Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 μM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 μM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.     

An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers

An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l^?1) and produced (4.56 ± 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0–2.0 mg l^?1) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05–0.4 mg l^?1). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l^?1) and IAA (0.1 mg l^?1) formed the maximum number of shoots (68.40 ± 2.74 per culture bottle) with an average height (6.59 ± 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 ± 0.64 roots per shoot and an average length of 2.76 ± 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.     

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.     

Molecular analysis of micropropagated neem plants using aflp markers for ascertaining clonal fidelity

Summary The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation 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.     

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.     

Micropropagation of <Emphasis Type="Italic">Sapindus trifoliatus</Emphasis> L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis

An efficient in vitro propagation system has been developed for rapid micropropagation of Soapnut (Sapindus trifoliatus Linn.), a medicinally and economically important tree from nodal (axillary bud) segments of seedlings. The frequency of shoot regeneration from seedling node explant was influenced by the age of the seedlings, growth regulators and successive transfer of the mother explant. Explants from 4-week-old seedlings yielded the maximum shoot regeneration frequency (97.22%) on full-strength MS medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP). After harvesting the newly formed shoots, the mother explants transferred to same medium subsequently produced a maximum of 5.16 shoots per explant after third passage. Further improvement in the morphogenic response occurred when the nodal explants excised from in vitro regenerated shoots were employed, and 6.89 shoots per explant were obtained on the same medium after the third subculture. Optimal rooting (91.67%) was obtained by placing the micro-shoots in liquid MS medium with 1.0 mg l⁻¹ IBA for 24 h and then transferring to the agar solidified MS medium devoid of IBA. The micropropagated shoots with well-developed roots were acclimatized and successfully transplanted to soil with 90% survival rate. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants. This is the first report of an efficient protocol for regeneration of S. trifoliatus through organogenesis, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

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     

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.     

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.     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

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.     

Genotypic fidelity of micropropagated pineapple (Ananas comosus) plantlets assessed by isozyme and RAPD markers

In the present work, pineapple plantlets (cv. `Amarelinho') micropropagated by stationary (S) and temporary immersion (T) systems were evaluated in terms of genotypic fidelity by isozyme and RAPD markers. Neither isozymes (average 0.67%) nor RAPDs (average 7.5%) alone detected significant differences between the two micropropagated systems. However, when combined isozymes and RAPDs data more somaclonal variants were detected in S than T, with RAPDs revealing more variation than isozymes. In the T system, the treatment with PBZ 6.0 M gave the greatest occurrence of variants (8.9%), although significant statistical differences (; p>0.05) between presence and absence of PBZ or GA were not detected. Temporary immersion in comparison with the stationary system resulted in the lowest proportion of somaclonal variants (1.9% vs. 3.9%). Although it does not represent an in depth genome analysis, ours is the first work that has attempted to evaluate the genotypic fidelity of micropropagated pineapple plantlets.     

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.     

Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.     

Varietal identification and assessment of genetic relationships in Hippeastrum using RAPD markers

The National Botanical Research Institute (NBRI) in Lucknow, India, maintains germplasm of Hippeastrum, a beautiful summer blooming ornamental. Germplasm collections comprise NBRI hybrids developed through selective breeding, hybrids with unknown parentage, local species, and Dutch hybrids for research purposes. Considering the importance of protecting plant breeders’ rights for commercial exploitation of hybrids, a PCR-based technique (random amplified polymorphic DNA—RAPD) was used to correctly identify known and unknown hybrids and to determine cultivar relatedness. RAPD profiles were used very successfully to trace and confirm the parentage of all the hybrids tested and to determine clear molecular relationships among varieties.     

Using morphological diagnosis and molecular markers to assess the clonal fidelity of micropropagated Echinacea purpurea regenerants

Both morphological characteristics and amplified fragment length polymorphism (AFLP) markers were used to validate the genetic fidelity of 1 080 field-grown Echinacea purpurea plants regenerated from leaf explants of donor T5-9. Morphological diagnosis revealed that 1 067 out of 1 080 regenerants were normal, while 13 regenerants were aberrant. AFLP analysis was further performed to assess DNA variations among donor, 43 sampled normal regenerants and all 13 aberrant regenerants. Seven primer combinations generated 471 fragments among donor and normal regenerants, of which 9 fragments were polymorphic. The same primer pairs generated 484 fragments for aberrant regenerants, of which 417 fragments were polymorphic. UPGMA clustering indicated that 42 normal regenerants and donor fell into same cluster at similarity scale of > 0.99, while all 13 aberrant regenerants and one morphologically normal regenerant comprised the other clusters. AFLP analysis indicated that these 14 regenerants are off-types.     

An assessment of genetic relationships within the genus Digitalis based on PCR-generated RAPD markers

RAPD markers were used to study inter-specific variation among six species of the genus Digitalis: D. obscura, D. lanata, D. grandiflora, D. purpurea, D. thapsi and D. dubia, and the hybrid D. excelsior (D. purpurea×D. grandiflora). A total of 91 highly reproducible bands amplified with four arbitrarily chosen decamer primers were obtained. Homology of the co-emigrating RAPD markers was tested by blot hybridisation and sequencing of selected bands. The application of a range of statistical approaches for RAPD data analysis, including distance and parsimony methods, family clustering and the analysis of molecular variance (AMOVA), indicated that these molecular markers were taxonomically informative in Digitalis. The species relationships revealed were fully consistent with those previously obtained using morphological affinities. The hybrid D. excelsior seems to have stronger affinity to the section Digitalis than to Grandiflorae. This is the first known report of the application of RAPD markers for the study of genetic relationships among species of the genus Digitalis. Received: 20 October 1999 / Accepted: 9 November 1999