Organogenesis and evaluation of genetic homogeneity through SCoT and ISSR markers in Helicteres isora L., a medicinally important tree

South African Journal of Botany 106 (2016) 204–210

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Organogenesis and evaluation of genetic homogeneity through SCoT and ISSR markers in Helicteres isora L., a medicinally important tree Mariappan Muthukumar a, Thiruppathi Senthil Kumar b,⁎, Mandali Venkateswara Rao a a b

Department of Plant Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620024, India Department of Industry University Collaboration, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620024, India

a r t i c l e

i n f o

Article history: Received 9 May 2016 Received in revised form 19 July 2016 Accepted 21 July 2016 Available online xxxx Edited by E Balazs Keywords: Helicteres isora Organogenesis Glutamine Microshoots SCoT ISSR

a b s t r a c t The present study develops the highly reliable and reproducible protocol for indirect organogenesis of Helicteres isora L., a medicinally important multipurpose plant of Sterculiaceae family. Murashige and Skoog (MS) medium supplemented with different plant growth regulators induced callus with different type and texture. The combination of 10.74 μM naphthalene acetic acid (NAA) and 5.71 μM indole-3-acetic acid (IAA) fortified with MS medium resulted best response (100%) in callus induction with fresh weight from leaf (3.75 ± 0.11 g) and internodal (3.30 ± 0.14 g) explants. Green compact nodular callus (GCNC) was transferred to shoot regeneration medium for shoot regeneration. The multiplication rate of adventitious shoots was influenced by various factors like explant type, media composition, plant growth regulator combinations and concentrations. MS medium supplemented with 2.69 μM NAA, 0.57 μM IAA and 4.92 μM N6-(2-isopentenyl) adenine (2ip) combination resulted in 80.95 ± 4.76 percentage of response with 8.43 ± 0.43 number of shoots per piece of callus derived from leaf explant. This combination was expressed 76.19 ± 4.76 percentage of response with 7.28 ± 0.28 number of shoots per internode-derived callus respectively. The addition of 50 mg l−1 glutamine in 2.69 μM NAA, 0.57 μM IAA and 4.92 μM 2iP combination enhanced the shooting frequency with 12.14 ± 0.83 and 9.14 ± 0.51 shoots from leaf and internodal explant-derived callus. Elongated shoots [4.92 μM 2iP and 1.44 μM of gibberellic acid (GA3)] were achieved rooting in half-strength MS medium fortified with 4.90 μM indole-3butyric acid (IBA) with 582.84 μM activated charcoal. The genetic fidelity between mother plant and in vitro plants was assessed through SCoT and ISSR marker systems. Both these analysis revealed that all the samples were found monomorphic in nature. This suggests that the current study standardised the true-to-type culturing protocol and authenticated the in vitro raised plantlets are remain free from somaclonal variations. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Helicteres isora L. (Sterculiaceae) is commonly known as East Indian screw tree and widely distributed throughout India in dry deciduous forests up to 1500 m on hill slopes. It is a medicinally important multipurpose subdeciduous shrub or a small tree. Various parts of H. isora possess pharmacological activities, including antidiabetic (hypoglycaemic and hyperglycaemic), anticancer, antimicrobial, antispasmodic and to treat snake bite, dog bite, diarrhoea and blood purification from ancient days till nowadays (Pramod et al., 2012; Kumar and Singh, 2014). Also the plant has various bioactive compounds, such as cucurbitacin- B, isocucurbitacin- B, diosgenin, daucosterol, hibifolin, trifolin, rosmarinic acids and many from different parts of plant (Pramod et al., 2012; Kumar and Singh, 2014). In vitro propagation studies most often widely used for ex situ conservation, mass propagation and also the bioactive compound ⁎ Corresponding author. Fax: +91 0431 2407045. E-mail address: [email protected] (T. Senthil Kumar).

