ISSR and RAPD based evaluation of genetic fidelity and active ingredient analysis of regenerated plants of Picrorhiza kurroa

Genetic stability and phytochemical analysis of in vitro established plants of Picrorhiza kurroa Royle ex Benth, have been carried out. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of tissue culture products including three adventitious shoots from three calli and 6 months old tissue culture raised plants growing in green house condition with mother plant. Apparent genetic variation was detected in the five types of plant materials. The percentage of polymorphic bands in the RAPD and ISSR analysis were 16.25 and 14.54 %, respectively. The genetic similarity was calculated on the basis of RAPD and ISSR data among the five types of plant materials and were ranged from 0.5 to 1.0 (mean 0.75) and 0.47 to 1.0 (mean 0.73), respectively. The similarity coefficient by both RAPD and ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, but notable differences were observed among three adventitious shoots regenerated from three calli. The phytochemical analysis of tissue culture raised products showed higher secondary metabolite (picrotin and picrotoxinin) content as compare to mother plant. The information gained on genetic stability/variability will be valuable for the large scale propagation and secondary metabolite production of P. kurroa.     

Micropropagation of Tectona grandis: Assessment of Genetic Fidelity

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

An Assessment of Genetic Integrity of Micropropagated Plants of Plumbago Zeylanica by RAPD Markers

Clones of Plumbago zeylanica were micropropagated using nodal culture. The application of random amplified polymorphic DNA (RAPD) in assessing the genetic integrity of the micropropagated plants was evaluated by polymerase chain reaction. Twenty arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic fidelity. All RAPD profiles from micro-propagated plants were monomorphic and similar to those of field grown mother plants. No polymorphism was detected within the micropropagated plants.     

Evaluation of genetic stability using FRAPD markers as novel method along with antioxidant and anti-diabetic properties of micropropagated <Emphasis Type="Italic">Salacia chinensis</Emphasis> L.

Salacia chinensis L., a perennial medicinal plant, is well-known for its well-documented anti-diabetic properties. The daily growing demand in pharmaceutical industry is stimulating the conservation and wide-ranging production of the plant using plant tissue culture techniques (micropropagation). In the present study, the plants generated by direct micropropagation from nodal explants were assessed using fluorescently labeled RAPD (FRAPD) primers. Although standard RAPD primer bands in agarose gel showed genetic stability, using FRAPD analysis in genetic DNA sequencer as a novel strategy showed more accurate and reliable method has indicated by the evidence in 5% genetic variation. Antioxidant and anti-diabetic activities of micropropagated plants versus mother plant were examined using DPPH, FRAP, α-amylase, and α-glucosidase assays. The results showed that the micropropagated plants, which are able to produce higher amount of secondary metabolites than the mother plant, possess higher in vitro antioxidant and anti-diabetic properties.     

Assessment of genetic fidelity of micropropagated plants of <Emphasis Type="Italic">Simmondsia chinensis</Emphasis> (Link) Schneider using RAPD and ISSR markers

RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) markers were screened to test the genetic integrity of jojoba (Simmondsia chinensis) plants multiplied through axillary bud multiplication from nodal segments. The in vitro raised plantlets were maintained for up to 12 in vitro subcultures. During the study a total of 48 (32 RAPD and 16 ISSR) primers were screened, out of which 24 RAPD and 13 ISSR primers produced a total of 191 (126 RAPD and 65 ISSR) clear, distinct and reproducible amplicons. The amplified products were monomorphic across all the selected micropropagated plants and were similar to the mother plant. The micropropagation protocol developed by our group for rapid in vitro multiplication is appropriate for clonal propagation of jojoba. The outcome supports the fact that axillary bud multiplication can also be used as one of the safest modes for the production of true-to-type plants.     

