukumar1, Selvaraj Muthukrishnan1,2, Thiruppathi Senthil Kumar1*, Mandali

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Venkateswara Rao1.

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1- Department of Botany, Bharathidasan University, Tiruchirappalli, Tamil Nadu– 620024, India

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2- Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu– 626124, India

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E-mail: [email protected]; telephone: +919442156480; telefax: 0431- 2407045

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Abstract

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Helicteres isora L., is an endangered valuable medicinal tree species. Direct shoot regeneration was initiated

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from cotyledonary node (CN) and axillary nodal explants (AN) of in vitro raised seedlings using the plant

11

growth regulators (PGRs) and organic additives. A combination of 4.92 µM N6- (2- isopentenyl) adenine

12

(2iP), 9.08 µM thidiazuron (TDZ) and 2.69 µM naphthaleneacetic acid (NAA) in Murashige and Skoog (MS)

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medium resulted with 16.1 and 11.4 shoots from CN and AN explants respectively. Maximum shoot numbers

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(CN: 21.3; AN: 16.9) were recorded by addition of 342.1 µM glutamine with 4.92 µM 2iP, 9.08 µM TDZ and

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2.69 µM NAA combination. Basal callus associated shoot regeneration problems were successfully overcome

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through altering sucrose concentration (20 g l-1) in culturing media. Pre-treated microshoots were elongated

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(10.9 and 9.8 cm) when cultured on half-strength MS medium fortified with a combination of 4.92 µM 2iP and

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1.44 µM gibberellic acid (GA3). Elongated shoots were best rooted (12.9 per shoot), in half-strength MS

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medium containing 4.90 µM indole-3-butyric acid (IBA). Plantlets were successfully acclimatised (71.4 %) in

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poly tunnel chamber. Genetic homogeneity was successfully confirmed by SCoT and ISSR marker systems. In

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both techniques 63 and 27 monomorphic bands were resolved from SCoT and ISSR primers respectively.

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Developed micropropagation protocol could be a reliable method for clonal propagation of H. isora.

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Keywords Helicteres isora. Microshoot. Basal callus. Genetic fidelity. SCoT. ISSR.

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1 Introduction

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Helicteres isora L. (Sterculiaceae) is commonly known as East Indian screw tree and widely distributed

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throughout India in dry deciduous forests up to 1500 m altitude on hill slopes. It is a medicinally important

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multipurpose sub deciduous shrub or a small tree. Various plant parts of H. isora possess pharmacological

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activities

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antispasmodic and to treat snake bite, dog bite, diarrhoea and blood purification from ancient days to till date.

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Also the plant has various bioactive compounds, such as cucurbitacin- B, isocucurbitacin- B, diosgenin,

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daucosterol, hibifolin, trifolin, rosmarinic acids and many from different parts of plant (Sabale et al., 2012;

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Kumar and Singh, 2014). Hard seed coat leads to seed dormancy (Badave and Jadhav, 2014) and inadequate

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availability of natural pollinators resulted in reduced fruit production in H. isora (Atluri et al., 2000). Further,

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indiscriminate harvesting of plants from wild for its purported medicinal uses have indeed become a serious

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threat to the survival of H. isora, moreover becomes endangered and possibly leading to the extinction

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(Kumar, 2005; Bagul and Yadhav, 2007). Hence it is necessary to develop in vitro propagation protocol for H.

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isora for mass clonal propagation, conservation and production of secondary metabolites to examine the

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pharmacological activity as well as to confirm traditional medicinal usage.

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There are few earlier reports of in vitro shoot regeneration from nodal explants and shoot tip explants derived

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from both seedling and field grown trees. Direct regeneration and indirect organogenesis of H. isora has been

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reported by various groups with variety of explants. Shriram et al. (2007) first initiated direct regeneration of

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nodal explants procured from 25 days old seedlings and recorded about 79 % response with 7.1 shoots using 6-

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Benzylaminopurine (BA) and Kinetin (KN) combination. Subsequently, Shriram et al. (2008) reported indirect

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organogenesis using seedling derived nodal explants, at different concentrations of BA and KN combination

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helped to raise callus as well as adventitious shoot regeneration with 67 % response and 3.2 average shoot per

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0.5 g callus. Chawla and Bansal (2014) also reported direct regeneration with seedling derived axillary bud

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explant, which had responded to BA, KN and Silver nitrate (AgNO3) (94 % response; 4.14 shoots per explant).

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However, George et al. (2007) tried with shoot tip explant of field grown tree and reported only response rate

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of 100 % without mentioning shoot numbers using BA and KN separately. Moreover, no successful high

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throughput micropropagation protocol has been reported till date in H. isora.

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Micropropagation is an alternative method for clonal propagation of medicinally important plant species

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(George et al., 2007). Various degrees of difficulty can be encountered during the in vitro culturing of hard

including

antidiabetic

(hypoglycaemic

and

2

hyperglycaemic),

anticancer,

antimicrobial,

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wood species like H. isora, as it is non-responsive to tissue culture. Recalcitrance effect of low shoot initiation,

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basal callus formation and subsequent inhibition of shoot multiplication remain major bottlenecks to in vitro

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manipulation for the micropropagation of woody plant species (Moyo et al., 2011). Likewise, donor plant’s

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physiology, phenolic exudation, explant browning and subsequent coloration of culture media are major causes

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for in vitro culturing system (Benson, 2000; Huang et al., 2002). Supplementation of suitable PGRs at optimal

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levels with balanced media components and proper environmental provisions reduces the risks mentioned

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above. Complex combinations of plant growth regulators (PGRs) and organic additives will induce multiple

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shoots in short period of time. However, in mass multiplication genetic stability of micropropagated plants are

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really matters. Thus the obvious choice of explants could be meristematic tissue rich cotyledonary node (CN)

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and axillary node explants (AN) for the clonal propagation of H. isora.

