Assessment of genetic stability of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Dictyospermum ovalifolium</Emphasis>

In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of 2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from 200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92 %) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups. Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred fifty plants were successfully established.     

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.     

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.     

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.     

Determination of genetic stability of long-term micropropagated plantlets of Platanus acerifolia using ISSR markers

Inter-simple sequence repeat (ISSR) markers were used to assess the genetic stability of long-term micropropagated plantlets of London plane tree (Platanus acerifolia Willd.). Twenty micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 8 years, as achieved by axillary branch multiplication. Out of 38 ISSR primers screened, 16 primers were found to produce clear reproducible bands resulting in a total of 103 distinct bands with an average of 6.44 scorable bands per primer. Of these 103 bands, 86 were monomorphic across all 20 of the plants tested and 17 showed polymorphisms (16.5 % polymorphism). Based on the ISSR band data, similarity indices between the plantlets ranged from 0.92 to 1.00. These similarity indices were used to construct an UPGMA dendrogram and demonstrated that all 20 micropropagated plants grouped together in one major cluster with a similarity level of 91 %. A total of 1771 scorable bands were obtained from the full combination of primers and plantlets and only 51 (2.88 %) were polymorphic across the plantlets which indicates that this micropropagated line of P. acerifolia is genetically stable.     

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.     

Evaluation of clonal integrity in desert date tree (Balanites aegyptiaca Del.) by inter-simple sequence repeat marker assay

Axillary shoot bud multiplication as a safest mode of micropropagation to obtain clonal progeny was revealed through the application of molecular marker technique in Balanites aegyptiaca. Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic constancy of micropropagated plantlets chosen from a clonal collection of shoots that originated from mature nodal explants (mother plant). Out of 20 ISSR primers screened, ten primers yielded reliable and reproducible patterns of amplified products in all the tested plants. In this study, on an average, 11.7 bands were amplified per primer. A total of 117 bands were scored for the tissue culture-raised plantlets; 115 amplification products were monomorphic and 2 bands were polymorphic. Based on the ISSR band data, 98.2 % genetic uniformity was detected among the regenerants. Thus, the amplification products validated that the plantlets were true-to-type in morphological or growth characteristics when compared with the mother plant.     

Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis

A newly developed and novel DNA marker technique, i.e. start codon targeted (SCoT) polymorphic markers that target plant gene regions were used to assess genetic stability of in vitro raised plants of Cleome gynandra multiplied by enhanced bud proliferation from nodal segments. Seven randomly selected micropropagated plants, following at least 2 months of growth in the greenhouse along with mother plant were subjected to molecular analysis. Of 24 primers screened, 15 primers produced unambiguous and reproducible bands. All 15 primers generated a total of 65 fragments, with a mean of 4.3 ranging 2–7 per primer. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. To verify the results of SCoT analysis, random amplified polymorphic DNA (RAPD) markers were also used for the assessment of genetic fidelity of tissue culture raised plants. The monomorphic banding pattern in micropropagated plants and the mother plant obtained from SCoT and RAPD analysis confirms the genetic stability of the in vitro raised plants and demonstrates the reliability of our micropropagation system for C. gynandra, an important C₄ plant.     

Micropropagation of Daylily (<i>Hemerocallis fulva</i>) from Crown-Tip Explants and Assessment of Somaclonal Variation of <i>in Vitro</i>-Propagated Plants Using SCoT Markers

Determination of the somaclonal variation of in vitro-propagated plants is crucial to determine the appropriate micropropagation protocol and growth regulators for commercial scale multiplication. In this research, nine multiplication media (MM) augmented with different concentrations of 6-benzyl adenine (BA), Kinetin (Kin), and Thidiazuron (TDZ), Three rooting media (RM) supplemented with three levels of α-naphthalene acetic acid (NAA) and three types of soil mixtures (v/v); Coco peat/Vermiculite/Sand (CVS), Peat moss/Perlite/Sand (PPS) and Peat moss/Perlite (PP) were used in the micropropagation protocol of daylily plants. MM2 showed the maximum shoot length and the number of leaves, while MM9 showed the maximum number of shoots. The RM1 showed the maximum root length and the number of roots. During acclimatization, CVS, PPS, and PP soil mixture showed similar performance except the CVS mixture showed lower performance regarding plant height and diameter. The genetic fidelity of micropropagated plants was evaluated using Start Codon Targeted (SCoT) Markers. Six SCoT primers amplified 51 scorable bands with an approximate range from 146 bp to 1598 bp size. Thirty one out of 51 loci were presented in the mother plants. 40 loci were polymorphic, 11 were monomorphic and 7 were unique. The amplification patterns of the micropropagated plants demonstrated genetic integrity to the mother plant ranging from 84.32 to 47.06 and somaclonal variations ranging from 52.94 with 5 mg/l BA pathway to 15.68 with 1mg/l TDZ pathway, thus demonstrating that the homogeneity and the variation of the micropropagated plants affected by the type and the quantity of the plant growth regulator used during multiplication subcultures. This research can be successfully used for other ornamental and medicinal plants’ bulk multiplication, germplasm conservation, and future genetic improvement.     

