Introduction

Tecomella undulata (Sm.) Seem (family Bignoniaceae), commonly known as Rohida, Desert Teak, or Marwar Teak, is a medicinally and economically important timber tree of hot arid regions (state flower of Rajasthan, India). It has been used in indigenous medicinal systems for a wide range of therapeutic activities like hepatoprotective, antibacterial, antimicrobial and antifungal immunomodulatory, anticancer, cytotoxic, analgesic, anti-inflammatory, anti-obesity, etc. (Kalia et al. 2014). Its other uses include phytoremediation of chromium-contaminated soil (Mathur et al. 2010) and rehabilitation of lignite mine backfills (Kumar et al. 2011). It is a widely accepted tree species in arid zone agroforestry but is heading towards extinction due to overexploitation and has been designated as “threatened” in Rajasthan, India (Tripathi and Jaimini 2002). The older trees are dying due to infestation of wood by borers and wood-decaying fungi while young plantlets are seldom found growing in the field. It is usually desirable to multiply elite trees having desirable silvicultural traits; however, no vegetative propagation methods are available for this species. In vitro propagation has emerged as a powerful technique for large-scale propagation of this slow-growing tree. Attempts have been made to propagate this tree through in vitro approaches using seedling (Nandwani et al. 1995, 1996; Aslam et al. 2006; Singh et al. 2009; Varshney and Anis 2012) as well as mature explants (Arya and Shekhawat 1986; Rathore et al. 1991; Bhansali 1993; Kumari and Singh 2012; Tyagi and Tomar 2013; Chhajer and Kalia 2016).

In vitro methods are being used routinely for mass multiplication of selected genotypes, conservation of rare and threatened species, and species with no or recalcitrant seeds and large-scale production of phytochemicals (Pal et al. 2009; Kalia 2015). However, occurrence of somaclonal variation during in vitro propagation is a serious limitation in the practical applications of the plant tissue culture technique. The primary factors controlling induction of somaclonal variation in vitro include culture method, explants source, ploidy level, and in vitro culture age (Rani and Raina 2000); high concentrations of growth regulators used (Venkatachalam et al. 2007); cell cycle disturbances caused by exogenously applied growth regulators (Peschke and Phillips 1992); increased mutation rate per cell-generation over time and accumulation of mutations over a period of time (Rodrigues et al. 1998); alteration in DNA methylation patterns, DNA damage, and mutation (Phillips et al. 1994); and alteration of cell’s ability to repair damaged and mutated DNA (Leroy et al. 2000). The variability detected is a combined effect of genetic and epigenetic variations occurring during the course of culture and the genetic heterogeneity of the cells of explants (Bhojwani and Dantu 2013).

Assessment of genetic homogeneity of in vitro-raised plants at initial stage of ex vitro transfer can help in minimizing the propagation of somaclones. Therefore, suitability of developed micropropagation protocol must be scrutinized critically especially in perennials where commercial success of micropropagation depends solely on the maintenance of clonal uniformity (Heinz and Schmidt 1995). Morphological and physiological traits have been used for fidelity testing in many species (Agnihotri et al. 2009; Singh et al. 2012a, b); however, these morphological evaluations require field maintenance of plants till maturity with extensive observations. Further, the morphological and physiological differences may disappear over the growing seasons or the initially uniform looking plants may behave differently during flowering/fruiting stages due to genetic aberrations. Also, some changes induced during in vitro culture may not be apparent under ex vitro conditions (Palombi and Damiano 2002). Nowadays, more efficient DNA markers are available which can detect variations irrespective of the age or tissue of the plant, growth stage, or the prevailing environmental conditions. In the last two decades, many DNA-based markers like RAPD, ISSR, SCoT, AFLP, SAMPL, and SSR have been successfully used for assessment of genetic homogeneity of tissue culture-raised plants. It has been suggested to use a combination of two or more marker types for genetic fidelity testing of plants so as to target a larger part of the genome under study (Singh et al. 2013a, b). Therefore, in the present study, four marker systems viz. RAPD, ISSR, SCoT, and SSR were used to assess genetic homogeneity of tissue culture-raised plants of an endangered tree species, T. undulata. To the best of our knowledge, there is no report on the application of ISSR, SCoT, and SSR markers in the analysis of genetic homogeneity of micropropagated plants of T. undulata.