Evaluation of the genetic fidelity of in vitro-propagated gerbera (<Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus) using DNA-based markers

The genetic fidelity of in vitro-raised gerbera clones was assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 35 RAPD and 32 ISSR primers screened, only 12 RAPD and 10 ISSR primers produced clear, reproducible and scorable bands. The 12 RAPD primers produced 54 distinct and scorable bands, with an average of 4.5 bands per primer. The number of scorable bands for ISSR primers varied from 3 (ISSR-14) to 9 (ISSR-07), with an average of 5.5 bands per primer. The number of bands generated per primer was greater in ISSR than RAPD. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix based on Jaccard’s coefficient revealed that the pair-wise value between the mother and the in vitro-raised plantlets was 1, indicating 100% similarity. This confirmed the true-to-type nature of the in vitro-raised clones.     

Detection of genetic variation among micropropagated tea [Camellia sinensis (L.) O. Kuntze] by RAPD analysis

Summary Randomly amplified polymorphic DNA (RAPD) techniques were applied to assess genetic instability among micropropagated tea [Camellia sinensis (L.) O. Kuntze] eultivar ‘T-78’. Out of 49 random 10-mer primers, 11 generated polymorphism in four out of 17 micropropagated plants and one mother plant. A total of 221 bands, ranging from 525 bp to 2.5 kb, were produced by the 49 primers. Twenty-four were polymorphic for those four plants. However, the remaining bands were monomorphic among all plants. Polymorphism among those four plants showed an identifical banding pattern suggesting the occurrence of a single mutation. Our results demonstrated that RAPD can be used successfully to determine the genetic instability among micropropagated plants which otherwise were morphologically indistinguishable.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh

RAPD markers were used to assess genetic fidelity of 23 micropropagated plants of a single clone (L34) of Populus deltoides. Eleven arbitrary 10-base primers were successfully used to amplify DNA from in vivo and in vitro material. Of these, 5 distinguished a total of 13 polymorphisms common across 6 micropropagated plants. Apart from these 6 plants, the amplification products were monomorphic across all the micropropagated plants, the mother plant and 4 additional field-grown control plants. Our results show that RAPD markers can be used to gain rapid and precise information about genetic similarities or dissimilarities in micropropagation systems that might not be so easily evident from other commonly used techniques.     

In vitro propagation and assessment of clonal fidelity of Nepenthes khasiana Hook. f.: a medicinal insectivorous plant of India

An efficient in vitro protocol for large-scale multiplication of Nepenthes khasiana, a threatened insectivorous plant of India, has been developed from nodal stem segments. The highest shoot proliferation of 19.16 ± 0.23 shoots/explant was recorded in half-strength Murashige and Skoog (MS) medium supplemented with 2.5 mg/l kinetin, 2.0 mg/l 6-benzyl aminopurine, 3 % sucrose and 0.8 % agar. The best rooting was achieved in half-strength MS medium supplemented with 2.0 mg/l α-naphthalene acetic acid with an average of 9.04 ± 0.46 roots/shoot. The plantlets were successfully transferred to the greenhouse with survival rate of 92 %, exhibiting normal development. Cytological and random amplified polymorphic DNA (RAPD) analyses were carried out to assess the genetic integrity of the regenerated plantlets. Cytological analysis revealed no change in chromosome number with cells studied showing 2n = 80. Of the 80 primers screened for RAPD analysis, 14 primers resulted in clear and scorable bands. A total of 72 amplification products were obtained out of which only 4.1 % bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient ranging from 0.98 to 1.0, thus suggesting genetic stability in the micropropagated plants of N. khasiana.     

RAPD Fingerprint to Appraise the Genetic Fidelity of In Vitro Propagated <Emphasis Type="Italic">Araucaria excelsa</Emphasis> R. Br. var. glauca Plantlets

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the genetic fidelity of in vitro propagated Araucaria excelsa R. Br. var. glauca with explants taken from orthotropic stem along with their related mother plants after treatment with kinetin, 2iP, BA (0.02–0.26 mg/l) and TDZ (0.001–1 mg/l) to produce axillary shoots. TDZ and kinetin induced more shoot and higher length per explant. Results showed a total of 1,676 fragments were generated with 12 RAPD primers in micropropagated plants and their donor mother plants. The number of loci ranged from 6 in OPB 12–18 in OPY 07 with a size ranging from 250 bp in OPH 19–3500 bp in OPH 11. Cluster analysis of RAPD data using UPGMA (unweighted pair group method with arithmetic average) revealed more than 92% genetic similarities between tissue cultured plants and their corresponding mother plant measured by the Jaccard’s similarity coefficient. Similarity matrix and PCoA (two dimensional principal coordinate analysis) resulted in the same affinity. Primers had shown 36% polymorphism. However, careful monitoring of tissue culture derived plants might be needed to determine that rooted shoots are adventitious in origin.     