http://dx.doi.org/10.1016/j.sajb.2016.07.017 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

production (Sajc et al., 2000; Nasim et al., 2010). H. isora has problem with seed setting as well as seed dormancy causes regeneration difficulties in vivo (Badave and Jadhav, 1998; Atluri et al., 2000). H. isora has been harvested indiscriminately for its extensive medicinal uses have made serious survival threat also the further possibility of field extinction. Indirect organogenesis is a better way of getting callus biomass as well as adventitious shoot regeneration protocol. Earlier investigation on indirect organogenesis of H. isora reported low frequency of response (b 70%) with limited number of shoots using nodal explants (Shriram et al., 2008). Various factors such as age, season and explant type causes low responsiveness for woody plant species at in vitro conditions (Purohit and Kukda, 2004). However, in adventitious shoot culture system, the selection of explant is important criteria; hence, we have chosen leaf and internodal explants. Early reports of in vitro raised seedlingbased explants were found successful for indirect organogenesis in many woody plant species (Lavanya et al., 2014; Piatczac et al., 2015). Somaclonal variation during the in vitro regeneration causes serious limitations over this techniques (Bhojwani and Dantu, 2013). Explant nature, genotype, PGR type then concentration, methodology and

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repeated subculture play a vital role in genetic variations (Bairu et al., 2011). Likely disorganised growth phase and long-term maintenance and repeated subculture can cause variation among propagules (Rani and Raina, 2000; Kuznetsova et al., 2006). Lakshmanan et al. (2007) recommended variety of DNA-based marker system for the analysis of genetic homogeneity as it will have different target regions in that genome. Start codon targeted polymorphism (SCoT) and inter-simple sequence repeats (ISSR) are the two highly reproducible marker systems widely utilised to analyse the genetic homogeneity between mother plants and tissue cultured plants (Bekheet et al., 2015; Bhattacharyya et al., 2016). There was no report on genetic variation studies for indirect organogenesis of H. isora; hence, it makes necessitate to check genetic fidelity. The present study investigates the highly reproducible protocol for H. isora through indirect organogenesis and validates the genetic homogeneity between mother plants and in vitro raised plantlets. 2. Materials and methods 2.1. Plant collection and explant selection H. isora L. fruits were collected from Kolli hills on Western Ghats of Tamil Nadu, India. Plants were identified by Botanical survey of India, Coimbatore (Specimen No.177312). Matured dry fruits of H. isora were untwisted to dehisce the seeds. Seeds were acid treated (H2SO4, 98%) for 1 min, then the seeds were washed with running tap water for 10 min to remove debris followed by detergent wash (Teepol™, Sigma- Aldrich, India) for 1 min and thoroughly washed through tap water and then rinsed with ethanol for 30 s, then 3% NaOHCl for 1 min and 0.1% w/v HgCl2 for 5 min. Finally, the seeds were five times washed with sterile distilled water. Treated seeds were further inoculated into sterile wet cotton bed for seed germination. Leaf and internode of 4- to 6-week-old seedlings were used as explants. 2.2. Media selection and culture conditions Murashige and Skoog medium (MS) (Murashige and Skoog, 1962) was used as basal medium with sucrose (20.0 g l−1) and agar (0.7%, Agar Agar type II) for callus initiation and further proliferation, whereas shoot elongation and rooting was performed with 0.8% agar. The cultures were incubated in culture room maintained at 25 ± 2 °C, under 16 h photoperiod with a light intensity of 35 μΣ m−2S−1 from Philips cool white fluorescent tubes with 55–60% relative humidity. 2.3. Callus initiation and proliferation Explants including internode (IN) (1 cm in length) and leaf (L) (0.5 sq. cm) were excised from 35-day-old in vitro germinated seedlings and placed horizontally on MS medium. The effects of both auxins and cytokinins were studied individually also in combination for callus initiation and proliferation. Auxins like 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), indole-3-butyric acid (IBA) and picloram (PIC) then different cytokinins, 6-benzyl adenine (BA), 6-furfuryl amino purine (KN) and thidiazuron (TDZ) were fortified at various concentrations either alone or in combination. The frequencies of response, fresh weight and nature of callus were recorded after 40 days. 2.4. Shoot regeneration from callus Green compact organogenic callus (~0.5 g fresh weight) were selected and further maintained in PGRs free MS basal medium for 1 week then subjected to shoot regeneration (SR) medium, MS medium (20 g l− 1 sucrose) supplemented with different cytokinins [BA, N6-(2isopentenyl) adenine (2ip) and TDZ] with the best suited auxins at a concentration of 2.69 μM NAA and 0.57 μM IAA for shoot initiation