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In vitro regeneration and its associated genetic variation has been reported by many. But it causes limitation as

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well as beneficial based upon the experimental utility. Earlier reports have been indicated that somaclonal

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variants appear depends upon variety of factors like explant type, PGRs, regeneration method, repeated sub-

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culturing and genotype (Goto et al., 1998; Martins et al., 2004). Hence, the genetic fidelity analysis is essential

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to ascertain the true-to-type production of in vitro generated propagules. Direct regeneration of tree or woody

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plant species via, germinated seedlings is quite easy due to adequate availability and extensive regeneration

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capacity. The seedlings of outcrossed plant species are genetically heterogeneous or heterozygous and might

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not retain the desirable characteristics of the donor plant, thus limiting the use for micropropagation

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(Bramhanapalli et al., 2016). However, open pollinated seeds also producing true to true type of regenerants

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through in vitro culture.

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Genetic variation is analysed using different genetic marker systems such as Start Codon Targeted

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polymorphism (SCoT) and Inter Simple Sequence Repeats (ISSR). SCoT is a highly reproducible marker

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system, it targets the sequences either derived from gene itself or its immediate flanking sequences (Collard

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and Mackill, 2009), whereas ISSR markers targeted the sequences in non-coding regions of palindromic

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microsatellite DNA, also produces medium to highly reproducible band patterns (Zietkiewicz et al., 1994;

78

Agarwal et al., 2015). The main objective of the present study is to develop a simple and efficient protocol for

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mass propagation of H. isora for clonal propagation and to determine the genetic fidelity of regenerated plants

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compared with mother plants.

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2 Materials and Methods

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2.1 Plant collection and explant selection

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Helicteres isora juvenile twigs and fruits were collected from Kolli hills on Western Ghats of Tamil Nadu,

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India, and plants were identified (Specimen No.177312) by Botanical survey of India, Coimbatore. Stem

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explants were collected in wet polythene bag and then kept in ice box for 3 h. Thereafter the in vivo AN

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explants were washed under running tap water for 10 min to remove debris. The explants were decontaminated

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with commercial TeepolTM (Sigma- Aldrich, India) for 2- 3 min followed by ethanol for 30 sec, 3% NaOHCl

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for 1 min and 0.1% (w/v) HgCl2 for 5 min. The explants were washed five times with sterile distilled water.

89

In addition, in vitro germinated seedlings were also used as source of explants. Seeds were germinated as per

90

procedure of (Muthukumar et al., 2016, 2017). Germinated seeds were transferred from sterile wet cotton bed

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to half-strength MS (Murashige and Skoog, 1962) medium with 20 g l-1 sucrose for seedling development.

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Cotyledonary nodes and axillary nodes were excised out from one month old seedlings and used as explants.

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2.2 Shoot induction and multiplication

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In vivo AN explants and in vitro seedling derived CN and AN explants were cultured on MS medium (2 %

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sucrose) supplemented with various concentrations of cytokinins [BA, KN, N6- (2- isopentenyl) adenine (2iP),

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thidiazuron (TDZ)] and auxins [indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA) and IBA], either

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individually or in combination for shoot induction and multiplication. Combination of 4.92 µM 2iP, 9.08 µM

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TDZ and 2.69 µM NAA with different concentrations of organic additives viz., polyamines (Putrescine and

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Spermidine), amino acids (glutamine and proline), Adenine sulphate and sodium citrate were tested for further

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enhancement of shoot multiplication. The MS medium devoid of plant growth regulators and organic additives

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were used as a control.

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2.3 Microshoot recovery, shoot elongation and rooting

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Microshoots (≤1 cm) obtained from shoot multiplication treatments were further transferred to shoot recovery

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medium, half-strength MS basal medium containing 2.0 % sucrose for a period of two week time. Recovered

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shoots (2- 3 cm with intact leaves) were inoculated into elongation medium containing half-strength MS and

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full-strength MS media supplemented with 2iP (2.46- 9.84 µM) alone or in combination with gibberellic acid

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(GA3) (0.29- 5.77 µM). Elongated shoots (> 5- 6 cm) were transferred to half-strength MS medium containing

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auxins (IAA and IBA) and activated charcoal (83.26- 832.63 µM).

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2.4 Acclimatisation

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Regenerated plantlets were transferred to sterilised paper cups (6 x 8 cm) containing sterile red soil: sand:

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compost (120 g/cup) (1:1:1 v/v) and then irrigated with half-strength MS without sucrose for 2 weeks,

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followed by regular watering two days interval. Initially paper cups were covered with transparent polythene

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bags to avoid immediate desiccation of in vitro cultured plants and maintained in culture room, where the

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cultures were maintained at 25 ± 2˚C, under a 16 hrs photoperiod with a photosynthetic photon flux density of

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35 µmol m-2s-1 from cool white fluorescent tubes (Philips, India) with 55- 60% relative humidity. Gradually

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the humidity was reduced to transfer the plants into pots (10 x 16 cm) containing 2:1:1 (v/v) mixture of red

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soil, sand and manure (500 g/pot) for 30 days. Then the plants were maintained in a poly tunnel chamber

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(Saveer Biotech, New Delhi), where the plantlets were maintained at 28 ± 3˚C with 35 ± 5% relative humidity

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and exposed to natural photoperiod.

