Assessment Of Clonal Fidelity Research Articles

In vitro propagation and clonal fidelity assessment of Rhynchostylis retusa (L.) Blume: a threatened ornamental and medicinal orchid

In vitro propagation and clonal fidelity assessment of Rhynchostylis retusa (L.) Blume: a threatened ornamental and medicinal orchid

Indirect shoot regeneration from root explants, assessment of clonal fidelity of regenerated plants using SCoT primers and antioxidant analysis in Thottea siliquosa (Lamk.) Ding Hou

Indirect shoot regeneration from root explants, assessment of clonal fidelity of regenerated plants using SCoT primers and antioxidant analysis in Thottea siliquosa (Lamk.) Ding Hou

Control of media browning during micropropagation and assessment of biochemical and clonal fidelity of in vitro-derived and mother plants in Thottea siliquosa (Lamk.) Ding Hou., an important ethnomedicinal shrub

BackgroundThottea siliquosa (Lamk.) Ding Hou., an important medicinal shrub, is widely used in both ayurvedic and indigenous systems of medicine. Root being the most useful part, the plant is constantly uprooted and thus puts pressure on the natural population. Until date, no micropropagation study is available in this plant. The objective of the study is to develop an efficient in vitro propagation protocol and assessment of clonal fidelity of T. siliquosa. ResultsMedia browning was a serious issue during micropropagation, and the addition of 40.0 mg/L ascorbic acid reduced the media browning. For direct shoot regeneration, the optimum response (92% frequency with 20.9 shoots per explant) was obtained when 7-day-old cotyledons were cultured on WPM supplemented with 1.0 mg/L thidiazuron and 0.25 mg/L α-naphthalene acetic acid. The cultures were transferred to WPM augmented with 0.4 mg/L thidiazuron for shoot elongation and growth. On this medium, 100% of cultures responded with a mean number of 27.6 shoots. For callus induction, MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L N6-benzylaminopurin was used. Shoot organogenesis was initiated on the same medium, and calli with minute shoots were transferred to MS medium fortified with 0.5 mg/L N6-benzylaminopurin and 0.25 mg/L α-naphthalene acetic acid for highest shoot regeneration (100% cultures responded with a mean number of 26.5 shoots per explant). Maximum rooting frequency (82%) and number (20.8) were obtained on half-strength MS medium with 1.0 mg/L indole-3-butyric acid. The rooted plants were acclimatized and transferred to the field. The HPTLC and SCoT analysis revealed the phytochemical and clonal similarity between the in vitro propagated plants and mother plant. ConclusionsIn this study, it is confirmed that cotyledon is an excellent explant for direct and indirect shoot organogenesis in T. siliquosa. For direct shoot induction WPM and indirect organogenesis, MS medium was found to give better response. The true-to-type nature of in vitro-derived plants were confirmed by phytochemical and SCoT analysis. The protocol described here could be used for the large-scale propagation of elite clones of T. siliquosa.

Transverse thin cell layer (tTCL) technology: a promising tool for micropropagation of Centratherum punctatum Cass.

The present study describes an efficient protocol for the micropropagation of Centratherum punctatum Cass. through transverse thin cell layers (tTCLs). The effect of plant growth regulators (PGRs), thickness of tTCLs, and source of tTCLs were evaluated. The tTCLs of varying thickness (0.5 to 5.0 mm) were excised from 10-d-old leaf and 45-d-old node and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of PGRs (BAP, TDZ, KN; 0.1 to 2.0 mg L−1), alone or in combination with NAA (0.1 to 1.0 mg L−1) for shoot induction. The leaf and node tTCLs responded with direct shoot regeneration. A significant effect of thickness or width on shoot induction was observed. For leaf, 1.0-mm-wide and for node 2.0-mm-thick tTCLs showed a maximum response. MS medium supplemented with 1.5 mg L−1 TDZ and 1.0 mg L−1 BAP in combination with 0.2 mg L−1 NAA was found to be optimum for shoot induction from tTCLs of leaf (96% response with 41.5 shoots per explant) and node (87% response with 11.9 shoots per explant), respectively. Rhizogenesis was obtained when micropropagated shoots were transferred to half strength MS medium supplemented with various concentrations (0.25 to 2.0 mg L−1) of IBA. The rooted plantlets were eventually acclimatized and transferred to soil. The clonal fidelity assessment by SCoT markers of the mother plant and micropropagated plants revealed their genetic similarity. Chlorophyll content estimation indicated lower levels in in vitro plants, which increased to a comparable level to that of field-grown plants after 3 mo of acclimatization, showing absence of stress during acclimatization. The procedure described here is a promising tool for micropropagation of C. punctatum as it produces high-frequency healthy shoots from a minimum explant source.

