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.     

Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISR markers

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

Assessment of genetic fidelity of micropropagated <Emphasis Type="Italic">Swertia chirayita</Emphasis> plantlets by ISSR marker assay

Inter simple sequence repeat (ISSR) marker assay was employed to validate the genetic fidelity of Swertia chirayita plantlets multiplied in vitro by axillary multiplication upto forty-two passages. Sixteen ISSR primers generated a total of 102 amplicons among the tissue-cultured plants. Forty-eight amplicons were amplified in the outlier (a Swertia species). The outlier (negative control) was employed to rule out the possibility that the invariant fingerprint was due to chance alone and that the ISSR technique employed was not discriminatory enough to detect the off-types. A homogenous amplification profile was observed for all the micropropagated plants. The results confirmed the clonal fidelity of the tissue culture-raised S. chirayita plantlets and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

Genetic stability of micropropagated almond plantlets,as assessed by RAPD and ISSR markers

Almond shoots produced by axillary branching from clone VII derived from a seedling of cultivar Boa Casta were evaluated for somaclonal variation using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) analysis. To verify genetic stability we compared RAPD and ISSR patterns of plantlets obtained after 4 and 6 years of in vitro multiplication. A total of 64 RAPD and 10 ISSR primers gave 326 distinct and reproducible band classes, monomorphic across all 22 plantlets analysed. Thus, a total of 7,172 bands were generated, exhibiting homogeneous RAPD and ISSR patterns for the plantlets tested. These results suggest that the culture conditions used for axillary branching proliferation are appropriate for clonal propagation of almond clone VII, as they do not seem to interfere with the integrity of the regenerated plantlets. These results allowed us to establish the use of axillary branching plantlets (mother-plants) as internal controls for the analysis of somaclonal variation of shoots regenerated from other in vitro culture processes performed with clone VII (adventitious regeneration, regeneration from meristem culture, virus sanitation programs and genetic engineering).M. Martins and D. Sarmento contributed equally to this paper     

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

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.     

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.     

Study of genetic variation among parsley (<Emphasis Type="Italic">Petroselinum Crispum</Emphasis> (Mill.) Nym.) samples using RAPD and ISSR markers

RAPD and ISSR markers have been used to classify 32 samples of parsley (Petroselinum Crispum (Mill.) Nym. ex A. W. Hill) including the root and leaf plants. In total, 51 RAPD and 66 ISSR markers were amplified by five RAPD and six ISSR primers. On average, the ISSR and RAPD spectra had 11 and 10.2 bands, respectively. The matrix of genetic distances between the samples was constructed using Jaccard coefficients. The dendrogram constructed by the UPGMA method graphically shows the values of genetic distances. The distance between root and leaf forms of parsley was 0.292.     

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.     

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.     

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.     

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.     

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.     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

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.     

R-ISSR marker as a useful tool for detection of new genomic loci in <Emphasis Type="Italic">Arthrocnemum macrostachyum</Emphasis>

Arthrocnemum macrostachyum, is a perennial halophytic shrub typical of Mediterranean salt marshes. The present study aims to investigate some combinations of inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) primers applied in real PCR. Thereby, the potential of R-ISSR markers to detect new genomic loci in 3 genotypes of A. macrostachyum grown in the Western coast of Syria was examined. Different combinations of RAPD and ISSR primers produced bands that were absent when single ISSR or RAPD primers were used. The results have demonstrated that ISSR primer (AG)₈TC gave more informative pattern when combined with different RAPD primers comparing to other tested primers. In contrast, the tested ISSR primer (GACA)₄ gave less informative pattern when used alone. These combinations were successfully applied in real PCR to detect new genomic variability in A. macrostachyum genotypes.     

PCR-based molecular markers for assessment of somaclonal variation in <Emphasis Type="Italic">Pinus pinea</Emphasis> clones micropropagated <Emphasis Type="Italic">in vitro</Emphasis>

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.     

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.