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and multiplication. The effect of organic additives like amino acids (glutamine and proline), polyamines (putrescine and spermidine), sodium citrate and adenine sulphate were tested at various concentrations in best responded shoot regeneration medium. 2.5. Shoot elongation, rooting and acclimatisation Microshoots (≤ 1 cm) were excised from 40-day-old cultures and transferred to microshoot recovery medium, half-strength MS medium with 20 g l−1 sucrose and devoid of any PGRs. Recovered shoots with 3– 4 leaves were further introduced into shoot elongation medium, halfstrength MS medium (20 g l−1 sucrose) fortified with 4.92 μM 2iP and various concentrations of GA3. Developed shoots above 5–6 cm were introduced into root induction medium, half-strength MS medium containing various concentrations of auxins (IAA, IBA and NAA) and activated charcoal. Plantlets with developed roots were transferred to sterile paper cups (6 × 8 cm) consists of sterile red soil: sand: compost (120 g/cup) (1:1:1 v/v) and then transferred to pots containing red soil, sand and manure (2:1:1, v/v). 2.6. Isolation of genomic DNA Seven acclimatised plants (60 days old), derived from in vitro cultures, were randomly selected for genetic homogeneity studies. Young leaves (0.1 g) grounded with liquid nitrogen in a pestle and mortar, then further processed to acquire genomic DNA using HiPurA plant genomic DNA miniprep purification kit (MB507-Himedia). Quantity and quality of isolated DNA's were recorded with Biophotometer (Eppendrof BioPhotometer Plus, Germany). The final quantity of isolated DNA was adjusted to 50 ng/μl. 2.7. SCoT analysis Seventeen SCoT primers (Collard and Mackill, 2009) were initially scrutinised for reproducible multiple band formation, and those responded positively were trailed to analyse genetic homogeneity between mother plants and acclimatised plants. Final volume of 20 μl reaction mixture consists of 4 μl template DNA, 3 μl of SCoT primer (GeNei™, Bangalore, India), 3 μl of sterile distilled water and 10 μl of 2× PCR master mix [Tris–HCl pH 8.5, (NH4)2SO4, 4 mM MgCl2, 0.2% Tween 20, 0.4 mM dNTPs, 0.2 U/μl Taq DNA polymerase, inert red dye and stabiliser] (Ampliqon, Denmark), used for amplification in thermal cycler (Eppendrof AG, Germany). The PCR programmed with initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C (60 s), annealing for 60 s at Ta, extension at 72 °C (120 s) and final extension for 5 min at 72 °C. The products of PCR were resolved at 50 V for 3 h in 1.2% Agarose gel with 1 × TAE buffer and stained with ethidium bromide. Gels were photographed using gel documentation system (Alpha Imager EP). 2.8. ISSR analysis Ten ISSR primers [Department of Biotecnology laboratory, University of British Colombia (UBC Set 09] were trailed initially to perform ISSR analysis. Final volume of 20 μl reaction mixture consists of 4 μl template DNA, 3 μl of SCoT primer (GeNei™, Bangalore, India), 3 μl of sterile distilled water and 10 μl of 2× PCR master mix (Ampliqon, Denmark) were used for amplification in thermal cycler (Eppendrof AG, Germany). The PCR programmed with initial denaturation at 94 °C for 5 min followed by 45 cycles of denaturation at 94 °C (45 s), annealing for 45 s at Ta, extension at 72 °C (90 s) and final extension for 7 min at 72 °C. The products of PCR were resolved at 50 V for 3 h in 1.2% agarose gel with 1× TAE buffer and stained with ethidium bromide. Gels were photographed using gel documentation system (Alpha Imager EP).