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2.5 Genomic DNA extraction

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Randomly selected two months-old seven acclimatised plants and mother plants were selected for genetic

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fidelity studies. Leaves (100 mg) were ground in liquid nitrogen using a pestle and mortar, and genomic DNA

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was extracted using HiPurA plant genomic DNA miniprep purification kit (MB507- Himedia). Isolated DNA’s

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quantity and purity were analysed using a Biophotometer (Eppendrof BioPhotometer Plus, Germany).

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2.6 SCoT analysis

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A total of 17 SCoT primers [17] were selected to analyse the genetic fidelity between acclimatised tissue

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cultured plants and mother plants. Amplification was performed in a 20 µl reaction mixture containing 4.0 µl

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of template DNA (50 ng/µl), 10 µl of 2X master mix [Tris-HCl pH 8.5, (NH4)2S04, 4 mM MgCl2, 0.2% Tween

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20, 0.4 mM dNTPs, 0.2 units/µl Taq DNA polymerase, Inert red dye and stabilizer] (Ampliqon, Denmark), 3

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µl of SCoT primer (GeNeiTM, Bangalore, India) and 3 µl of sterile distilled water. In a thermal cycler

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(Eppendrof AG, Germany), the PCR programme was conducted with initial denaturation for 3 min at 94⁰C,

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then 35 cycles of 1 min denaturation at 94⁰C, 1 min annealing at annealing temperature (Ta) and 2 min

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extension at 72⁰C with a final extension at 72⁰C for 5 min. The PCR products were resolved on 1.2 % Agarose

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gel with 1 X TAE buffer and stained with ethidium bromide at 50 V for 3 h. Gels were visualised and photo

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captured using gel documentation system (Alpha Imager EP).

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2.7 ISSR analysis

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Ten ISSR primers [Department of Biotecnology laboratory, University of British Colombia (UBC Set 09] were

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used to perform ISSR analysis. Amplification was performed in a 20 µl reaction mixture containing 4.0 µl of

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template DNA (50 ng/µl), 10 µl of 2X master mix [Tris-HCl pH 8.5, (NH4)2S04, 4 mM MgCl2, 0.2% Tween

140

20, 0.4 mM dNTPs, 0.2 units/µl Taq DNA polymerase, Inert red dye and stabilizer] (Ampliqon, Denmark), 3

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µl of ISSR primer (GeNeiTM, Bangalore, India) and 3 µl of sterile distilled water. In a thermal cycler

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(Eppendrof AG, Germany) the PCR programmed as follows; initial denaturation for 5 min at 94⁰C, then 45

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cycles of 45 sec denaturation at 94⁰C, 45 sec annealing at annealing temperature (Ta) and 1.5 min extension at

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72⁰C with a final extension at 72⁰C for 7 min. The PCR products were resolved on 1.2 % Agarose gel with 1

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X TAE buffer and stained with ethidium bromide at 50 V for 3 h. Gels were visualised and photo captured

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using gel documentation system (Alpha Imager EP).

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2.8 Data analysis

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2.8.1 Plant tissue culture experiments

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Minimum seven replicates were used per experiment and all the experiments were repeated thrice. The

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experimental design was random and factorial. Data were recorded for shoot and root regeneration after 40

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days of culture. The data were subjected to mean and mean separation analysis by using Duncan’s Multiple

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Range Test (DMRT) (IBM SPSS Statistics 23.0, Armonk, NY).

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2.8.2 Genetic fidelity

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Minimum seven replicates of in vitro regenerants [derived from both CN (T1- T4) and AN (T5- T7) explants)

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and one sample of mother plant (M) were utilised per experiment and all the experiments were repeated thrice.

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SCoT and ISSR marker-derived gel images were manually compiled to binary matrix, based on the presence

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(1) and absence (0) of particular selected band. Moreover, the reproducible and clear bands were selected,

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while ambiguous, weak and smeared bands all neglected for data scoring. Similarity index between mother

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plant and each individual in vitro raised plants were analysed using Jacarrd's coefficient using the following

160

formula: Jab = Nc/Na + Nb + Nc, where Na is the number of elements positive in set А and negative to set B, Nb

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is the number of elements in positive in set B negative to set A and Nc is the number of elements positive in

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intersecting set.

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3 Results

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3.1 Explant selection and culture establishment

6

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Axillary node explants collected from in vivo plants were failed to induce shoots due to necrosis at preliminary

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cytokinin treatments (data not shown). Seedlings from matured seeds were successfully germinated using acid

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scarification and successive imbibition and germination at sterile wet cotton bed. Then the germinated

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seedlings were transferred to half-strength MS medium for further development. One month old seedling

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consists CN and 2- 3 AN were excised and used as explants for further experiments. In vitro raised seedlings

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derived CN and AN explants were found more responsive for shoot induction than field grown plant materials.

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3.2 Shoot induction and multiplication

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CN and AN were cultured on MS basal medium (control) remained green and produced least number of

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shoots, however the explants treated with plant growth regulators induced multiple shoots at varying numbers

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(see supplementary materials). Among the four cytokinins tested, 4.92 µM 2iP produced significant number of

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shoots (6.0 shoots per CN and 4.7 shoots per AN) with responsive rate of 66.67 % and 61.90 % from CN and

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AN explants respectively. TDZ treatment was also responded equally or little less with 9.08 µM concentration

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by producing 61.90 % and 52.38 % response with 5.29 and 4.57 shoots in both CN and AN explants

178

respectively. Over all in all PGRs of individual cytokinin treatment responded 19- 66 % and 14- 61 %

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frequency of response in CN and AN explants respectively. Between the different explant types, CN exhibited

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better frequency of response and shoot numbers than other explant, AN in 4.92 µM 2iP.