Evaluation of genetic homogeneity of in vitro-raised plants of <Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem. using molecular markers

Tecomella undulata (Sm.) Seem (family Bignoniaceae) is an economically and pharmaceutically important timber tree of arid regions of India. Overexploitation of natural stands coupled with minimal conservation and reforestation efforts has led to its incorporation in list of endangered species. This monotypic genus can be propagated only through seeds as no methods are available for its vegetative propagation. Therefore, protocol for multiplication of T. undulata via direct regeneration using nodal segments from mature trees has been standardized. Authentication of genetic homogeneity of these in vitro-raised plants is necessary for commercial-scale application of the developed micropropagation protocol. PCR-based molecular markers which have emerged as simple, fast, reliable, and labor-effective tools for testing the genetic homogeneity of in vitro-raised plants were used in the present study. Arbitrary (random amplified polymorphic DNA, RAPD), semi-arbitrary (inter-simple sequence repeat, ISSR; start codon targeted (SCoT) polymorphism), and sequence-based (simple sequence repeat, SSR) markers were used. DNA samples of shoots maintained in vitro for 2 years collected after every 4 subculture cycles (of 3 weeks each) and field-transferred plantlets were compared with the mother tree DNA using 131 primers (25 each of RAPD, ISSR, SCoT and 56 SSR). Scorable unambiguous and reproducible DNA fragments were produced by 77 (21 RAPD, 20 ISSR, 22 SCoT and 14 SSR) primers. A total of 71, 93, 94, and 42 distinct and scorable DNA fragments were produced by RAPD, ISSR, SCoT, and SSR primers respectively with an average of 3.38, 4.65, 4.27, and 3.0 DNA fragments per primer. The true-to-type nature of the in vitro-raised plants of T. undulata undergoing up to 32 subculture passages over a period of approximately 2 years was authenticated by monomorphic DNA fragments amplified with all primer combinations. Therefore, the developed micropropagation protocol can be safely used on a commercial scale for multiplying T. undulata plants.     

RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-India type)

An efficient in vitro propagation method using enhanced axillary branching cultures produced plants from nodal explants of three mature, elite tea clones: diploid UPASI 26 and UPASI 27 (2n=2x=30) representing Camellia sinensis (China type) and triploid UPASI 3 (2n=3x=45) representing C. assamica ssp. assamica (Assam-India type). The genetic fidelity of the micropropagated plants of these three tea clones was assessed by analysing their nuclear, mitochondrial (mt), and chloroplast (cp) genomes using multiple molecular DNA markers. A total of 465, 446 and 462 genetic loci were produced with RFLP, RAPD and ISSR fingerprinting in the micropropagated plants and the corresponding mother plant of C. sinensis clone U (UPASI) 26, and C. assamica ssp. assamica clones U3 and U27, respectively. RFLP fingerprinting was performed using six restriction endonuclease digests and 14 mt and cp gene probes in 84 enzyme-probe combinations. For PCR fingerprinting, 50 RAPD and SSR primers were used for amplifications. The micropropagated plants of both the U3 and U27 clones revealed complete stability in the 462 and 446 genetic loci analysed. In comparison, 36 (7.7%) of the 465 loci were polymorphic among micropropagated plants of the U26 clone. The observed polymorphic loci were not restricted to a particular genome (nuclear or organellar), although a relatively low (7.43%) level of polymorphism was observed in the nuclear as compared to the mt genome (16.3%). ISSR fingerprinting (12.8%) detected more polymorphic loci than RAPD fingerprinting (4.28%). No polymorphism was observed in the cp genome of the micropropagated plants of the three tea clones. The rigorous screening of nuclear and two organellar genomes has demonstrated, for the first time, subtle genetic variation at the DNA sequence level in organized meristem-derived micropropagated plants of tea. Clearly, this is another example demonstrating that organized meristem cultures are not always genetically true-to-type. The genomic changes in tea clones are genotype dependent rather than culture condition dependent.     

