Single Primer Amplification Reaction Research Articles

Cross section of genetic diversity in mainland and insular populations of Costus speciosus (Koen ex. Retz.) Sm. using SPAR markers reveal patterns linked to allopolyploidy and biogeography

Costus speciosus (Koen ex. Retz.) Sm. is a major source of diosgenin, used for the commercial synthesis of cortisone, sex hormones and contraceptives. The genetic diversity analysis in wild populations of C. speciosus from 3 biogeographic regions viz., Western Ghats (WG), Eastern Ghats (EG) and Andaman and Nicobar Islands (AN) were done using 2 different Single Primer Amplification Reaction (SPAR) methods. A total of 70 accessions spanning these regions were used in the present study. The assay yielded a total of 314 amplicons of which 268 were polymorphic, exhibiting 85.35% of polymorphism. The prevalence of high rate of genetic differentiation (mean Gst = 0.90) and low gene flow (mean Nm= 0.06) are the main attributes of the observed low diversity in these populations. The accessions clustered broadly under 2 major groups corresponding to the three biogeographic zones with insular populations diverse from the mainland. This was further resolved by AMOVA analysis. C. speciosus is found to exist in different cytotypes exhibiting allopolyploidy. The differences in distribution and genetic fitness of the population from EG and WG may be attributed to the allopolyploid nature of the taxa. In the present study, Island populations comprise very low heterozygosity (Ht = 0.10) suggesting that the rate of fixation is more in these populations. Similarly, the rate of gene flow was almost absent (Nm = 0.02). The higher levels of genetic similarity (0.99) may be due to an increase in fixation of the genes resulting from allopolyploidy. This is the first study on comparative genetic diversity of C. speciosus using SPAR markers.

Morphological and molecular markers based assessment of genetic diversity of a valuable endemic plant Lasiurus sindicus Henr. in the arid region of Rajasthan, India

Morphological and molecular markers are useful in genetic diversity assessment and improvement of plants. In the present study, five morphological traits, random amplified polymorphic DNA (RAPD) and Start codon targeted (SCoT) markers were used for genetic diversity evaluation of Sewan grass (Lasiurus sindicus Henr.), a valuable endemic plant of Indian Thar desert. Accessions collected from 14 different sites were used for measuring morphological traits, namely plant height (PH), number of tillers (NT), number of nodes (NN), leaf length (LL) and spike length (SL) which showed large variations among populations; particularly PH, NT and LL. Ward’s linkage hierarchical cluster analysis exhibited two major clusters and Cluster I contained two minor clusters. The percentage polymorphic bands (PPB) produced were 89.64% and 64.51% for RAPD and SCoT respectively. RAPD markers based dendrogram revealed three clades while there was no clear clade formation with SCoT markers. Single primer amplification reaction (SPAR) analysis with combined data of RAPD + SCoT showed more similarity with SCoT than with RAPD clusters. AMOVA revealed higher variation within population (90%) than among populations (10%) of L. sindicus. The Principal coordinate analysis (PCoA) also supported this result. There was no correlation between morphological and molecular data confirming environmental impact on morphological variation. SCoT and SPAR markers have clearly identified a novel genotype (LS JS 3) which has high values of PH, NN and LL. This study suggests that morphological data combined with molecular data can alone help in the identification of novel genotypes of L. sindicus among its populations.

Genetic Diversity Analysis, Population Structure and Gene-flow Estimates among Citrus Macroptera Mont. Collections from Garo Hills, Meghalaya using SPAR Approach