RAPD based genetic stability analysis among micropropagated,synthetic seed derived and hardened plants of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.): a threatened Indian medicinal herb

Bacopa monnieri (L.), a highly endangered miracle medicinal herb with global interest, is one of the popular ancient Indian ayurvedic plants. With ever increasing demand for Bacopa based formulations in pharmaceutical industries, there is a need to preserve the stocks of the plant through biotechnological approaches. Randomly amplified polymorphic DNA (RAPD) fingerprinting approach was applied to analyze the genetic stability of 19 different B.monnieri plants randomly selected after micropropagation, regrowth from alginate encapsulated uninodal cuttings (before and after storage at 4°C) and hardening with the mother plant (wild type). 16 arbitrary decamer primers amplified a total of 334 reproducible distinct DNA fragments ranging from 180 to 1,500 bp, of which 262 (78.4%) were monomorphic and the rest (21.5%) were polymorphic with an average of 20.8 bands per primer. The extent of polymorphism was low to moderate. Primers OPAK 14, OPM 15 and OPD 13 generated 69, 46 and 42% polymorphic patterns. Primers OPA 04, OPU 13 and OPD 08 generated 100% monomorphic pattern. Similarity matrix based on Jaccard’s coefficient revealed that pair wise values between the wild type and its analyzed plants ranged from 0.00 to 0.92 and among the micropropagated, synthetic seed derived and hardened plants, the range of genetic distance is from 0.67 to 0.92. Unweighted pair group method with arithmetic averages cluster analysis resulted in one loose group of the wild type with three subgroups. The present study paves the way for the identification and maintenance of genetically uniform B. monnieri plants micropropagated in the lab, plants regrown from synthetic seeds and hardened in the field.     

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.     

ISSR and RAPD Based Evaluation of Genetic Stability of Encapsulated Micro Shoots of Glycyrrhiza glabra Following 6?Months of Storage

In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6?months of normal storage at 25?±?2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30?days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500?bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR.     

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.     

Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers

Dendrocalamus asper, an edible bamboo is valued for its tender edible shoots in the food industry. However, overexploitation of natural stands of D. asper coupled with minimal conservation and reforestation efforts has led to its rapid depletion in nature. Therefore protocol for rapid multiplication of D. asper via direct regeneration using nodal segments from mature clumps was standardized and more than 25,000 plants were transferred to the field (Singh et al. 2012a). However, genetic fidelity of these in vitro raised plants needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plants. This study report the genetic fidelity analysis of in vitro raised plants of D. asper for the first time using arbitrary (Random Amplified Polymorphic DNA, RAPD), semi-arbitrary (Inter-Simple Sequence Repeat, ISSR; Amplified Fragment Length Polymorphism, AFLP), and sequence-based (Simple Sequence Repeat, SSR) markers. Bulked DNA samples of 20 in vitro raised shoots (collected after every three subculture cycles starting from 3rd to 30th passage) and field transferred plantlets were compared with the mother plant DNA using 90 primer combinations (25 each of RAPD, ISSR, SSR, and 15 AFLP) and scorable bands were produced by 78 (22 RAPD, 24 ISSR, 21 SSR, and 11 AFLP) primers. A total of 146 distinct and scorable bands were produced by 22 RAPD primers with an average of 6.6 bands per primer while the number of bands for ISSR primers varied from 3 (ISSR-4 and 9) to 13 (ISSR-17), with an average of 7.1 bands per primer. Similarly, SSR markers also showed wide variation in number of bands, ranging from 2 (RM 261) to 12 (RM 44, 140, and 224) with an average of 7.8 bands. AFLP primer combinations could generate 35–72 bands with an average of 48.7 bands per primer pair. Amplification of monomorphic bands with all primer combinations authenticated the true to type nature of the in vitro raised plants of D. asper which underwent up to 30 subculture passages over a period of approximately 2 years thereby supporting the commercial utilization of the developed micropropagation protocol.     

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.     

Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC

In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia 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 in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.     

Genetic analysis of enhanced-axillary-branching-derived Eucalyptus tereticornis Smith and E. camaldulensis Dehn. plants

In a culture method for enhanced axillary branching functional plants of Eucalyptus tereticornis and E. camaldulensis are efficiently regenerated. To assess the genetic integrity among the regenerants, we employed multiple analytical tools including cytochemical and molecular assays. The 2C DNA amounts were estimated in the meristematic zones of root and shoot tips of 250 micropropagated plants, collected at various cycles of tissue culture from multiplication to field transfer, and compared to the corresponding mother plants. The culture conditions did not induce amplification or deletion of DNA sequences, nor were there drastic change(s) in chromosome number, since all the micropropagated plants of E. tereticornis (1.2 pg) and E. camaldulensis (1.4 pg) maintained the same DNA amounts as the mother plant. Total DNA of 46 micropropagated and mother plants digested with eight restriction enzymes and hybridized to 13 nuclear, mitochondrial, and synthetic oligonucleotide DNA probes yielded 82 bands. Hybridization patterns indicated that the variation observed was minor. To further confirm the genetic fidelity, 12 arbitrary 10-base primers and six synthetic oligonucleotide sequences, successfully used to amplify genomic DNA from in vivo and in vitro materials, produced 133 fragments that were monomorphic across the plants tested. The present results demonstrate that enhanced-axillary-branching culture of mature trees could be utilized commercially for mass clonal propagation of these two important Eucalyptus species that have been recalcitrant to vegetative propagation. The results also provide novel insights into the genetic differences between E. tereticornis and E. camaldulensis. Received: 8 October 1996 / Revision received: 22 July 1997 / Accepted: 30 July 1997     

Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers

Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability of long-term (more than 10 years) micropropagated shoots of Japanese black pine (Pinus thunbergii Parl.). Thirty-six shoots consisting of three morphotypes (short, medium, and long needles) were randomly chosen from about 4,000 micropropagated shoots regenerated from the explants of a single nematode-resistant mother plant. Out of 126 primers screened, 30 gave 134 clear reproducible bands. A total of 4,824 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested shoots. Our results show that regenerants from our plant micropropagation system are genetically stable. Received: 5 December 1997 / Revision received: 17 May 1998 / Accepted: 1 June 1998     

Plant regeneration of <Emphasis Type="Italic">Erigeron breviscapus</Emphasis> (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 μM 6-benzylaminopurine (BAP) and 5 μM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5–10 μM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.