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To enhance shoot multiplication and proliferation, series of experiments were performed, where the explants

182

were cultured on MS medium containing 4.92 µM 2iP with other cytokinins at different concentrations (2- 14

183

µM) alone or in combination with different auxins (0.4- 12 µM). Combination between these cytokinins quite

184

enhanced the frequency of response and shoot numbers in all treatments. Combination of 4.92 µM 2iP and

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9.08 µM TDZ was produced 11.3 shoots per CN (Figure 1a) and 8.1 shoots per AN (Figure 1c). CN and AN

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explants topped with 95.2 % response in 4.92 µM 2iP and 9.08 µM TDZ combination. This combination (4.92

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µM 2iP and 9.08 µM TDZ) had been further tested with individual auxins, whereas NAA inclusion in medium

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enhanced the shoot numbers in both the explants. Increase in concentration of NAA in medium decrease the

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frequency of response and shoot numbers, whereas lower level concentration were found productive in both

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response and shoot multiplication. Other auxin combinations were not practicable for shoot proliferation in

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both explants (see supplementary materials). Combination of 4.92 µM 2iP, 9.08 µM TDZ and 2.69 µM NAA

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supplemented in MS medium produced high frequency of response (100 %) and number of shoots [16.1 shoots

193

per CN and 11.4 shoots per AN].

7

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To obtain higher shoot multiplication various organic additives were included in MS medium fortified with

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best combination of PGRs (2iP, TDZ and NAA). Among the different additives tested, L- glutamine 342.1

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µM produced the maximum shoot number in the present study. L- Glutamine (342.1 µM) supplemented in MS

197

medium fortified with the following PGRs combination (4.92 µM 2iP, 9.08 µM TDZ and 2.69 µM NAA)

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produced significantly the highest number of shoots (21.3) in CN explant (Figure 1b) and the same

199

combination also recorded shoot promoting effect in AN explant with 16.9 shoots (Figure 1d; Table 1). Other

200

organic additives like spermidine, putrescine, proline and adenine sulphate were reduced the efficiency of

201

frequency and shoot multiplication. Whereas sodium citrate exhibited neither reduction nor increment in

202

response of both explants, but the produced shoots were stayed intact after repeated sub-culturing.

203

3.3 Microshoot recovery and shoot elongation

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Basal callus was observed frequently in higher concentration of sucrose (3.0%) however, 2.0% of sucrose

205

induced lower basal callus (see supplementary materials). Over the period of time and successive subculture

206

basal callus reached up to regenerated shoots and hindered further proliferation and then enhance the existing

207

shoots to induce callus. In addition higher concentration of TDZ rather responded with high shoot

208

multiplication rate than employing shoot elongation, which resulted much shunted shoots were produced. To

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overcome callus induction and indefinite morphology of the microshoots (≤ 0.3- 0.5 cm), they were transferred

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to recovery medium (Figure 1e). About 60-70 % of the microshoots were recovered to normal (2-3 cm) on

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half-strength MS medium devoid of any growth regulators or additives (Figure 1f).

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Despite the microshoots recovery into definite shoot morphology, the shoot elongation was failed in this phase

213

of study. Hence the different basal media like half-strength and full strength MS media were used either as

214

such or fortified with PGRs like 2iP and GA3. Basal media or 2iP combination not likely to elongate the shoots

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in both half-strength and full strength MS media, whereas inclusion of GA3 enhanced the quick elongation

216

performance in the recovered shoots. Over all half-strength MS medium supplemented with 4.92 µM 2iP and

217

1.44 µM GA3 promoted higher shoot length (10.9 cm for CN and 9.8 cm for AN based shoots) (Figure 1g;

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Table 2).

219

3.4 Rooting and acclimatisation

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Earlier in elongation experiments, regenerated shoots tend to produce rooting in control as well as PGRs

221

included media. However poor response were noticed for rooting, which needs further improvement.

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Elongated shoots of different explant origin exhibited no marked difference in rooting, hence the experiment

8

223

results were compiled together. Half-strength MS medium fortified with IBA and IAA promoted rooting

224

response, whereas other auxins had not found helpful. However, half-strength MS medium fortified with 4.90

225

µM IBA produced higher response rate (100%), root number (12.9), root length (6.1 cm) and secondary roots

226

(158.6) (Figure 1h & i; Table 3). IBA was found efficient, though the IAA produced normal averaged lengthy

227

shoots with reduced frequency of response. Activated charcoal was supplemented to enhance the rooting,

228

whereas its addition improved secondary root numbers (196.9), however root number and root length were not

229

significantly different from treatment of IBA (Table 3).

230

Rooted plantlets were cautiously transferred to paper cups without damaging to roots and maintained in culture

231

room for 4-6 weeks. The healthy plantlets were acclimatised for living room condition then translocated to

232

poly tunnel chamber for another 4-6 weeks. In vitro regenerated plantlets rejuvenated new leaves after

233

withered existing leaves and new branching to grow further (Figure 1j). Acclimatised plants were grown

234

normally and to reach the height of above 25 cm in 5-6 weeks with survival rate of 71.4 %.

235

3.5 Genetic Fidelity

236

Purified genomic DNA was successfully isolated and spectral readings suggested that the samples were pure

237

enough to perform the PCR reactions, then the working concentration of samples were adjusted to 50 ng/µl.

238

All the gel images were thoroughly analysed to identify the bands presence or absence manually. Reproducible

239

unambiguous bands were scored, whereas smeared and weak bands were neglected. In the present study, SCoT

240

and ISSR based molecular markers were employed to evaluate the genetic homogeneity between in vitro and

241

in vivo plants of H. isora.

242

Unique band pattern was observed between all 13 primers out of the 17 primers tried, products size ranges

243

from 0.3 to 3.0 kb. Number of scorable bands from each primer varies from 3 (SCoT06) to 7 (SCoT12 and

244

SCoT16). These 13 primers produced total number of 63 bands with average of 4.8 bands per primer (Table 4).