Materials and methods

Plant material and culture conditions

The plant material consisted of in vitro-raised shoots maintained on cytokinin-supplemented multiplication medium for 3 months to 2 years and in vitro-raised plants transferred to the field after rooting, hardening, and acclimatization. These plants were regenerated via enhanced axillary branching of nodal explants collected from a 31-year-old tree of T. undulata (Chhajer and Kalia 2016). The micropropagation procedure consisted of culturing the nodal explants on BAP-supplemented MS medium, transfer of shoot clumps to multiplication medium (SH + 5 μM BAP + 5 μM Kinetin), rooting of shoots on auxin-supplemented media (SH + 10 μM IBA), and finally hardening and acclimatization of in vitro-raised plants in the greenhouse before field transfer. Details of the micropropagation procedure are summarized in Fig. 1. The shoot cultures were maintained continuously on multiplication medium for approximately 2 years (32 passages) with regular subculturing to fresh medium every 3 weeks. Shoots were randomly collected from 5 culture flasks after every four subcultures starting from the 4th to 32nd subculture passage for testing the effect of continuous cytokinin exposure duration on genetic homogeneity of shoots. In addition, leaf samples of two randomly selected field-transferred plants (4 months after transfer) were also collected for analysis.

Fig. 1
figure 1

Micropropagation protocol for in vitro multiplication of Tecomella undulata through axillary bud proliferation from mature nodal explants

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DNA extraction and PCR amplification

Total genomic DNA of the mother plant, in vitro-raised shoots, and field-transferred plants was extracted using the modified CTAB method (Doyle and Doyle 1990). The yield and purity of the isolated DNA was determined electrophoretically on 0.8 % agarose gel and spectrophotometrically at the wavelength of 260 nm using Biophotometer (D30, Eppendorf, Germany).

In order to target a wider coverage of the genome, four sets of primers including arbitrary (RAPD), semi-arbitrary (ISSR, SCoT), and sequence based (SSR) markers were used in the present study. Twenty-five primers each of RAPD, SCoT (Integrated DNA Technologies Inc.), and ISSR (Life-Technologies, India Pvt. Ltd.) and 56 SSR (Sigma Aldrich Chemicals Pvt. Ltd., Banglore, India) were used for genetic homogeneity testing. In this study, 25 out of 36 SCoT primers (developed by Collard and Mackill (2009)) were screened to evaluate the genetic fidelity of micropropagated plants. Due to absence of SSR sequences of T. undulata in the public domain, cross-species SSR markers available from other members of the family Bignoniaceae (Arrabidaea chica (Figueira et al. 2010—9 primers); Incarvillea sinensis (Yu et al. 2011—8 primers); Jacaranda copaia (Vinson et al. 2008—8 primers), (Jones and Hubbell 2003—10 primers); Tabebuia aurea (Braga et al. 2007—21 primers)) were used.

DNA amplification was performed in an Eppendorf thermocycler (Mastercycler Nexus GSX1). Details of PCR reaction mix and PCR thermal cycler conditions are given in Table 1A and B, respectively. The agarose (Amresco, USA) gels with 1× TAE buffer were stained with ethidium bromide (10 μl/100 ml gel; Sigma-Aldrich Chemicals Pvt. Ltd., Banglore, India), run under a steady voltage of 110 V for 120 min and then photographed under the gel documentation system (Alphaimager EC, Protein Simple, CA, USA). In all agarose gels, GeneRuler 100 bp plus DNA ladder (Thermo Scientific, EU, Lithuania) was used as size marker.