Biotechnological interventions and indole alkaloid production in Rauvolfia serpentina.

Rauvolfia serpentina (L). Benth. ex Kurz. (Apocynaceae), commonly known as Sarpagandha or Indian snakeroot, has long been used in the traditional treatment of snakebites, hypertension, and mental illness. The plant is known to produce an array of indole alkaloids such as reserpine, ajmaline, amalicine, etc. which show immense pharmacological and biomedical significance. However, owing to its poor seed viability, lesser germination rate and overexploitation for several decades for its commercially important bioactive constituents, the plant has become endangered in its natural habitat. The present review comprehensively encompasses the various biotechnological tools employed in this endangered Ayurvedic plant for its in vitro propagation, role of plant growth regulators and additives in direct and indirect regeneration, somatic embryogenesis and synthetic seed production, secondary metabolite production in vitro, and assessment of clonal fidelity using molecular markers and genetic transformation. In addition, elicitation and other methods of optimization of its indole-alkaloids are also described herewith. KEY POINTS: • Latest literature on in vitro propagation of Rauvolfia serpentina • Biotechnological production and optimization of indole alkaloids • Clonal fidelity and transgenic studies in R. serpentina.

Callus mediated in-vitro plant regeneration and clonal fidelity assessment of Lilium longiflorum cv. “Pavia” by ISSR markers

Aim: Developing a protocol for mass multiplication of Lilium (Lilium longiflorum) bulbs via indirect organogenesis and somatic embryogenesis for in-vitro plant regeneration and ISSR marker-based mapping to assess the fitness of true to type. Methodology: To induce organogenic callus, six combinations of 2,4-D and BAP were tested; while nine combinations of picloram and NAA were used for somatic embryogenesis. NAA and IBA were tested for root induction. The clonal fidelity of the in-vitro regenerated plantlets from both calluses were tested using polymerase chain reaction (PCR)-based ISSR markers analysis. All experiments were arranged in a complete randomized block design and replicated three times. Results: Two methods (direct and indirect organogenesis) were investigated to assess the best callus mediated plant regeneration for Lilium bulb multiplication. The maximum organogenic callus induction and the highest regeneration percent was recorded with BAP (0.5 mg l-1) + 2,4-D (3.0 mg l-1). However, MS medium containing 0.50 mg l-1 picloram and 0.20 mg l-1 NAA was found best for initiation of embryogenic callus and proliferation and was found superior over direct organogenesis. Clonal fidelity was assessed through ISSR markers comparing the mother plant and regenerated plantlets. Interpretation: Both organogenic callus andembryogenic callus are capable of developing true to type in-vitro plants and can be explored for mass multiplication of Lilium bulbs. Embryogenic callus can further be utilized in liquid suspension based bioreactor system. The present protocol has potential applications in micropropagation and genetic transformation studies in other Lilium spp.

Genetic clonal fidelity assessment of rhizome-derived micropropagated Acorus calamus L. – A medicinally important plant by random amplified polymorphic DNA and inter-simple sequence repeat markers

Background: Acorus calamus – a critical medicinal plant, is overexploited, leading to population reduction. Establishing an efficient in vitro protocol is essential for the large-scale production of genetically identical plants. Objectives: Development of fast and reliable in vitro regeneration protocol for A. calamus and clonal fidelity assessment of the regenerants using molecular markers. Materials and Methods: Plants were regenerated on Murashige and Skoog medium with different concentrations of growth regulators in two phases – shooting and rooting. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic stability of in vitro clones. Results: 6 Benzylaminopurine (BAP) at 1.6 and 2.4 mgL−1 was effective for shoot induction, while root induction was superior in indole-3-butyric acid-incorporated medium at 2.5 mgL−. Thirteen RAPD and 16 ISSR primers produced 59 and 96 clear, unambiguous, and reproducible bands, respectively. Both the markers revealed a high monomorphism of 96.79% and 95.63% among the regenerants. Nei's genetic distance analysis disclosed a close genetic association (0.000–0.068) among the genotypes. Conclusion: ISSR was better than RAPD markers in clonal fidelity assessment of the regenerants. The in vitro protocol developed is reliable and suitable for the rapid propagation of true-to-type A. calamus plants.