245

There were no polymorphic bands observed in any of the primer products with respective to in vivo and in

246

vitro plants. Monomorphic band pattern of SCoT11 and SCoT12 were showed in Figure 2a, b respectively.

247

Five ISSR primers produced unique band pattern out of 10 primers. Five ISSR primers produced a total of 27

248

reproducible bands, individual primer bands range between 2 (UBC828) to 8 (UBC810) with the average of

249

5.4 bands per primer. Its size varies from 0.3- 2.2 kb (Table 4). All the 27 resolved bands were monomorphic

250

to all 7 in vitro raised plants as well in vivo based mother plant. No polymorphic bands were observed, after

9

251

repeating the experiment thrice. ISSR amplification pattern of primers UBC810 and UBC864 were shown in

252

Figure 2c, d respectively.

253

The 17 SCoT primers amplified a total of 504 bands across all the 8 samples tested. Likewise, 5 ISSR primers

254

generated 216 bands from all the plant samples. Jaccard’s coefficient was indexed with both SCoT and ISSR

255

band scoring, which revealed that the high similarity index (1) was observed among the regenerants and

256

mother plant. All the samples were produced monomorphic band patterns irrespective to any marker system,

257

this suggests that the micropropagated plantlets were true-to-type nature.

258

4 Discussion

259

4.1 Explant selection and culture establishment

260

In vitro response of woody plant species is comparatively less successful due to various factors such as age,

261

season and explant types (Purohit and Kukda, 2004). In the present study field grown plants based explants

262

were found to be recalcitrant towards in vitro manipulation. Hence the in vitro derived seedlings were used as

263

explant source. Acid scarification treatment successfully germinated the seeds for many hard coated seeds as

264

well as for H. isora (Muthukumar et al., 2016). In vitro derived explants had been successfully used in

265

previous studies of H. isora and other woody species (Badave and Jadhav, 1998; Panda and Hazra, 2010).

266

Explant selection is important task before initiating the experiment. Because suitable explants are essential for

267

successful establishment of the culture. Likewise, in the present study in vitro seedlings derived CN and AN

268

explants were responded well and CN explant was found to be superior in response compared to AN explant.

269

Whereas, all the reports of previous experiments of H. isora on direct regeneration was either carried with

270

nodal explant or shoot tip explants. This is the first report that utilised the CN as suitable explant for direct

271

regeneration studies.

272

4.2 Shoot induction and multiplication

273

Multiple shoots were initiated at all the cytokinins used, but the frequency of response and number of shoot

274

numbers varied (14- 66 %; 1- 6). Cytokinins like 2iP and TDZ performed well compared to other cytokinins

275

used in this study. Though the response levels were slightly varied, both the results were statistically come

276

under same group between 4.92 µM 2iP and 9.08 µM TDZ treatments. All the previous reports were

277

performed with BA and KN either alone or in combination, in the present study we had used 2iP and TDZ for

278

the first time in H. isora regeneration. Both these PGRs are strong cytokinins has been well reported for its

10

279

shoot promotory effect (Huetteman and Precce, 1993; Jaakola et al., 2001). When series of experiments

280

conducted to enhance the shoot induction property, different combination of PGRs were tested. TDZ and 2iP

281

combination quite enhanced the multiple shoot regeneration capacity from 60- 66 % to 95 % in both explants.

282

Similarly 2iP and TDZ supplementation in culture medium enhanced the adventitious shoot proliferation in

283

Pongamia pinnata (Sujatha et al., 2008). Maximum frequency of response rate with high shoot numbers were

284

recorded when the minimal concentration of NAA (2.69 µM) was supplemented with other two cytokinins in

285

previously prescribed concentration (4.92 µM 2iP and 9.08 µM TDZ). Shriram et al. (2007) demonstrated that

286

high concentration of BA and KN combination resulted effective shoot regeneration in H. isora, whereas when

287

they were further sub-cultured with less concentrated cytokinins (BA and KN) with high concentration of IAA

288

further proliferated to attain maximum shoot multiplication. This shows that multiple cytokinins with auxin

289

combination is essential for effective regeneration of H. isora. George et al. (2007) clearly illustrated that low

290

level of auxins in combination with high concentration of cytokinins causes axillary shoot proliferation. The

291

present study demonstrates the positive effect on 2iP, TDZ and NAA combination upon shoot proliferation for

292

the first time between these three growth regulators and to this Sterculiaceae family.

293

Organic additives are complex materials, precursors, stabilisers and elicitors, which facilitates the immediate

294

and enhanced response when supplemented in the culture medium. Additives like poly amines, amino acids

295

and few others were employed to enhance the shoot multiplication. Out of which L- glutamine (342.1 µM)

296

showed promising effect in the current culturing system of H. isora. It has been enhanced the shoot numbers to

297

nearly 30-40 % increase when supplemented with previously formulated PGRs. Glutamine is well known for

298

its ready to act form of nitrogen source, which helps in immediate synthesis of amino acids followed by

299

protein assimilation used for many biosynthesis pathways of shoot morphogenesis. Glutamine has been

300

reported to promote the shoot proliferation in many plant species like Ficus religiosa (Siwach and Gill, 2011)

301

and Hildegardia populifolia (Lavanya et al., 2012), by aiding the utilization of both nitrate and ammonium

302

nitrogen and its conversion into amino acids (George et al., 2007).