Table 1 PCR conditions for different molecular markers assayed
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To ensure the consistency of results, all reactions were performed twice. Only consistently produced, unambiguous, and well-resolved amplicons produced through amplification of RAPD, ISSR, SCoT, and SSR markers were scored manually. All markers were treated as dominant for the purpose of scoring which was done on the basis of presence (“1”) or absence (“0”) of amplicons in the gel. NTSYS-pc version 2.1 (Rohlf 2000) was used to evaluate genetic associations by calculating the Jaccard’s similarity coefficient for pairwise comparisons based on the proportion of shared bands produced by the primers.

Results and discussion

Micropropagation protocol was developed for clonal multiplication of T. undulata via axillary bud proliferation from mature nodal explants (Figs. 1 and 2; Chhajer and Kalia 2016). Morphological aberrations like leaf size, shape and color, branching pattern, or change in multiplication rate were not observed during propagation of shoots maintained on cytokinin-enriched medium for over 2 years. In vitro regeneration through axillary bud proliferation has minimum risk of generation of somaclonal variations as organized (pre-existing) meristems develop into shoots without involving dedifferentiation or redifferentiation of cells or tissues into meristems (de novo organogenesis). Morphological stability of the in vitro-raised plants was also reported by Singh et al. (2012b, 2013b) in Dendrocalamus asper; however, Lakshmanan et al. (2007) documented morphological variation (e.g., hyperhydricity) in banana due to use of high levels of cytokinins. Release of cytotoxic by-products by various hormones in tissue culture media act as a stress which induces programmed loss of cellular controls (Phillips et al. 1994).

Fig. 2
figure 2

Micropropagation of Tecomella undulata through axillary bud proliferation from mature nodal explants a mother tree, b nodal explants, c induction of axillary buds, d multiplication of shoots, e rooting, and f 1-month-old pot-transferred plantlet

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DNA-based molecular markers have emerged as a powerful technique for the purpose and therefore are being used in many crops and trees (Singh et al. 2013a, b; Rathore et al. 2014; Agarwal et al. 2015). Previously, only Kumari and Singh (2014) have reported the assessment of genetic stability of tissue culture-raised shoots of T. undulata using RAPD markers. However, chances of occurrence of mutations outside the priming sites of RAPD markers cannot be ruled out. A better analysis of genetic stability of plants can be achieved by using more than one DNA amplification technique allowing increased possibilities for identification of genetic variations. Therefore, a set of 131 primers involving four marker systems (RAPD, ISSR, SCoT, and SSR) were used in the present study. Screening for variations in the whole genome was ensured by selection of marker systems having the ability to target different regions of the genome including coding as well as non-coding regions. RAPD (arbitrary, dominant, decamers) and ISSR (semi-arbitrary, medium to highly reproducible, dominant, and more stringent) markers are capable to scan the whole genome randomly and quickly while SSRs, the highly polymorphic and reproducible markers being sequence based, can detect variation at pre-determined sites such as repetitive regions of the genome (Singh et al. 2013b). On the other hand, SCoT being a gene-targeted DNA marker based on the short conserved region flanking the ATG translation start codon (Collard and Mackill 2009) specifically targets only the coding regions. Rathore et al. (2011) and Lakshmanan et al. (2007) suggested the use of more than one marker system for better analysis of genetic stability of plants to target different regions of the genome. Cuesta et al. (2010) and Singh et al. (2013a, b) also used four marker systems for ascertaining the genetic fidelity of micropropagated Pinus pinea and bamboos, respectively.