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2.9. Data analysis

3.2. Adventitious shoot regeneration

2.9.1. Plant tissue culture experiments Minimum seven replicates were used per experiment, and all the experiments were repeated thrice. The experimental design was random and factorial. Data were recorded for callusing, shoot and root regeneration after 40 days of culture. The data were subjected to mean and mean separation analysis by using Duncan's multiple range test (DMRT) (IBM SPSS Statistics 23.0, Armonk, NY).

A piece of green compact nodular callus (~0.5 g) was maintained in MS basal medium before introduced into SR medium. To induce morphogenesis in callus, auxin levels were reduced to minimum either individually or combined with high concentrations of different cytokinins. Shoot morphogenesis on organogenic callus was observed after 3 weeks transferred to SR medium (Fig. 1c). The combination of 2.69 μM NAA, 0.57 μM IAA and 4.92 μM 2iP resulted 80.95 ± 4.76 percentage of response with 8.43 ± 0.43 number of shoots per piece of callus derived from leaf explant. This combination expressed 76.19 ± 4.76 percentage of response with 7.28 ± 0.28 number of shoots per piece of internode-derived callus (Fig. 1d; Table 2). Shriram et al. (2008) reported about only 3 shoots with the BA and KN combination. Well this is the first report as far as our concern in this family that 2iP had been successfully promoted shoot morphogenesis along with two auxins, NAA and IAA. Organic additive glutamine inclusion enhanced the shoot regeneration frequency (90.48 ± 4.76 % & 95.24 ± 4.76 %) as well as adventitious shoot numbers (12.14 ± 0.83 & 9.14 ± 0.51) from both leaf and internode-derived callus. The combination of 2.69 μM NAA, 0.57 μM IAA, 4.92 μM 2iP and 50 mg l−1 glutamine resulted 90.48 ± 4.76 percentage response with 12.14 ± 0.83 shoots per piece of callus derived from leaf explant (Fig. 1e), whereas 95.24 ± 4.76 % responsiveness with 9.14 ± 0.51 shoots in internode-derived callus (Table 2). The PGR combinations help the callus to attain morphogenic changes to produce adventitious shoots, whereas the glutamine inclusion helped the callus to assimilate more nitrogen source readily to accelerate the metabolic activities and bring out rapid shoot regeneration compared to PGRs treatments. Glutamine and glutamic acid are directly involved in the assimilation of NH4+. A direct supply of these amino acids should therefore enhance the utilisation of both nitrate and ammonium nitrogen and its conversion into amino acids (George et al., 2008). Earlier glutamine is found promotory in shoot proliferation for many woody species (Annapurna and Rathore, 2010; Siwach and Gill, 2011).

2.9.2. Genetic fidelity SCoT and ISSR marker-derived gel images were manually compiled to binary matrix, based on the presence (1) and absence (0) of particular selected band. Moreover, the reproducible and clear bands were selected, while ambiguous, weak and smeared bands all neglected for data scoring. Similarity index between mother plant and each individual in vitro raised plants were analysed using Jacarrd's coefficient using the following formula: Jab = Nc/Na + Nb + Nc, where Na is the number of elements positive in set А and negative to set B, Nb is the number of elements in positive in set B negative to set A and Nc is the number of elements positive in intersecting set.

3. Results and discussion 3.1. Callus initiation and proliferation Internode and leaf explants were successfully derived from in vitro raised seedlings. Callusing response was initiated by the second week onwards at edges and cut ends of leaves and internodes, then further extended to whole explants. Irrespective of PGRs and concentration, all treatment produced callus formation in both the explants, and so the differences had been observed at frequency of response, callus nature and colour (see supplementary materials). PGRs like auxins and cytokinins were individually tested, the treatment of 10.74 μM NAA resulted top with high callus frequency (100%) and fresh weight (2.79 ± 0.07 g, L; 2.45 ± 0.26 g, IN) in both explants (Table 1). The combination of 10.74 μM NAA with 5.71 μM IAA quite enhanced the callus fresh weight to 3.75 ± 0.11 g and 3.30 ± 0.14 g in L and IN explants, respectively (Table 1). Shriram et al. (2008) earlier reported that the individual treatment of PGRs provided only creamish and granular callus mass in nodal explants. In our study, the individual treatment of NAA produced green compact callus in both IN and L explants. Earlier, the same result was obtained in Japanese raisin tree by Ribeiro et al. (2015). However, NAA combined with IAA developed green compact nodular callus (GCNC) in both explants (Fig. 1a, b), which is suitable for callogenesis.