North East India is extremely rich in natural resources and is a biodiversity hotspot. Many plant species, including Citrus are known to have originated from this region. Citus macroptera Mont. is a wild, endangered species which have been found to exist naturally in various parts of this region, including Meghalaya. To access the genetic variability among genotypes and their phylogeny, 30 genotypes of wild C. macroptera Mont. were collected from Garo Hills of Meghalaya. Single primer based DNA markers viz. RAPD, ISSR, DAMD were utilized to ascertain genetic diversity. The percentage polymorphic bands for RAPD, ISSR, DAMD were found to be 97.71%, 94.67% and 100% respectively. ISSR showed the highest values for both RP (7.67) and MI (5.03) highlighting its efficacy in determining genetic variations. A concatenated approach, Single Primer Amplification Reaction (SPAR) was also followed to assess their genetic diversity. Dendogram generated from SPAR data showed that the South-West Garo Hills population is the most recently evolved amongst all others while West Garo Hills collections retain an ancestral position in the evolutionary time-frame. Population genetics parametres such as Gene flow (Nm) and the diversity among populations (GST) were found to be 1.9894 and 0.2009 respectively. Gene flow estimates (Nm>1) suggests appreciable gene flow in the populations. AMOVA data further supported this with high percentage of variations (92%) within populations whereas variations among populations were about 8% only. Shannon’s information index (I) values and Nei’s gene diversity (h) varied between 0.303-0.423and 0.201-0.285 respectively. The use of SPAR method yields a clear and concise picture of the underlying genetic variabilities, and a detailed and comprehensive data analysis will help conceive efficient and sustainable conservation strategies for this important plant.

SPAR methods reveal high genetic diversity within populations and moderate gene flow of pointed gourd (Trichosanthes dioica Roxb.) germplasm

Abstract Pointed gourd (Trichosanthes dioica Roxb.) is one of the most nutritive cucurbitaceous vegetable crops cultivated in tropical and subtropical regions around the world. Poor characterization of the available germplasm, vegetative means of propagation and dioecy are the main constraints in developing a systematic breeding programme in pointed gourd. In this investigation, genetic variability of pointed gourd accessions belonging to four agro-climatic zones of Bengal region of India was evaluated by employing polymerase chain reaction (PCR) based single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and directed amplification of minisatellite DNA (DAMD). Percentage of polymorphic loci, Nei’s gene diversity index (h) and Shannon’s Information index (I) indicated a moderate level of genetic heterogeneity in studied germplasm. The mean coefficient of genetic differentiation (Gst) between populations for RAPD, ISSR and DAMD were 0.419, 0.305 and 0.315 respectively, indicating that 58.1%, 69.5% and 68.5% of genetic variability prevailed within the population. Similarly, analysis of molecular variance (AMOVA) revealed that majority of the genetic variation were partitioned among the individuals within a population. Gene flow estimate (Nm) calculated for RAPD, ISSR and DAMD marker were 0.692, 1.139 and 1.086 respectively indicating a moderate gene flow among the populations. The clustering pattern based on the combined marker systems was primarily related to the geographical distribution of the accessions. This study clearly demonstrates the efficiency of single primer based amplification reactions (SPARs) in duplicate identification, core collection and improvement of pointed gourd germplasm.

Molecular analyses of genetic variability in the populations of Bergenia ciliata in Indian Himalayan Region (IHR).

Bergenia ciliata is an important medicinal plant species of Indian Himalayan Region (IHR). Genetic variability and population genetic structure of B. ciliata sampled from IHR was studied using two single primer amplification reaction (SPAR) methods (DAMD: Directed Amplification of Minisatellite region DNA; ISSR: Inter Simple Sequence Repeats). To provide a reasonable scientific basis for management and conservation of B. ciliata populations in IHR, genetic diversity analysis of 11 populations with 24 SPAR markers (15 ISSR and 9 DAMD) revealed significantly high level of (90.03%) polymorphism at species level. However, genetic variability was low at population level and KUL and BWS populations showed maximum while SHM population revealed least genetic diversity among the 11 populations. Analysis of molecular variance revealed highest percentage of variation (73%) within populations, followed by 17% among populations and least (10%) among the Himalayan regions. Clustering pattern obtained from UPGMA dendrogram was supported by STRUCTURE and principal coordinate analysis, segregating all the 11 natural populations of B. ciliata into two genetic clusters: Eastern and Western Himalayan populations. The clustering patterns of all the three statistical methods indicated that populations of B. ciliata have structured in response to the local micro-climates of the habitats in IHR, and therefore, it can be concluded that genetic variability is in congruence with the geographical diversity.