The carry-over effect of growth regulators is a well-known phenomenon; therefore, the in vitro-raised shoots maintained for more than 2 years on cytokinin-enriched medium, and also plants after 4 months of field transfer, were tested to address the issue of carry-over effect of growth regulators on genetic integrity, if any. Shoots were collected after every 4 subculture cycles starting from the 4th cycle to the 32nd cycle (3–24 months). The fingerprinting profiles of the in vitro shoot cultures, regenerated plantlets, and the mother plant generated using the RAPD, ISSR, SCoT, and SSR markers are shown in Fig. 3, and their scoring data is summarized in Tables 2, 3, 4, 5, and 6. A total of 21 (out of 25) random decamer oligonucleotides showed amplification of 71 scorable DNA fragments, in the size range of 200 to 3000 bp (Table 3; Fig. 3a). The number of fragments for each primer varied from 1 to 6, with an average of 3.38 DNA fragments per RAPD primer. All the fragments were monomorphic in nature. Out of 25 ISSR primers used for the genetic homogeneity testing, 20 primers produced amplification with 93 scorable fragments in the size range of 200–2000 bp (Table 4). Figure 3b is a representative example of monomorphic fragments obtained with ISSR primers. More fragments were amplified by ISSR primers (4.65/primer) compared to RAPD (3.38/primer), which is in line with results of Singh et al. (2013a, b) but contrary to the results of Cuesta et al. (2010) wherein ISSR markers amplified fewer bands compared to RAPD markers. Singh et al. (2013a, b) and Cuesta et al. (2010) reported a DNA fragment size range of 200 to 3000 bp and 500 to 3000 bp respectively for both RAPD and ISSR primers while in the present study size of fragments varied from 200 to 3000 bp with RAPD and 200–2000 bp for ISSR primers.

Fig. 3
figure 3

Genetic homogeneity testing of in vitro-raised shoots/plants of Tecomella undulata. Amplification of DNA with a RAPD (OPA 04), b ISSR (ISSR 44), c SCoT (SCoT 28), and d SSR (Jc 28) primers. Lanes: M 100 bp plus DNA ladder; 1 mother plant, 2–9 in vitro cultured shoots from the 4th (3 month) to 32nd (24 months) passage after every 4 subculture cycles, 10–11 in vitro-raised plants transferred to pots (4-months old)

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Table 2 Summary of results obtained with various molecular markers assayed for evaluating the genetic homogeneity of micropropagated plants of Tecomella undulata
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Table 3 Random amplified polymorphic DNA (RAPD) primers used for testing the genetic homogeneity of micropropagated plants of Tecomella undulata
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Table 4 Inter-simple sequence repeat (ISSR) primers used for testing the genetic homogeneity of micropropagated plants of Tecomella undulata
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Table 5 Start codon targeted (SCoT) primers used for testing the genetic homogeneity of micropropagated plants of Tecomella undulata
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Table 6 Simple sequence repeat (SSR) primers used for testing the genetic homogeneity of micropropagated plants of Tecomella undulata
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SCoT primers (22 out of 25 tested) amplified a total of 94 DNA fragments with an average of 4.27 fragments per primer. The number of fragments in the selected primers varied from one (SCoT 31) to nine (SCoT 21) in the size range of 200 to 3000 bp (Fig. 3c; Table 5). No polymorphic fragments were observed with any of the SCoT primers used. This start codon targeted polymorphism detection technique reported by Collard and Mackill (2009) for the first time has been used in genetic fidelity testing in some recent reports only (Rathore et al. 2014; Agarwal et al. 2015). Rathore et al. (2014) reported amplification of 2–7 fragments (average 4.3/primer) with SCoT primers in the size range of 200–1500 bp in Cleome gynandra while Agarwal et al. (2015) reported amplification of 2–10 fragments (average 5.3/primer) in the size range of 100–1100 bp. Information generated by SCoT primers is usually derived either from the gene itself or its immediate flanking sequences compared to RAPD or ISSR where information is usually based on the non-coding regions of DNA. Therefore, SCoT marker technique being correlated to functional genes and their corresponding traits (Collard and Mackill 2009; Xiong et al. 2011) can be of better use for gene tagging studies. The microsatellite markers used in the present study were adopted from other members of the family Bignoniaceae (Figueira et al. 2010; Yu et al. 2011; Vinson et al. 2008; Jones and Hubbell 2003; Braga et al. 2007) in the absence of T. undulata sequences in public domain. Amplification was obtained with 14 out of the 56 cross-species SSR markers used. A total of 42, all monomorphic, DNA fragments ranging in size from 100 to 1100 bp were produced (Fig. 3d; Table 6). Various workers have already proved the suitability of genomic and cross-species SSRs (Pandey et al. 2012; Singh et al. 2013a) for genetic fidelity studies.