3.3. Microshoot recovery, elongation and rooting The SR medium induced the morphogenesis in GCNC, yet the callus proliferation also occurs subsequently in reduced rate. This had caused the developed shoots either covered by callus or converted into callus. Pre-existing PGRs in SR medium does not help the regenerated shoots to elongate and requires further treatment. Regenerated microshoots (≤ 0.3–1.0 cm) were carefully excised out of callus culture and transferred to recovery medium for the period of 2 weeks time (Fig. 1f), until it attained reformed structure with 2–4 intact leaves (Fig. 1g). Recovered microshoots were failed to elongate in same medium, whereas

Table 1 Callus induction and proliferation using different PGRs form leaf and internodal explants of H. isora. Concentration (μM)

Leaf

NAA

Response (%)

2.69 5.37 10.74 16.11 21.48 10.74 10.74 10.74 10.74 10.74

IAA

2.85 5.71 11.42 17.13 22.84

61.90 ± 4.76 76.19 ± 4.76 100.00 ± 0.00 95.24 ± 4.76 90.48 ± 4.76 90.48 ± 4.76 100.00 ± 0.00 100.00 ± 0.00 80.95 ± 4.76 61.90 ± 4.76

Internode Fresh weight (g) gh

1.03 ± 0.04 1.69 ± 0.06c 2.79 ± 0.07a 2.45 ± 0.10b 1.86 ± 0.20c 2.63 ± 0.06b-d 3.75 ± 0.11a 2.96 ± 0.29b 2.64 ± 0.15bc 2.54 ± 0.09b-d

Type of callus* GFC GCC GCC GWC YFC GCC GCNC GCNC GCC GWFC

Response (%) 66.67 ± 4.76 95.24 ± 4.76 100.00 ± 0.00 90.48 ± 4.76 80.95 ± 4.76 95.24 ± 4.76 100.00 ± 0.00 90.48 ± 4.76 76.19 ± 4.76 66.67 ± 4.76

Fresh weight (g) e-k

0.99 ± 0.08 1.43 ± 0.17c-e 2.45 ± 0.26a 2.35 ± 0.31a 2.11 ± 0.17ab 2.68 ± 0.13bc 3.30 ± 0.14a 2.89 ± 0.11b 2.61 ± 0.16b-d 2.48 ± 0.18b-e

Type of callus* GFC GCC GCC YFC YFC GCC GCNC GCNC GWFC GWFC

* GFC—green friable callus; GCC—green compact callus; GWC—green watery callus; YFC—yellow friable callus; GCNC—green compact nodular callus; GWFC—green watery friable callus. Values with the same superscript are not significantly different at 5% probability level according to DMRT. Data recorded after 40 days of culture.

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Fig. 1. Organogenesis from leaf and internodal explants of H. isora: (a) Green compact nodular callus initiation and proliferation from internodal explant in MS medium fortified with 10.74 μM NAA and 5.71 μM IAA. (b) Green compact nodular callus initiation and proliferation from leaf explant in MS medium fortified with 10.74 μM NAA and 5.71 μM IAA. (C) occurrence of morphogenic changes at leaf-derived callus on SR medium. (d) Shoot regeneration from internodal explant-derived callus at MS medium fortified with 20 g l−1 sucrose, 2.69 μM NAA, 0.57 μM IAA and 4.92 μM 2iP. (e) Shoot regeneration form leaf-derived callus at MS medium fortified with 20 g l−1 sucrose, 2.69 μM NAA, 0.57 μM IAA, 4.92 μM 2iP and 50 mg l−1 glutamine. (f) Microshoot recovery in half-strength MS medium with 20 g l−1 sucrose. (g) Recovered shoots with definite structure. (h) Elongated shoots in rooting development on half-strength MS medium with 20 g l−1 sucrose and 4.90 μM IBA. (i) Acclimatisation of in vitro raised plantlets in poly tunnel chamber. Bar: 5 mm.