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

Augmenting a competent in vitro organogenesis etiquette from leaf base of country mallow, Abutilon indicum L. sweet: An ethno-botanically valuable medicinal plant

Abstract An easy, efficient and highly reproducible regeneration system was established through organogenesis from leaf base mediated callus of an ethnobotanical hairy shrub Abutilon indicum L. sweet, which is documented to possess pharmaceutically important phytochemicals. Among various explants, leaf bases produced significant callus induction (89%) with 140 mg fresh weight on Murashige and Skoog (MS) medium supplemented with 11.31 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D). Combinations of 2, 4-D and kinetin (KIN) increased the biomass (191 mg) of embryogenic calli. Regeneration medium with a combination of 8.88 μM of 6-amino benzyl purine (BAP) and 8.06 μM of naphthalene acetic acid in MS basal media significantly influenced the initiation of shoot primordium with 91% of regeneration. Proficiently regenerated shoot apical meristems (SAMs) produced higher frequency of multiple shoot induction on 4.10 μM of zeatin. The fully regenerated mature shootlets produced high root biomass (791.67 ± 48.05 mg) on half - strength MS with 4.54 μM indole-3-butyric acid (IBA) with an excellent mat root system for efficient acclimatization. In vitro established plantlets were successfully acclimatized under ex vitro conditions with high frequency. Presence of homologous DNA banding pattern with Single Primer Amplification Reaction markers confirmed the genetic similarity. FTIR and GC-MS spectrum showed similar metabolic profiles between wild and in vitro regenerated plantlets. Thus the protocol is reliable for in vitro regeneration through leaf base derived callus which could pave a way to the large scale production of secondary metabolite through biotechnological approaches.

Thidiazuron induced in vitro morphogenesis for sustainable supply of genetically true quality plantlets of Brahmi

An effective and rapid method for propagating genetically true plantlets of a pharmaceutically important plant Bacopa monnieri L. was developed from thidiazuron (TDZ) pulse treated nodal segment explants. Explants treated for 24 h with different concentration of 0.0, 5.0, 10, 20 or 40 μM thidiazuron (TDZ) prepared in liquid Murashige and Skoog (MS) medium induces multiple shoot on MS basal medium without growth regulators. The highest frequency 95.3 ± 2.3% of regeneration and maximum number 43.7 ± 2.0 of shoots per explants with an average shoot height of 5.4 ± 0.29 cm were achieved after 08 weeks of culture from the explants treated with 20 μM TDZ. Simultaneously, the regenerated microshoots were rooted on the same growth regulator free media. Elongated shoots with roots were successfully acclimated to ex vitro condition with a 97% survival rate. Genomic stability of in vitro developed plants of B. monnieri was first time determined using flow cytometry and single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA (RAPD), intersimple sequence repeat polymorphic DNA (ISSR) and directed amplification of minisatellite DNA (DAMD) and compared with donor plant. Nuclear DNA (nDNA) content of in vitro propagated B. monnieri plants has been estimated as 2.08 pg/2C based on the flow cytometric investigation of nuclei isolated from leaf tissues. No significant variations in nDNA content, plant genome size and ploidy level were observed between micropropagated plants and field grown donor plants. Likewise, the SPAR markers also revealed monomorphic banding profiles in micropropagated plantlets of B. monnieri and similar with to that of the donor plants verifying their genomic stability.

Assessment of genetic variation in Linum L. using SPAR markers

Flax (Linum usitatissimum subsp. usitatissimum) is an important crop plant cultivated both for its fiber and seed oil content. Uniform cultivation and selection practice may lead to genetic erosion and reduced genetic variability of Linum cultivars, which in turn makes the crop vulnerable to diseases and pathogens. Therefore we performed a genetic diversity analysis of few populations including both Linum usitatissimum subsp. usitatissimum L. and L. Bienne L. We used a combination of single primer amplification reaction markers, including RAPD, ISSR, and RAMPO molecular markers for genetic variability study. In general, all three molecular markers revealed a low degree of genetic variability in the materials studied; however, L. bienne had a higher level of genetic variability. Clustering and network analyses grouped the studied plants inter-mixed and did not separate the studied species or populations. STRUCTURE plot and K-Means clustering also identified these populations as genetically homogenous. These results indicate the need for introducing new linseed cultivars into the country for future hybridization and breeding programmes.