303

Shriram et al. (2007) utilised same sort of PGR combination with different PGRs and the multiple shoots

304

productivity was around 7. In our study we produced over 2 fold increase over shoot numbers (maximum of

305

16.9 shoots) in AN explant. Moreover the choice of explant, PGRs selection and inclusion of additives played

306

a vital role in the present study, as we had used CN, different PGRs (like 2iP, TDZ and NAA) and L-

307

glutamine which responded with 3 fold increase over the previous study of (Shriram et al., 2007).

11

308

4.3 Microshoot recovery and shoot elongation

309

Most of the above mentioned shoot multiplication treatments ended up with producing microshoots, short

310

sized or fasciated shoots. This may be due to the high concentration of TDZ supplemented in the medium

311

throughout the shoot initiation and multiplication period. TDZ proliferates existing meristems into buds but it

312

suppressed the differentiation of buds into shoots (Sujatha et al., 2012). But this TDZ induced shoot anomalies

313

are not permanent. Similarly, basal callusing was the predominant problem in establishment of shoot culture in

314

the present study. There has been no clear cut protocol to overcome this basal callus problem. In order to cut

315

this basal callus, reduction of sucrose concentration had been employed and found that frequency of basal

316

callus was reduced directly proportional to sucrose concentration. However the shoot induction frequency

317

along with shoot numbers also reduced. In this due course, sucrose concentration 2.0% ameliorates the basal

318

callusing by delayed response and slow callus proliferation, moreover does not disturb the shoot initiation and

319

multiplication. This gives an adequate time to establish the multiple shoot culture from explants. The

320

exogenous cytokinins may have converted to N7- and N9-glucoside conjugates, an inactive form and also

321

irreversible to active form of free base (Van Staden and Crouch, 1996; Auer, 1997). These cytokinins

322

conjugates were sunk into basal callus then inhibit the shoot proliferation. The observed callus induction was

323

may be due to that of unutilised auxins existed in the culture medium after the active cytokinins were

324

quenched. The recalcitrance effect of this basal callus had been clearly mentioned in reports of (Moyo et al.,

325

2011). Hence the microshoots were excised out of the explant and inoculated to PGR free half-strength MS

326

medium for acclimatise before transferring to elongation treatment. However, no microshoots later on

327

produced any basal callus even it was further developed on same concentration of sucrose. Sucrose

328

concentration as well as PRGs inclusion played important role in producing basal callus, which is later on

329

controlled when the PGRs are withdrew from culture medium. Nevertheless, PGRs alone not sole responsible

330

for basal callus, even in its absence higher concentration of sucrose triggered basal callusing in the present

331

study.

332

Microshoots and fasciated shoots were brought back to normal structure. It has been reported that the

333

prolonged exposure of TDZ produced fasciated, distorted shoots, indicating an inhibitory effect of TDZ on

334

shoot elongation (Huetteman and Preece, 1993; Ahmed et al., 2013). Recovered microshoots were grown

335

normally in the elongation medium, but the shoots did not elongated much or slower in response. However

336

half-strength MS medium supplemented with 2iP and GA3 facilitates the optimal shoot elongation. Effective

12

337

elongation was observed in the presence of GA3 which accordance with other woody plant species of

338

Sterculiaceae family members (Hussain et al., 2008; Lavanya et al., 2012).

339

4.4 Rooting and acclimatisation

340

Elongated shoots produced few roots while treating for elongation as well as control treatment of rooting

341

medium. Minimal level of auxin concentration tends to induce rooting response in well-developed shoots. IBA

342

(4.90 µM) recorded its best response over root initiation and development. Shriram et al. (2007) documented

343

that combination of half-strength MS medium with IBA (4.14 µM) found to be suitable for rooting of nodal

344

explant derived shoots. Recently, Sadeghi et al. (2015) reported the combination of half-strength MS medium

345

plus IBA supplied with 20 g l-1, exhibited beneficiary rooting. In addition low salt concentration of culture

346

medium supplemented with IBA were recorded for better response of rooting for shoots of many woody plants

347

species (Rai et al., 2010; Phulwaria et al., 2012). Activated charcoal treatment was not beneficial to enhance

348

rooting rather it helped to increase secondary root induction in this study. All the healthy plantlets were

349

carefully acclimatised by properly exposing them to normal temperature and humidity levels. Through the

350

series of changes in environmental conditions and proper nutritive supply enlisted ≥70 % of plants successfully

351

grown at poly tunnel chamber.

352

4.5 Genetic Fidelity

353

Meristematic culture system like direct regeneration studies generally produce true-to-type plants. But there

354

are cases where genetic variation occurs for the micropropagated plantlets of tree species (Goto et al., 1998;

355

Martins et al., 2004). Moreover the present study was carried out with in vitro germinated seedlings based

356

explants, where most of the seeds could be produced by cross pollination. In addition many PGRs with variety

357

of combinations were employed to produce the plantlets. Hence it is necessary to validate the genetic similarity

358

among the in vitro raised plantlets and mother plants. Recently more than one marker systems are used to

359

assess the genetic fidelity for tissue culture raised plants (Lakshmanan et al., 2007; Rathore et al., 2011). In the

360

present study two type of marker systems utilised to prove the genetic similarity, viz, SCoT and ISSR. Both

361

these systems are highly reliable and reproducible systems employed widely in recent days. Thirteen suitable

362

SCoT primers were identified for H. isora and produced total 63 monomorphic bands between in vitro

363

regenerants and source plant. ISSR marker system also produced 27 reproducible monomorphic bands, which

364

implies that no polymorphism detected in this study. There are many reports extensively utilised the open

365

pollinated seed source for seedling development and used as explants, yet there is no polymorphism observed

13

366

in those reports (Gaderi and Jafari, 2014; Bramhanapalli et al., 2016). Likely, micropropagated plants of

367

Cleome gynandra (Rathore et al., 2014), Aloe vera (Rathore et al., 2011) and Alhagi maurorum (Agarwal et

368

al., 2015) also proved to be genetically stable using SCoT and ISSR marker system. In the present

369

investigation both these marker system produced only monomorphic bands to ensure the genetic fidelity of

370

micropropagated plants towards field grown mother plants.