Assessment of clonal fidelity and phytomedicinal potential in micropropagated plants of Bulbophyllum odoratissimum - An endangered medicinal orchid of Indo Burma megabiodiversity hotspot

Bulbophyllum odoratissimum (Sm.) Lindl. ex Wall. is widely used as traditional medicine for the treatment of tuberculosis, chronic inflammation and is listed as an endangered orchid due to over-exploitation and habitat destruction. The present study was carried out to establish a genetically stable in vitro regeneration protocol for mass multiplication using nodal segments and to determine phytochemical constituents, the antioxidant capacity of different parts (leaf, pseudobulb and root) of mother plants and acclimatized in vitro raised plants of B. odoratissimum. The synergistic effect of BA (4.0 mgl−1) and IBA (0.5 mgl−1) was found to be optimum for producing maximum shoot proliferation. IBA was found to be more effective for root proliferation than IAA, NAA and an average (3.08 roots/shoot) was obtained in 0.5 mgl−1 IBA. The well rooted plantlets were successfully acclimatized (more than 90% survival rate) in greenhouse, exhibiting normal development. The ISSR banding profiles confirmed high degree of genetic homogeneity among the clones. Extraction of all plant material from both mother plant and in vitro raised plant possess relevant TPC, TFC, radical scavenging activity (DPPH and ABTS) as well as total reducing power ability. HPLC chromatogram exhibited that phytochemical compounds detected in regenerated plants were similar to those present in mother plant and there was no remarkable difference. High regeneration frequency along with its genetic stability and secondary metabolite production validate the efficiency of this micropropagation protocol of B. odoratissimum for species recovery program as well as a potential source of commercial application.

Micropropagation and clonal fidelity assessment of acclimatized plantlets of Crotalaria longipes Wight & Arn. using ISSR markers

An efficient protocol for micropropagation of Crotalaria longipes Wight & Arn., an endemic and endangered Indian legume, was developed. Nodal explants cultured on Murashige and Skoog (MS) supplemented with Thidiazuron (TDZ) 1.0 mg L−1 and NAA 0.5 mg L−1 showed maximum shoot induction. The multiplied shoots were subsequently sub-cultured every 3 weeks on the same medium. The separated microshoots cultured on half-strength MS basal salts supplemented with 0.5 mg L−1 indole-3-butyric acid (IBA) showed maximum root induction. After 4 weeks of culture, the rooted shoots (> 6 cm) were planted in pots containing coco peat and garden soil (1:1) covered with plastic domes and maintained at 25 oC for 2 weeks. After 2 weeks, the potted plants were transferred to a glasshouse, maintained at 25 °C with a relative humidity of 80%. The acclimatized plants were then successfully established in soil with an 80% survival rate. Clonal fidelity assessment of acclimatized plantlets was confirmed using inter simple sequence repeat (ISSR). All the in vitro developed plants showed monomorphic banding pattern similar to the mother plant, thus ascertaining the true-to-type nature of the in vitro raised plants.

In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.)

In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.)

Recurrent secondary embryogenesis in androgenic embryos and clonal fidelity assessment of haploid plants of Tea, Camellia assamica ssp. assamica and Camellia assamica ssp. lasiocaylx