Table 2 Shoot regeneration from leaf and internode explant-derived callus of H. isora using PRGs and glutamine. Concentration (μM)

Leaf

NAA

IAA

BA

2.69 2.69 2.69 2.69 2.69 2.69 2.69 2.69 2.69 2.69 2.69 2.69 2.69

0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57

2.22 4.44 8.87

2IP

TDZ

Response (%)

No. of shoots

Response (%)

No. of Shoots

25 50 100 150

66.67 ± 9.52 71.43 ± 8.25 38.10 ± 4.76 80.95 ± 4.76 80.95 ± 4.76 76.19 ± 4.76 57.14 ± 8.25 33.33 ± 4.76 – 23.81 ± 4.76 90.48 ± 4.76 76.19 ± 4.76 38.10 ± 9.52

5.71 ± 0.36c–f 7.14 ± 0.40b 5.86 ± 0.51c–e 6.14 ± 0.51b–d 8.43 ± 0.43a 6.57 ± 0.61bc 4.57 ± 0.30e–g 3.14 ± 0.40i – 4.00 ± 1.09g–k 12.14 ± 0.83a 9.43 ± 0.84b 6.57 ± 0.53c–f

71.43 ± 8.25 71.43 ± 0.00 42.86 ± 8.25 76.19 ± 4.76 76.19 ± 4.76 80.95 ± 4.76 57.14 ± 8.25 38.10 ± 4.76 – 28.57 ± 8.25 95.24 ± 4.76 80.95 ± 4.76 47.62 ± 9.52

4.57 ± 0.30c–e 6.28 ± 0.18b 4.86 ± 0.14c–e 5.57 ± 0.20b–d 7.28 ± 0.28a 5.42 ± 0.36cd 3.14 ± 0.26fg 2.71 ± 0.36g – 3.29 ± 0.61hi 9.14 ± 0.51a 7.86 ± 0.74ab 6.43 ± 0.61b–e

2.46 4.92 9.84 2.25 4.54 9.08 4.92 4.92 4.92 4.92

Internode

Glu*

* Glutamine's (Glu) concentration mentioned as mg l−1. Values with the same superscript are not significantly different at 5% probability level according to DMRT. Data recorded after 40 days of culture.

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Table 3 Shoot elongation of microshoots of H. isora on half-strength MS medium supplemented with 4.92 μM 2iP with various concentration of GA3. Leaf`

Internode

GA3 concentration (μM)

Percentage of response

Shoot length (cm)

No. of nodes/shoot

Percentage of response

Shoot length (cm)

No. of nodes/shoot

0.29 1.44 2.89 5.77

85.71 ± 8.25 100.00 ± 0.00 85.71 ± 0.00 80.95 ± 4.76

6.29 ± 0.39de 9.13 ± 0.78a 7.50 ± 1.19c 5.13 ± 0.61f

6.00 ± 0.31bc 7.71 ± 0.36a 6.43 ± 0.20b 4.29 ± 0.18ef

90.48 ± 9.52 100.00 ± 0.00 90.48 ± 4.76 85.71 ± 8.25

6.13 ± 0.14e 9.14 ± 0.14a 7.23 ± 0.28c 5.06 ± 0.21f

4.86 ± 0.14d-f 7.29 ± 0.52a 6.00 ± 0.22bc 4.43 ± 0.20d-g

Values with the same superscript are not significantly different at 5% probability level according to DMRT. Data recorded after 40 days of culture.