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods.

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.

Assessment of genetic variation and population structure in Indian Gladiolus cultivars inferred from molecular markers

Genetic variability and relationships in 54 genotypes belonging to nine Gladiolus cultivars were determined using single primer amplification reaction methods (Random Amplified Polymorphic DNA—RAPD; Directed Amplification of Minisatellite DNA—DAMD; and Inter Simple Sequence Repeats—ISSR). A total of 30 primers (10-RAPD, 10-DAMD and 10-ISSR) resulted into 230 DNA fragments, of which 177 (75.8 %) were polymorphic in nature. The polymorphic information content (PIC = 0.18), resolving power (RP = 11.2) and marker index (MI = 11.4) was found to be highest in ISSR markers. To understand the extent of genetic variability among the Gladiolus cultivars, the cumulative dataset were used to generate UPGMA dendrogram. All the 54 genotypes clustered together in nine broad clusters, revealing that the clustering pattern of the genotypes were as per their cultivars. PCoA analyses also revealed similar clustering patterns along the axis and therefore corroborating the grouping of genotypes in UPGMA dendrogram. The STRUCTURE analysis showed the admixture of alleles and resulted into eight genetic populations. The present study demonstrates the efficiency and discriminatory potential of an individual as well as cumulative analysis of RAPD, DAMD and ISSR markers to estimate the genetic variability and inter-relationships among indigenously developed Gladiolus cultivars. Furthermore, documentation and preservation of genetically diverse Gladiolus germplasm could be an important source for improvement of this floricultural crop.

Analysis of intraspecific genetic variation in Musa balbisiana Colla from Meghalaya as revealed by Single Primer Amplification Reaction approach

The Indian subcontinent is considered to be the major centre of hybridization of the wild Musa progenies, as it forms the centre of secondary origin of the taxa and therefore expected to exhibit a high degree of diversity. Musa balbisiana Colla is known to be one of the wild progenitors of cultivated bananas and plantains. Knowledge of genetic variability is very important for contemplating any conservation and management programmes. Single Primer Amplification Reaction (SPAR) approach i.e., a combination of random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and directed amplification of minisatellites DNA (DAMD) markers function as important tools for analyzing genetic variation in plants. They collectively provide a comprehensive description of the nature and the extent of existing natural genetic diversity, as the primers target different regions of the genome. A total of 47 SPAR primers (RAPD-25, ISSR-12 and DAMD-10) with high reproducibility were used for analysis of intraspecific genetic variation of the 12 genotypes of M. balbisiana, which yielded 331 polymorphic bands. The RAPD, ISSR and DAMD markers revealed 82.63 %, 90.36 % and 84.72 % of polymorphic bands respectively. The polymorphic information content (PIC) values were almost identical for each marker system while the resolving power (Rp) was found to be highest in DAMD (4.10). The dendrogram obtained showed the presence of two main clusters in all the analyses, with some genotypes (MB10-MB11 and MB5-MB7) always maintaining and manifesting their strong genetic relationship by clustering together in almost all the analyses. Combinations of such markers can be considered as more effective and promising tool for assessing genetic variation in other Musa species as well.