Globular androgenic haploid embryos of TV21 and TV19 cultivars of Camellia ssp., obtained on embryo induction medium (EIM), Murashige and Skoog medium with 10 µM 6-benzylaminopurine (BAP), 3 µM gibberellic acid (GA3), 800 mg l−1 L-glutamine and 200 mg l−1 L-serine, were pretreated with 18 µM abscisic acid (ABA) and 25 g l−1 mannitol for 20 days in liquid medium to attain maturation. The pre-treatment favoured the primary globular embryos to grow and pass through all the developmental stages from globular, heart-shape, torpedo shape to fully matured docotyledonous embryos. Fivefold and threefold increase in secondary embryogenesis (SE) was attained in Camellia assamica ssp. assamica (TV21; Assam Type) and Camellia assamica ssp. lasiocaylx (TV19; Cambod Type), respectively, when pre-treated primary embryos were transferred to the liquid embryo development (ED) medium, supplemented with tenfold reduced concentration of growth regulators that were present in EIM. Embryos were germinated well on 10 µM BAP, 0.5 µM GA3, 1 µM indole-3-butyric acid (IBA), 80 mg l−1 L-glutamine and 20 mgl−1 L-serine, following liquid-to-semi-solid transition method. Well developed shoots and roots were formed when embryos were initially kept for four weeks in liquid germination medium, followed by their transfer to semi-solid medium on similar medium composition. The method promoted recurrent secondary embryogenesis in lesser time with highest germination rate, 66.6% and 30.3% in TV21 and TV19 cultivars, respectively. Histology and field emission scanning electron microscopy (FESEM) were performed to determine ontological events during embryo development. Clonal fidelity of haploid plantlets was assessed using random amplified polymorphic DNA (RAPD) to ensure no somaclonal variations occurred during the multistep process of secondary embryogenesis, embryo maturation and germination. A two-step transition protocol from liquid-semi-solid medium is beneficial for embryos to overcome hyperhydricity. Secondary embryogenesis is the best mode to obtain genetically uniform true-to-type haploid plantlets.

Micropropagation through indirect organogenesis in Solanum capsicoides All., and assessment of clonal fidelity using ISSR markers

Solanum capsicoides All., is an important medicinal plant which is used as a source of ‘Kantakari’ (medicine for phlegm disorders) in India. The restricted distribution of the plant in Kerala along with its overexploitation necessitates the development of a protocol for in vitro propagation of S. capsicoides. The present study reports the effects of four different cytokinins (2 isopentenyl adenine (2iP), benzyl adenine (BAP), kinetin (KIN), and zeatin (ZEA)) in combination with indole 3-butyric acid (IBA) on indirect organogenesis of S. capsicoides from in vitro grown seedling explants such as cotyledon, shoot tip, and hypocotyl. Maximum shoot development was observed from cotyledon explants inoculated on MS medium supplemented with 2.46 μM IBA and 4.56 μM ZEA. An average of 17 ± 0.5 shoots per cotyledon with an average length of 0.53 ± 0.03 cm was observed. After subculture, an average of 20 ± 1 shoots was observed in cotyledon explants inoculated on Murashige and Skoog medium supplemented with 2.46 μM IBA and 6.84 μM ZEA. Clonal fidelity of regenerated plants was confirmed using DNA based molecular marker, intersimple sequence repeat (ISSR). Clonal fidelity test revealed 0% polymorphism and the PIC value 0 which indicate no diversity from seed-borne plants. The protocol thus developed can be used for rapid multiplication and conservation of S. capsicoides.

Shoot organogenesis and assessment of clonal fidelity of regenerated plants of Ocimum tenuiflorum L.: Queen of Herbs

An efficient and reliable shoot organogenesis protocol was developed from leaf explants taken from microshoots of Ocimum tenuiflorum L. Cultures were established using nodal explants taken from field grown mature plant on MS medium supplemented with 6-benzyladenine (BA; 2.5 μM). The effect of different cytokinins namely BA, kinetin (KIN) and thidiazuron (TDZ) in combination with 0.5 μM α-naphthalene acetic acid (NAA) was examined on shoot proliferation and elongation. Maximum number of shoots per culture vessel (38) was recorded on MS medium supplemented with 5.0 μM BA, whereas the maximum number of elongated shoots per culture vessel (15) along with maximum shoot length (4.80 cm) was observed on medium supplemented with 2.5 μM BA. Shoot organogenesis was attempted from the segments of fully expanded leafs and 34.45% explants regenerated shoots on MS medium supplemented with 5.0 µM each of BA and NAA. Incorporation of cefotaxime (300 mg/l) was found to be beneficial for shoot organogenesis and further increased shoot regeneration frequency to 36% explants. On the other hand, addition of carbenicillin into the medium was found to inhibit shoot organogenesis. For root induction in microshoots, among various auxins used, IBA was found to be the best. A pulse treatment of 50.0 μM IBA for 36 h improved rooting of microshoots and a maximum of 89% microshoots rooted. Rooted microshoots were successfully acclimatized under polyhouse conditions with a survival rate of 80%. Later acclimatized plants were established in their natural conditions. Random Amplified polymorphic DNA and Inter Simple Sequence Repeat markers established a clonal fidelity of micropropagated plants with that of mother plant.

