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.     

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.     

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.     

Application of rapd in the determination of genetic fidelity in micropropagated Drosera plantlets

Summary Random amplified polymorphic DNA (RAPD) markers were used to verify the clonal fidelity of two micropropagated Drosera species, D. anglica and D. binata, which were regenerated by adventitious budding from leaf explants and shoot tips, respectively. Twenty arbitrary decamers were used to screen 15 randomly selected plantlets of each species. No genetic variation was detected among D. binata regenerants, whereas a 0.08% polymorphism frequency was estimated for D. anglica plantlets. These results indicate that the regeneration of plants through shoot-tip culture is a low-risk method for generating genetic variability, whereas material regenerated through leaf explants requires further verification.     

An assessment of genetic fidelity of micropropagated plants of Chlorophytum borivilianum using RAPD markers

Rapid micropropagation was achieved in Chlorophytum borivilianum Santapau and Fernandes using shoot base as explants. Multiple shoots were induced on Murashige and Skoog’s (MS) medium supplemented with 3.0 mg dm⁻³ 6-benzylaminopurine, 0.1 mg dm⁻³ 1-naphthaleneacetic acid, 150 mg dm⁻³ adenine sulphates and 3 % saccharose. Rooting was readily achieved upon transferring the shoots onto half strength MS medium supplemented with 0.1 mg dm⁻³ indolebutyric acid and 2 % saccharose. Micropropagated plantlets were hardened in the greenhouse and successfully established in soil. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the micropropagated plants. Thirty one arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic stability. All RAPD profile analysis from micropropagated plants was genetically similar to mother plants.     

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, <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.     

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.     

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.     

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.     

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.     

RAPD Assessment for Identification of Clonal Identity and Genetic Stability of in vitro Propagated Chestnut Hybrids

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the clonal identity of four in vitro propagated chestnut rootstock hybrids (Castanea sativa × C. crenata) described as originally isolated from the same mother tree. To confirm genetic stability after in vitro multiplication for more than 4 years, RAPD patterns of in vitro and donor plants were compared. From 40 arbitrary 10-mer primers used to amplify DNA, 21 provided patterns and were chosen for comparisons. Although significant differences were found in growth parameters between in vitro material of the putative clones, RAPD profiling showed polymorphism in none but one. This accession may then be withdrawn from the same clonal origin as the other three. As expected, no polymorphism was detected between the material propagated in vitro and the donor plants they originated from.     

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.     

Assessments of somaclonal variation in micropropagated shoots of <Emphasis Type="Italic">Cedrus</Emphasis>: consequences of axillary bud breaking

Reversed-phase HPLC analysis and random amplified polymorphic DNA (RAPD) markers were used to monitor DNA methylation status and genetic stability of C. atlantica and C. libani shoots generated through axillary bud proliferation. Average DNA methylation in C. atlantica or C. libani seedlings and mature 200-year-old trees of C. libani was 19.8, 19.5 and 22.3%, respectively. These global amounts showed no significant variation after the in vitro establishment of seedling-originated shoot stocks. In contrast, in vitro culture caused a significant decrease in the amount of 5-methylcytosine in genomic DNA of the tissue culture (TC) progenies of one of the adult C. libani genotypes. This DNA demethylation event accompanied an enhancement of the regrowth capacity of this genotype. Detected RAPD variation between mother plants and their TC progenies was species-related, with C. libani TC progenies being genetically more stable than those of C. atlantica. Nevertheless, similarity indices ranged from 0.97 to 1 among mother plants and their TC progenies. Furthermore, the analyses of molecular variance (AMOVA) suggest that RAPD variation among the mother plants and their TC progenies might be considered as not significant. The application of various statistical approaches, including cluster-based genetic distance methods and AMOVA, demonstrates that RAPD markers discriminate C. atlantica and C. libani appropriately.     

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.     

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     

In vitro regeneration of an endangered medicinal plant Picrorhiza scrophulariiflora

A reproducible in vitro regeneration system for Nepalese kutki (Picrorhiza scrophulariiflora Pennell) was developed from in vitro leaf derived callus. Induction of more than seven shoot buds per explant was achieved on Woody plant medium (WPM) supplemented with 0.53 μM α-napthaleneacetic acid (NAA) and 0.23 μM kinetin (KIN). The shoots were elongated on WPM supplemented with 0.44 μM 6-benzylaminopurine (BAP) and rooted on WPM supplemented with 5.3 μM NAA within 2 weeks. The random amplified polymorphic DNA (RAPD) analysis indicated genetic uniformity of the micropropagated plants with its donor plants.     

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Summary Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 μM and 15 μM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 μM and 15 μM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N⁶-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 μM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred. Part of a thesis submitted by M. B. K. in partial fulfillment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.