Analysis of genetic variation in sorghum (Sorghum bicolor (L.) Moench) genotypes with various agronomical traits using SPAR methods

Genetic variation among 45 genotypes of sorghum (Sorghum bicolor L.) representing seven subpopulations was assessed using three single primer amplification reaction (SPAR) methods viz., inter-simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD) and directed amplification of minisatellite-region DNA (DAMD). Totally 15 ISSR, 8 RAPD and 7 DAMD primers generated 263 amplification products, accounting for 84.6% polymorphism across all the genotypes. The Mantel's test of correlation revealed the best correlation between ISSR and cumulative data with a correlation coefficient (r) of 0.84. Assessment of population diversity indicated that the maximum intra population genetic diversity was recorded among high FeZn lines (HFL) having maximum values of Nei's genetic diversity (h) (0.244), Shannon information index (I) (0.368) and the percentage of polymorphic loci (Pp) (72.65%) while the corresponding lowest values of 0.074, 0.109 and 17.95% respectively were observed among the members of MDT subpopulation. The mean coefficient of gene differentiation (GST) and the gene flow (Nm) between populations were observed to be 0.396 and 0.7680 respectively. The analysis of molecular variance (AMOVA) suggested that maximum genetic variation exists within populations (95%) than among populations (5%). Thus the information obtained from this study could be utilized in sorghum breeding programmes for the development of varieties with improved nutrition and agronomic values in future.

Clonal in vitro multiplication of grey mangrove and assessment of genetic fidelity using single primer amplification reaction (SPAR) methods

An efficient protocol for clonal multiplication of an important mangrove, Avicennia marina, was developed through in vitro culture of nodal segments obtained from a mature plant. The nodal explant induced multiple shoots when cultured on the Murashige and Skoog (MS) basal medium supplemented with varying concentrations and combinations of 6-benzyladenine (BA) and α-naphthalene acetic acid. The highest response in terms of per cent regeneration (73%), average number of shoots/explant (3.25 ± 0.25) and maximum shoot length (5.2 ± 0.27 cm) was obtained on the MS medium supplemented with BA 5.0 μmol/L + NAA 1.0 μmol/L + 3 g/L activated charcoal after 8 weeks of culturing. The regenerated shoots were rooted well in the MS medium supplemented with 1.0 μmol/L indole-3-butyric acid with an average of 2.9 ± 0.24 roots per microshoot. The rooted plantlets were successfully transferred to pots containing normal garden soil with 70% survival rate. The genetic stability of the regenerated plants was evaluated using single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA, directed amplification of minisatellite DNA and intersimple sequence repeat polymorphism. The SPAR analysis revealed monomorphic banding patterns in all in vitro regenerated plantlets of A. marina and similar with that of the mother tree confirming their genetic uniformity and clonal fidelity.

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

Micropropagation and assessment of genetic fidelity of Henckelia incana: an endemic and medicinal Gesneriad of South India.

Henckelia incana is an endemic medicinal plant used for the treatment of fever and skin allergy. In the present study shoot regeneration was evaluated on Murashige and Skoog's (MS) medium supplemented with auxins, Indole-3-acetic acid (IAA), Indole-3- butyric acid (IBA), 1-Naphthaleneacetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and cytokinins, 6-Benzylaminopurine (BAP) and Kinetin (Kn) at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0mgl(-1). MS medium with IBA (18.08), NAA (17.83) and IAA (17.58) at 0.5mgl(-1) concentrations showed efficient regeneration. Regenerated shoots were rooted on half-strength MS medium with and without 0.5mgl(-1) IBA or NAA. The plantlets were successfully hardened in rooting trays (peat, vermiculite and sand) and transferred to field mileu. The genetic fidelity of in vitro raised plants was assessed by using three different single primer amplification reaction (SPAR) markers namely random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of mini-satellite DNA region (DAMD). The results consistently demonstrated true-to-true type propagation. This is the first report of in vitro propagation and establishment of true-to-true type genetic fidelity in H. incana.

Efficacy of RAPD, ISSR and DAMD markers in assessment of genetic variability and population structure of wild Musa acuminata colla.