Biotechnological advancements in Catharanthus roseus (L.) G. Don.

Catharanthus roseus (L.) G. Don, also known as Madagascar periwinkle or Sadabahar, is a herbaceous plant belonging to the family Apocynaceae. Being a reservoir for more than 200 alkaloids, it reserves a place for itself in the list of important medicinal plants. Secondary metabolites are present in its leaves (e.g., vindoline, vinblastine, catharanthine, and vincristine) as well as basal stem and roots (e.g., ajmalicine, reserpine, serpentine, horhammericine, tabersonine, leurosine, catharanthine, lochnerine, and vindoline). Two of its alkaloids, vincristine and vinblastine (possessing anticancerous properties), are being used copiously in pharmaceutical industries. Till date, arrays of reports are available on in vitro biotechnological improvements of C. roseus. The present review article concentrates chiefly on various biotechnological advancements based on plant tissue culture techniques of the last three decades, for instance, regeneration via direct and indirect organogenesis, somatic embryogenesis, secondary metabolite production, synthetic seed production, clonal fidelity assessment, polyploidization, genetic transformation, and nanotechnology. It also portrays the importance of various factors influencing the success of in vitro biotechnological interventions in Catharanthus and further addresses several shortcomings that can be further explored to create a platform for upcoming innovative approaches. KEY POINTS: • C. roseus yields anticancerous vincristine and vinblastine used in pharma industry. •In vitro biotechnological interventions prompted major genetic advancements. • This review provides an insight on in vitro-based research achievements till date. • Key bottlenecks and prospective research methodologies have been identified herein.

Improvement of germination rate and in vitro multiplication of Lavandula angustifolia

Establishment of in vitro regeneration from petiole explants and assessment of clonal fidelity by ISSR markers in Luffa acutangula L.RoxbYashodhara Velivela, Muralikrishna Narra, Raghu Ellendula, Srinivas Kota, Sadanandam Abbagani

High-frequency direct shoot organogenesis from garlic (Allium sativum L.) inflorescence and clonal fidelity assessment in regenerants

Garlic (Allium sativum L.) is an important bulb vegetable with high culinary and medicinal values. It is cultivated by exclusively vegetative propagation which leads to complex virus infection, biodiversity reduction, inhibition of multiple breeding strategies. An efficient and widely applicable shoot regeneration system was established from garlic inflorescence as explant. By optimizing the basal medium type, pH value and explant size, a mean shoot regeneration rate of 97% and mean shoot number of 23.4 per explant were achieved in 14 commercial cultivars with various characteristics and origins. Histological observation revealed that shoots were regenerated through direct organogenesis when meristemoid initiated from subepidermal cells without callus formation followed by periclinal and anticlinal division of epidermal and subepidermal cells. No polymorphic bands were detected by simple sequence repeat (SSR) analysis between regenerants and donor plants. Furthermore, flow cytometry analysis indicated that there was no significant variability of genome size, and all plants maintained their ploidy. These results confirmed the clonal fidelity of regenerants. In conclusion, the present study provides a shoot regeneration system with great potential in micropropagation, germplasm preservation, genetic transformation and ploidy manipulation of garlic. Our study developed a widely applicable procedure for direct shoot regeneration via inflorescence of garlic and ascertained the clonal fidelity of regenerants by using simple sequence repeat (SSR) and flow cytometry.

TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry

In order to induce in vitro axillary shoot proliferation from single-node explants of Rhododendron mucronulatum Turcz., two techniques of thidiazuron (TDZ) application were tested: (i) two-step procedure including cultivation on Anderson medium (AM) supplemented with varying TDZ concentrations (0.1 μM; 0.25 μM; 0.5 μM; 1.0 μM; 2.5 μM) for 8 wk followed by cultivation on hormone-free medium (AM0) for 6 wk and (ii) 4-h liquid-pulse treatment with different TDZ concentrations (7.5 μM, 15.0 μM, or 30.0 μM) followed by cultivation on AM0 for 8 wk. The highest number of axillary shoots per explant was achieved with 0.1-μM TDZ after the two-step procedure. The best response in terms of percent regeneration (87%), shoot length (13 mm), absence of structure anomalies, and the shortest shoot production cycle (8 wk) was obtained with 30.0-μM TDZ liquid-pulse treatment for 4 h. The clonal fidelity of regenerated shoots was evaluated by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers and flow cytometry. Genetic similarity of all regenerants between themselves and with the mother seedlings was 99%. Flow cytometric analysis revealed that all samples studied were diploid. The nuclear DNA content of microshoots obtained under the TDZ treatments varied from 1.26 to 1.32 pg per 2C. There were no significant differences in DNA content among mother seedlings and in vitro developed shoots triggered by 0.1- and 2.5-μM TDZ nor by those triggered by the 30.0-μM TDZ pulse treatment.