Among the four marker systems used, ISSR primers produced the maximum average number (4.65) of DNA fragments per primer while SSR primers produced the minimum (3.0). The scoring data of well-resolved fragments of RAPD, ISSR, SCoT, and SSR markers when subjected to calculation of similarity matrix based on Jaccard’s similarity coefficient showed that pair-wise value of the in vitro cultures, regenerated plantlets, and the mother plant was 1, indicating 100 % similarity. This study confirmed that T. undulata cultures can maintain genetic homogeneity and remain free from somaclonal variations over a culture period extending up to 2 years, when multiplied through axillary bud proliferation. Organized (pre-existing meristems) cultures especially shoot tips and axillary buds are known to maintain strict genotypic and phenotypic stability compared to de novo originating meristematic structures like adventitious buds differentiating from callus or directly from cultured tissues (Singh et al. 2013b). Propagation by axillary buds circumvents the dedifferentiation or redifferentiation of cells or tissues, avoiding genomic aberrations and consequently maintenance of clonal fidelity of in vitro-raised plantlets (Negi and Saxena 2010). Length of in vitro culture maintenance has been variable in different plants—4 years (almonds; Martins et al. 2004), 44 months (Swertia chirayita, Joshi and Dhawan 2007), and 2 years (D. asper; Singh et al. 2013b). Initiation of in vitro cultures of T. undulata is difficult being season specific, prone to persistent contamination, physiological status of explants, etc. (Chhajer and Kalia 2016). Therefore, retention of clonal uniformity for prolonged period under in vitro conditions has immense commercial significance. The developed protocol can ensure continuous supply of genetically uniform plants over a prolonged duration without resorting to initiation of fresh cultures frequently, thus reducing the overall cost of plant production at commercial scale.

The four marker systems (RAPD, ISSR, SCoT, and SSR) used in this study targeted different regions of the genome; thus, amplification of 300 distinct and scorable fragments by 58.8 % of the assayed markers assured an extensive screening of the genome of T. undulata at various stages of in vitro propagation. The 100 % similarity index obtained between the mother plant, in vitro cultures, and regenerated plantlets with all the four DNA markers used revealed that these PCR-based cost effective, quick to use, easy to apply, and highly polymorphic markers separable on agarose gel can be effectively used for routine and rapid genetic homogeneity testing of in vitro-raised clones. Arbitary primers like RAPD and ISSR have been extensively used in genetic fidelity studies while SSR primers being sequence-based species-specific markers have found limited use. The recently developed SCoT marker is another marker of choice being based on start codon of functional genes. Cuesta et al. (2010) recommended the use of RAPD primers after comparison of RAPD, ISSR, AFLP, and SAMPL markers for assessment of somaclonal variation in micropropagated plants of stone pine.

Conclusions

The present study describes the use of four PCR based marker systems (RAPD, ISSR, SCoT, and SSR) for assessment of genetic stability of in vitro-raised plants of T. undulata, an endangered timber tree of arid regions. Production of monomorphic bands by in vitro cultures maintained on cytokinin-enriched medium for 3 months to 2 years, 4 months of field-transferred tissue culture-raised plants and the mother tree with all the DNA-based markers, confirmed the true-to-type nature of the plants produced. Method of regeneration primarily account for the appearance of somaclonal variations in vitro. Plantlets derived from organized cultures like shoot tips and axillary buds are usually more stable genetically than those obtained through indirect organogenesis from unorganized callus. Therefore, the developed micropropagation protocol based on axillary shoot proliferation can be used for large-scale micropropagation of T. undulata without any risk of genetic instability appearing for at least 32 passages of 3 weeks each or up to 2 years of continuous subculturing, thus can help to fill the gap of demand and supply of planting material. Detection of somaclonal variations at an early stage can help in getting rid of genetically instable plants, thus ensuring filed maintenance of only clonally uniform plants till maturity. This is the first study reporting genetic homogeneity testing of T. undulata maintained in vitro for more than 2 years using four DNA marker systems.