Table 4 Effect of auxins and AC on rooting of elongated shoots of H. isora. PGR and AC concentration (μM)

Percentage of response

No. of roots/shoot

Average root length/shoot (cm)

No. of secondary roots/shoot

IBA 4.90 4.90 4.90 4.90

71.43 ± 8.25 85.71 ± 8.25 100.00 ± 0.00 95.24 ± 4.76

6.14 ± 0.40d-h 6.71 ± 0.42d-f 11.71 ± 0.47a 7.43 ± 0.32c-e

4.73 ± 0.33e-g 4.81 ± 0.16d-g 7.87 ± 0.40a 5.61 ± 0.19b-d

59.86 ± 2.89de 63.29 ± 2.82d 162.14 ± 4.93a 92.14 ± 6.89c

AC 83.26 416.31 582.84 832.63

Values with the same superscript are not significantly different at 5% probability level according to DMRT. Data recorded after 40 days of culture.

half-strength MS medium (20 g l−1 sucrose) fortified with 4.92 μM 2iP and 1.44 μM of GA3 helped the microshoots with elongation to obtain 9.13 ± 0.78 and 9.14 ± 0.14 cm shoot length; 7.71 ± 0.36 and 7.29 ± 0.52 nodes per shoot in both leaf and internode-derived callus, respectively (Fig. 1h; Table 3). Philomina and Rao (2000) demonstrated that the GA3 was found effective in elongation while combined with the cytokinins. Also, woody species of same family needs GA3 treatment for better elongation as reported earlier (Hussain et al., 2008; Lavanya et al., 2012). Well-elongated shoots were transferred to root induction media, half-strength MS medium (20 g l−1 sucrose) fortified with 4.90 μM IBA with 582.84 μM activated charcoal resulted with 11.71 ± 0.47 roots per shoot with an average length of 7.87 ± 0.40 cm and 162.14 ± 4.93 secondary root formation (Table 4). Next to that 4.90 μM IBA produced 9.71 ± 0.29 number of roots with 6.16 ± 0.16 average root length and 125.14 ± 8.07 secondary root formation, followed by 5.71 μM IAA produced 8.14 ± 0.34 average roots per shoot, and NAA resulted in very poor comparing to control treatment (see supplementary material). Earlier report of Shriram et al. (2008) has been reported that 4.2 roots using the same PGR devoid of activated charcoal; however, root was weak in IAA treatment. The reduced sucrose concentration and addition of activated charcoal enhanced the rooting effect in IBA treatment, whereas IAA resulted next to best without adding activated charcoal. The sucrose played a vital role by reducing callus formation at shoot base at both PGRs treatment, whereas activated charcoal played supportive role by providing dark environment to induce more number of roots in IBA treatment. Many reports demonstrated that half-strength

MS medium supplemented with either IBA or IAA promoted rooting response (Koroch et al., 1997; Annapurna and Rathore, 2010). 3.4. Acclimatisation Well-rooted plantlets were taken out of culture media without causing any damage to roots and further transferred to paper cups containing sterile red soil: sand: compost (120 g/cup) (1:1:1 v/v) and covered with transparent polythene covers to avoid desiccation of in vitro habituated plantlets. Plantlets were initially irrigated daily with half-strength MS medium devoid of sucrose (~ 10 ml) for first 2 weeks, thereafter watered once in every 2 days for 30 days (Fig. 1i). Reducing the humidity in regular interval, survived plantlets were transferred to pots containing red soil, sand and manure (2:1:1, v/v) for 30 days, and then the pots were translocated to poly tunnel chamber (Saveer Biotech, New Delhi), where 76.19 ± 4.76 percentage of survival was observed. 3.5. Genetic fidelity SCoT marker system is recently developed methodology, which has the definite authenticity over other marker system, as it is targeting the functional genes or the immediate flanking sequences (Collard and Mackill, 2009). Seventeen SCoT primers were initially scrutinised for multiple and reproducible band formation, out of which 12 SCoT primers responded positively, which were further analysed for genetic homogeneity of mother plant and acclimatised plant. The variety of band patterning was observed in these 12 SCoT primers and their

Fig. 2. Evaluation of genetic fidelity of in vitro raised plants of H. isora using SCoT and ISSR markers: (a) SCoT primer 03. (b) ISSR primer UBC864. MP—mother plant; T1 to T7—in vitro raised plantlets; 1 KB—1 k base pair ladder; 100 bp—100 base pair ladder.