Musa sp. paradisiaca Cultivar Gopi and Clonal fidelity Assessment using ISSR Marker

Attempt was made towards optimizing an in vitro shoot multiplication of banana cultivar Gopi with subsequent assessment of genetic stability. Experiments on enhanced shoot multiplication were conducted in MS fortified with BAP, Kn and 2-iP (4 mg/l) for a period of 8 weeks. Administration of exogenous root inducing growth regulator was eliminated to reduce culture investment and economy. Treatment with BAP supplemented medium proved to be optimum for shoot multiplication whereas 2-iP produced satisfactory results for shoot elongation. Long term incubation in all the treatments had been favorable for developing efficient root system. Hardening of in vitro grown plantlets showed high rate of survival (95%) upon transfer to potted soil. Assessment of clonal fidelity through inter simple sequence repeats (ISSR) analysis revealed 100% uniformity.

Efficient slow-growth conservation and assessment of clonal fidelity of Ullucus tuberosus Caldas microshoots

Slow-growth is a biotechnological tool for medium-term conservation of plant germplasm under in vitro conditions. In the present study, we assessed six culture media supplements and two cultivation temperatures to develop a reliable in vitro conservation protocol for ulluco (Ullucus tuberosus Caldas), an Andean tuber crop rich in carbohydrates and vitamin C. One-nodal segments of ulluco were cultivated on half-strength Murashige and Skoog (MS) medium supplemented with mannitol (10–30 g l−1), sorbitol (10–30 g l−1), sucrose (10–120 g l−1), chlorcholinchlorid (CCC; 300–700 mg l−1), abscisic acid (ABA; 1–3 mg l−1), or maleic hydrazide (MH; 0.1–0.5 mg l−1). In vitro cultures were maintained at either 5 °C or 17 °C cultivation temperature. After 18-month cultivation, based on growth characteristics, survival percentage and form of survival (plant, live shoot + microtuber, microtuber + dead shoot), four treatments were selected: 30 g l−1 mannitol|17 °C, 30 g l−1 sorbitol|17 °C, 0.3 mg l−1 MH|17 °C and 60 g l−1 sucrose|17 °C. The plantlets from these treatments were tested for regrowth ability on half-strength MS medium. After 28 days of cultivation, survival percentage and morphological characteristics were evaluated. Plantlets originating from conservation medium supplemented with 30 g l−1 mannitol showed the fastest regrowth and provided overall superior characteristics over shoots from other treatments. These plants were subjected to inter simple sequence repeat analysis, which gave rise to monomorphic patterns indicating no detected genetic variation between plants recovered after conservation on this medium and proving the reliability of this conservation protocol. Slow-growth of ulluco plantlets at 17 °C and the use of mannitol as a culture medium supplement, represents a reliable system of conservation with minimal risk of somaclonal variation.

Assessment of clonal fidelity of doubled haploid line of marigold (Tagetes erecta) using microsatellite markers

In vitro multiplied plants of doubled haploid line of marigold (Tagetes erecta L.) derived through ovule culture, were subjected to evaluation of genetic uniformity using SSR markers in order to evaluate their genetic stability and/ or detect likely existing variations among in vitro derived plantlets. Leaf samples were taken from randomly selected 13 plants at post acclimatization stage, for checking the clonal fidelity of the in vitro derived regenerants by comparing them with the mother plant from which the leaf explants were taken. Out of 36 primers, 12 showed amplification in which clear and reproducible PCR profiles. Analysis of individual primers revealed that SSR primers produced same banding pattern which were shared by the in vitro leaf derived regenerants and the donor mother plant, which indicated that there was no variation observed within the tissue culture raised plants. All the banding profiles produced were monomorphic and were similar to those of the mother plant, and hence, showed that plants were true to type. Information presented in the study indicated that the in vitro regenerated plants were identical to the mother plant.