North east India is considered as one of the major biodiversity hotspots worldwide and centre of origin of several plant species including Musa. Musa acuminata Colla is known to be one of the wild progenitors of cultivated bananas and plantains. Three single primer based DNA marker techniques viz., random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and directed amplification of minisatellites DNA (DAMD) were used for diversity diagnostics among 25 genotypes of wild M. acuminata collected from Meghalaya province of north east India. A total of 58 primers (26-RAPD, 21-ISSR, and11-DAMD) yielded 451 DNA fragments, of which 395 (87.58%) were found to be polymorphic in nature. The polymorphic information content (PIC) values were almost identical for each marker system. The resolving power of the marker system was found to be highest in RAPD (3.96) whereas ISSR resolved highest marker index (16.39) in the study. Selected amplicon data obtained through single primer amplification reactions were utilized for determination of diversity within and among the populations of M. acuminata. Nei's genetic differentiation (Gst) value (0.451) indicated higher proportion of the genetic variation within the populations which is supported by the AMOVA analysis (88%). The study provides insight into the efficacy of RAPD, ISSR and DAMD to analyse the genetic variation existing in the wild Musa germplasm, which can furtherbe exploited for quality trait improvement and domestication of such important horticultural crops. The genetic diversity based population structure may shed light on the genetic basis of speciation and evolution of various species within the genus Musa.

An assessment of tissue culture-induced genetic variability in olive (Olea europaea L.) using chloroplast rpl16 intron sequences and single primer amplification reaction (SPAR) markers

SUMMARYOlive (Olea europaea L.) is an important tree crop grown throughout the Mediterranean Basin and Iran. Propagation of true-to-type olive plants is important for olive-growing companies and farmers. For this reason, we performed a genetic analysis of 49 randomly selected tissue culture-regenerated olive plants of the cultivar ‘Koroneiki’ and compared them with the mother plant. We used the sequence of the chloroplast rpl16 intron and ten single primer amplification reaction (SPAR) markers to test for genetic fidelity vs. somaclonal variation. A Neighbor-Joining dendrogram based on the RAPD data produced four major clusters. The mother plant, along with ten regenerated plants, were placed far from the others and formed Cluster I. Members of this Cluster had the highest genetic distance from members of Cluster III (0.266), followed by plants of Cluster IV (0.177). Analysis of molecular variance (AMOVA) produced significant differences (PhiPT value = 0.37; P≤ 0.01) among these clusters. The Neighbor-Joining tree, neighbor-Net method, and Tree analysis using New Technology (TNT) based on RAPD marker and rpl16 intron sequence analyses respectively, separated the mother plant and some of the regenerated plants in a separate cluster. Tajima's test produced a diversity (D) statistic value of = −2.17 (P ≤ 0.05) based on the intron sequence data. We also identified some regenerated plants that were true-to-type with the mother plant.

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.

Improved in vitro propagation, solasodine accumulation and assessment of clonal fidelity in regenerants of Solanum trilobatum L. by flow cytometry and SPAR methods

An efficient protocol for the development of genetically uniform clones of a valuable medicinal plant Solanum trilobatum L. has been established. An optimal shoot regeneration response was observed in a modified Murashige and Skoog medium (M-MS) containing 25 mM ammonium nitrate, 2 mg l−1 6-benzyl adenine and 0.1 mg l−1 indole-3-acetic acid using in vitro derived node and shoot tip explants. Consequently, the multiple shoot buds were elongated in MS medium supplemented with 0.5 mg l−1 Gibberellic acid. The in vitro regenerated shoots were rooted best in MS medium containing 1.5 mg l−1 indole-3-butyric acid and successfully acclimatized in the field. The single primer amplification reaction (SPAR) approach, including random amplified polymorphic DNA, inter simple sequence repeats and directed amplification of minisatellite DNA regions markers did not identify any genetic polymorphism among in vitro regenerants. Similarly flow cytometry analysis illustrated that the DNA content and genome size of micropropagated plants were equivalent to that of intact plants from field. In addition, the accumulation of solasodine in micropropagated plants was confirmed by thin layer chromatography and further quantified by high performance liquid chromatography analysis as 2.47 mg g−1 DW which is comparable to field grown plants. Thus the protocol can be effectively exploited for commercial propagation of this species to obtain solasodine and also in genetic transformation studies.