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Table 5 Details of SCoT and ISSR primers used for analysis of genetic fidelity for callus-derived plantlets and mother plants of H. isora. Primer code SCoT analysis SCoT01 SCoT02 SCoT03 SCoT04 SCoT07 SCoT11 SCoT12 SCoT16 SCoT17 SCoT26 SCoT32 SCoT36

ISSR analysis UBC810 UBC864 UBC808 UBC811

Primer sequence (5’ → 3’)

Tm (⁰C)

No. of amplicons

No. of monomorphic bands

No. of polymorphic bands

Size of amplicons (bp)

CAACAATGGCTACCACCA CAACAATGGCTACCACCC CAACAATGGCTACCACCG CAACAATGGCTACCACCT CAACAATGGCTACCACGG AAGCAATGGCTACCACCA ACGACATGGCGACCAACG ACCATGGCTACCACCGAC ACCATGGCTACCACCGAG ACCATGGCTACCACCGTC CCATGGCTACCACCGCAC GCAACAATGGCTACCACC Total no. of bands

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

4 4 6 4 4 6 7 4 5 7 5 4 60

4 4 6 4 4 6 7 4 5 7 5 4 60

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

800–2300 1000–2000 500–2000 1000–2500 1000–1500 500–2000 300–2500 600–1400 600–1600 600–3000 800–1700 800–2300

GAGAGAGAGAGAGAGAT ATATATATATATATATG AGAGAGAGAGAGAGAGG GAGAGAGAGAGAGAGAC Total no. of bands

50.4 59.4 52.8 50.4

8 7 5 5 25

8 7 5 5 25

0 0 0 0 0

350–2200 300–2100 300–1500 200–2100

band pattern lies between 300 bp and 3000 bp. Minimum 4 bands were set as par score, which was observed in SCoT01, SCoT02, SCoT04, SCoT07, SCoT16 and SCoT36, whereas maximum 7 reproducible bands were observed in SCoT10 and SCoT14. Overall 60 reproducible bands were resolved from total 12 SCoT primers. All those bands were highly monomorphic, and no dissimilarity between any samples of mother plant as well other tissue culture raised plantlets. Jaccard's coefficient value is “1” for the Scot analysis. DNA profile of SCoT03 band pattern is shown in Fig. 2a. ISSR marker system also one of the best marker system in analysis of genetic variability and characterised as medium to highly reproducible, more stringent, semi-arbitrary, dominant and also targets non coding regions of DNA (Agarwal et al., 2015). Total 10 ISSR primers were tested for band patterning and 4 ISSR primers showed promising band pattern, which were further trailed for analysis of genetic integrity between in vivo and in vitro plantlets. Unique band pattern was observed in all these 4 ISSR primers, and its size varies between 200 and 2200 bp. Total 25 reproducible bands were resolved in all the 4 ISSR markers and band patterning ranges from 5 (UBC808 and UBC811), 7 (UBC864) and 8 (UBC810). Band formation between the mother plant and other 7 acclimatised plantlets were found; monomorphic no polymorphic bands were observed in any samples. Jaccard's coefficient value is “1” for the ISSR analysis. DNA profile of UBC864 band pattern is shown in Fig. 2b. Primer sequences and its respective annealing temperature, total no of bands scored, monomorphic bands and polymorphic bands were tabulated (Table 5). The comparative analysis of both SCoT and ISSR marker system revealed that all the samples were found monomorphic in nature. Jaccard's coefficient analysis of both maker system-derived data were found similar (1), which means no polymorphism found in in vitro raised plants. This suggests that the current study standardised the true-to-type culturing protocol and authenticated the in vitro raised plantlets are remain free from somaclonal variations. PCR marker systems like SCoT and/or ISSR has been successfully validated genetic homogeneity in many tissue culture regenerated plantlets (Bhatia et al., 2011; Rathore et al., 2014; Agarwal et al., 2015). 4. Conclusion The present investigation describes the adventitious shoot regeneration from different explants, PGRs treatments and role of organic additives for H. isora. The microshoots were recovered with GA3 treatments and were successfully elongated and rooted. DNA-based markers, SCoT and ISSR were deployed to analyse the genetic homogeneity between

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