371

5 Conclusion

372

Helicteres isora direct regeneration protocol has been successfully developed with cotyledonary node and

373

axillary node of cultured tissues. Leading effect of 2iP, TDZ and NAA combination was found optimum for

374

multiple shoot induction. In addition L- glutamine inclusion in the culture medium along the side of PGRs

375

produced maximum shoot numbers in the present study. Shoot regeneration frequency was optimised to reach

376

maximum (100%) and shoot numbers quite enhanced (>20- CN; >15- AN). Basal callusing and shoot

377

elongation were the major difficulties encountered with plant regeneration. The current protocol circumvents

378

the problems associated by manipulating media, PGRs and sucrose concentrations and strengths. Microshoots

379

were successfully recovered to produce healthy plantlets. Frequency of shoot elongation and rooting was

380

recorded its best (100%). Genetic fidelity between the micropropagated plants and mother plants were assessed

381

through SCoT and ISSR marker systems. Both these marker systems, working on different targeting platform

382

distinctly reported that the cultured plants are genetically similar with mother plants. The developed

383

methodology in this study can be utilised in future for large scale cultivation of clonal plants of H. isora.

384

Appendix: Supplementary data of tables (all concentrations and combinations) and figures.

385

Conflict of Interest: The authors declare that they have no conflict of interest.

386

Acknowledgement

387

The authors are thankful to University Grant Commission (UGC) for providing financial support through UGC

388

Special Assistant Program (SAP) DRS II. Muthukumar M, thanks the DST for providing financial support in

389

DST-PURSE Phase II (SR/PURSE Phase 2/16(G) Dt. 21-02-2017) in manpower as well as departmental

390

assistance.

391

Author contribution statement

392

Muthukumar M, Rao MV and Senthil Kumar T designed the experiment. Muthukumar M and Muthukrishnan

393

S conducted field survey, sample collection, tissue culture and genetic fidelity experiment. Muthukumar M

14

394

prepared the manuscript. Rao MV, and Senthil Kumar T critically made corrections to bring out the

395

manuscript in submissive form.

396

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486 487 488 489 490

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491 492

Table 1 Shoot multiplication from cotyledonary node and axillary node explant of H. isora in MS medium supplemented with 4.92 µM 2iP, 9.08 µM TDZ, 2.69 µM NAA and organic additives. Concentration (mg l-1)

493 494 495

Cotyledonary Node Axillary Node Percentage of No. of shoots Percentage of No. of shoots Glutamine Sodium citrate response per explant* response per explant* (mean ± SE) (mean ± SE) (mean ± SE) (mean ± SE) 25 66.7±9.52 14.3±0.18cd 47.6±9.52 8.6±0.37d 50 100.0±0.00 21.3±0.99a 100.0±0.00 16.9±0.88a bc 75 90.5±4.76 15.9±0.63 81.0±4.76 10.6±0.90cd 100 33.3±4.76 7.4±0.30f 28.6±0.00 6.6±1.17e d 10 81.0±4.76 13.9±0.63 71.4±0.00 11.1±0.40c 20 100.0±0.00 16.1±0.51b 90.5±4.76 13.6±0.37b d 30 71.4±0.00 12.7±0.34 61.9±4.76 8.1±0.51d 40 47.6±9.52 9.1±0.96e 42.9±8.25 6.9±0.40e * Values with the same superscript are not significantly different at 5% probability level according to DMRT. Data recorded after 40 days of culture. Data of all concentrations and combinations are given in Supplementary files.

496

18

Table 2 Effect of different media and growth regulators on elongation of recovered microshoots of H. isora. Cotyledonary Node Shoot length No. of nodes / (cm) * shoots * (mean ± SE) (mean ± SE)

Axillary Node Shoot length No. of nodes / (cm) * shoots * (mean ± SE) (mean ± SE)

Root Initiation

Media type & PGR Concentration (µM) #

Percentage of response (mean ± SE)

½ MS Basal

81.0±4.76

4.2±0.07e

5.1±0.34cd

++

85.7±8.25

4.4±0.09e

4.6±0.20de

++

½ MS + 2iP 2.46

85.7±8.25

5.8±0.18d

5.3±0.18c

++

85.7±0.00

5.5±0.26d

4.9±0.26d

++

½ MS + 2iP 4.92

90.5±4.76

7.2±0.20

c

b

+

95.2±4.76

7.0±0.24

c

c

+

½ MS + 2iP 9.84

71.4±8.25

4.0±0.21e

4.3±0.18e

-

76.2±9.52

4.0±0.14f

4.3±0.29de

-

95.2±4.76

c

b

90.5±4.76

7.1±0.28

c

b

+

9.8±0.47

a

a

+

6.9±0.34

bc

-

6.4±0.20

bc

-

½ MS +2iP 4.92 + GA3 0.29 ½ MS +2iP 4.92 + GA3 1.44 ½ MS +2iP 4.92 + GA3 2.89 ½ MS +2iP 4.92 + GA3 5.77

100.0±0.00 85.7±0.00 76.2±4.76

7.5±0.19

10.9±0.36 8.7±0.29

a

b

7.2±0.16

c

6.4±0.20 6.6±0.30

9.0±0.31

a

7.1±0.34

b

6.9±0.26

b

Percentage Root of response Initiation (mean ± SE)

+ +

100.0±0.00

-

95.2±4.76

-

76.2±9.52

8.2±0.51

b

7.3±0.23

c

6.1±0.34 7.1±0.40

8.4±0.30

* Values with the same superscript are not significantly different at 5% probability level according to DMRT. ½ MS- half-strength MS. Data recorded after 40 days of culture. Data of all concentrations and combinations are given in Supplementary files. # 2iP concentrations were reduced to 0.98 µM with its respective GA3 concentration on first subculture onwards.