In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker

An efficient in vitro protocol has been established for clonal propagation of elite plant of Spilanthes calva DC., an important source of spilanthol, an antimalarial larvicidal compound. Nodal explants excised from field grown plant of S. calva DC. when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N⁶-Benzyladenine (BA), N⁶-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn), differentiated multiple shoots from the axils. BA at 10 μM proved optimum for elicitation of multiple shoots in 91.6 % cultures with an average of 7.12 shoots per explant within 6 weeks. The excised shoots rooted on half strength Murashige and Skoog’s medium supplemented with 0.1 μM IBA. Micropropagated plants were hardened and transferred to field for acclimatization, where 95 % plants survived and were phenotypically similar to the donor plant. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic fidelity amongst the regenerants. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 71 scorable bands, ranging in size from 100 bp to 1,100 bp were generated by a combination of the two markers in the aforesaid plants. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant. The similarity values amongst the aforesaid plants varied from 0.967 to 1.000. The dendrogram generated through UPGMA (Unweighted Pair Group Method with arithmetic mean) analysis revealed 98 % similarity amongst them, thus confirming the genetic fidelity of the in vitro clones.     

In vitro and ex vitro evaluation of long-term micropropagated turmeric as analyzed through cytophotometry,phytoconstituents, biochemical and molecular markers

Turmeric (Curcuma longa L.), a high valued medicinal plant, was micropropagated through induction of multiple shoots using latent axillary buds of rhizome. Cytophotometric and random amplified polymorphic DNA (RAPD) as well as inter simple sequence repeats (ISSR) analysis were used to periodically monitor the genetic stability of micropropagated clones of Curcuma longa conserved in vitro up to 7 years at every 6 months interval. A total of eighteen RAPD and eight ISSR primers gave 45,537 distinct and reproducible bands, monomorphic across all 353 plants analyzed. Micropropagated turmeric after being conserved for 7 years in vitro was transplanted into soil in field. Drug yielding potential of tissue culture derived plants was evaluated in field through estimation of phytoconstituents like curcumin and essential oil contents. The result of 2 years of field trial showed that micropropagated turmeric retained stability in all the characteristics examined when compared with the field performance of conventionally propagated plants. Thus long term conservation of an elite genotype of turmeric with epigenetic and genetic stability is significant for stable supply of drug i.e., curcumin and essential oil to the market.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.     

Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers

To evaluate genetic homogeneity of 1-year-old guava (Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300?bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200?bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava.     

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard’s coefficient classified the genotypes into five clusters (I–V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.     

Suitability of RAPD for analyzing spined and spineless variant regenerants of pineapple (Ananas comosus L., Merr.)

A random amplified polymorphic DNA (RAPD) analysis of spineless (variant phenotype) plants obtained from micropropagated dormant pineapple (Ananas comosus L., Merr.) axillary buds was performed using arbitrary 10-mer oligonucleotide primers. This was done to investigate the genetic fidelity of the regenerants and to distinguish these variants from regenerants bearing the normal spined phenotype. Of the 58 arbitrary primers used, 29 produced bands unique to the spineless phenotype, and 30 produced bands unique to the spined phenotype. A total of 914 bands were scored, 55 of which were polymorphic to the spineless phenotype and 51 of which were polymorphic to the spined phenotype. On the basis of RAPD amplification products, genetic similarity was estimated in both types of regenerants using similarity coefficients (Nei and Li, 1979). The characteristic finger-prints generated by each probe emphasize genetic variability of regenerants. This technique is suitable for analyzing variant regenerants induced in vitro.