19

Table 3 Effect of auxins and AC on rooting of elongated shoots of H. isora cultured on half-strength MS medium fortified with 4.92 µM 2iP + 1.44 µM GA3. Concentration(µM) Control IBA

Percentage of response (mean ± SE) 66.7±4.76

No. of roots / shoot (mean ± SE) 3.7±0.18f

Average Root length/ shoot (cm) (mean ± SE) 3.2±0.19b

No. of Secondary roots / shoot (mean ± SE) 45.3±3.78fg

0.49 57.1±0.00 6.1±0.34d 4.3±0.50ab 51.3±3.91f c a 5.1±0.65 93.6±5.82d 2.46 85.7±8.25 8.3±0.18 a a 4.90 100.0±0.00 12.9±0.63 6.1±0.79 158.6±4.08b e ab 4.1±0.34 62.7±1.64e 9.80 47.6±4.76 5.0±0.38 IBA 4.90 + AC 83.26 90.5±4.76 8.1±0.46c 5.0±0.69ab 94.7±2.97d a a 416.31 100.0±0.00 12.1±0.34 6.1±0.52 196.9±5.73a 582.84 100.0±0.00 10.7±0.61b 5.0±0.34ab 129.1±3.56c c a 832.63 81.0±9.52 7.7±0.47 5.3±0.71 95.4±5.15d * Values with the same superscript are not significantly different at 5% probability level according to DMRT. Data recorded after 40 days of culture. Data of all concentrations and combinations are given in Supplementary files. Table 4 Total number and size range of amplified fragments generated by SCoT and ISSR analysis in H. isora Primer code

Primer sequence (5’ → 3’)

SCoT analysis SCoT01 CAACAATGGCTACCACCA SCoT02 CAACAATGGCTACCACCC CAACAATGGCTACCACCG SCoT03 SCoT04 CAACAATGGCTACCACCT SCoT06 CAACAATGGCTACCACGC CAACAATGGCTACCACGG SCoT07 SCoT11 AAGCAATGGCTACCACCA ACGACATGGCGACCAACG SCoT12 SCoT16 ACCATGGCTACCACCGAC ACCATGGCTACCACCGAG SCoT17 SCoT26 ACCATGGCTACCACCGTC SCoT32 CCATGGCTACCACCGCAC SCoT36 GCAACAATGGCTACCACC Total No. of Bands ISSR analysis UBC810 GAGAGAGAGAGAGAGAT UBC864 ATATATATATATATATG UBC808 AGAGAGAGAGAGAGAGG UBC811 GAGAGAGAGAGAGAGAC UBC828 TGTGTGTGTGTGTGTGA Total No. of Bands

Tm (⁰⁰C)

No. of loci

53.7 56.0 56.0 53.7 56.0 56.0 53.7 53.7 58.2 58.2 58.2 60.5 56.0

50.4 59.4 52.8 50.4 52.8

20

No. of bands No. of across all polymorphic Size range (kb) plants bands

4 4 6 4 3 4 6 7 4 5 7 5 4 63

32 32 48 32 24 32 48 56 32 40 56 40 32 504

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.8- 2.3 1.0- 2.0 0.5- 2.0 1.0- 2.5 1.5- 2.5 1.0- 1.5 0.5- 2.0 0.3- 2.5 0.6- 1.4 0.6- 1.6 0.6- 3.0 0.8- 1.7 0.8-2.3

8 7 5 5 2 27

64 56 40 40 16 216

0 0 0 0 0 0

0.3- 2.2 0.3- 2.1 0.3- 1.5 0.2- 2.1 0.5- 1.2

Figure caption Figure. 1 Micropropagation from cotyledonary node and nodal explants of H. isora: a shoot initiation and multiplication from CN explant in MS medium containing 20 g l-1 sucrose and 4.92 µM 2iP and 9.08 µM TDZ. b multiple shoots of CN explant, MS medium containing 20 g l-1 sucrose 4.92 µM 2iP, 9.08 µM TDZ, 2.69 µM NAA and 342.1 µM glutamine. c multiple shoots of AN explant in MS medium containing 20 g l-1 sucrose, 4.92 µM 2iP, 9.08 µM TDZ, 2.69 µM NAA and 342.1 µM glutamine. d. induction and proliferation of basal callus (BC) and fasciated shoots (FS). e microshoot recovery in half-strength MS medium with 20 g l-1 sucrose devoid of PGRs and additives. f recovered shoots with definite structure. g elongated shoots with developed roots on half-strength MS medium with 20 g l-1 sucrose and 4.90 µM IBA. h acclimatisation of in vitro raised plantlets in poly tunnel chamber. Bar: 5mm Figure. 2 Assessment of genetic fidelity of in vitro raised plants of H. isora using SCoT and ISSR markers: a SCoT primer 11. b Scot primer 12. c ISSR primer UBC810. d ISSR primer UBC864. MP- mother plant. T1 to T7- micropropagated plants. 1KB- 1 kilo base pair ladder. 100bp- 100 base pair ladder.

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Highlights: N6- (2- isopentenyl) adenine (2iP) was found suitable cytokinin to enhance the direct shoot regeneration in H. isora Microshoots were successfully regenerated into well-developed shoots suitable for root induction SCoT and ISSR, the two different genetic marker systems were employed to ensure genetic fidelity of regenerants.