Soom Nath Raina

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.

Genetic fidelity of organized meristem-derived micropropagated plants: A critical reappraisal

SummaryThe commercial multiplication of a large number of diverse plant species represents one of the major success stories of urilizing tissue culture technology profitably. Micropropagation has now become a multibillion dollar industry, practised all over the world. Of the various methods used to micropropagate plants, somatic embryogenesis and enhanced axillary branching have become the principal methods of multiplication. Long-term benefits of this enterprise, however, lie in the production of clonally uniform plants. The concept of genetic uniformity among micropropagated plants derived through organized meristems was exploded by several convincing reports of the incidence of somaclonal variation at morphological, cytological (chromosome number and structure), cytochemical (genome size), biochemical (proteins and isozymes), and molecular (nuclear and organellar genomes) levels. Somaclonal variation is not limited to any particular group of plants; it has been reported, for example, in ornamentals, plantation crops, vegetable and food crops, forest species and fruit trees. The upsurge of these reports, facilitated to a large extent by the technical developments made in molecular biology, is a matter of great concern for any micropropagation system. The economic consequences of somaclonal variation can be enormous in forest trees and woody plants, as they have long life cycles. Therefore, somaclonal variation has to be dispensed with if large-scale micropropagation of diverse plant species is to become not only successful but also accepted by end-users. In the light of the various factors (genotype, ploidy level, in vitro culture age, explant and culture type, etc.) that lead to somaclonal variation of divergent genetic changes at the cellular and molecular levels, genetic analysis of micropropagated plants using a multidisciplinary approach, especially at the DNA sequence level, initially and at various cultural stages, is essential. The results obtained at early multiplication stages from these tests could help in modifying the protocol/s for obtaining genetically true-to-type plants, and ultimate usage by entrepneneurs without any ambiguity.

Genotyping safflower (Carthamus tinctorius) cultivars by DNA fingerprints

SummaryCarthamus tinctorius (2n = 2x = 24) (family Asteraceae), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia and the Americas as a source of high-quality vegetable and industrial oil. India ranks first in the production of safflower oil. Fourteen cultivars, widely cultivated in various agro-climatic regions of India, have been fingerprinted by RAPD, ISSR, and AFLP markers utilizing 36, 21 primers, and 4 primer combinations, respectively. On an individual assay basis, AFLP has proven to be the best marker system as compared with the other two markers applied as assessed by high discriminating power (0.98), assay efficiency index (33.2), marker index (18.2), resolving power (40.62), and genotype index (0.856). Thirty-six RAPD and 21 SSR primers could differentiate a maximum of eight and four cultivars, respectively, whereas, two AFLP primer combinations could fingerprint all the 14 cultivars. To understand genetic relationships among these cultivars, Jaccards similarity coefficient and UPGMA clustering algorithm were applied to the three marker data sets. Mean genetic similarities ranged from 0.689 (AFLP) to 0.952 (ISSR). Correlation coefficient comparisons between similarity matrices and co-phenetic matrices obtained with the three markers revealed that AFLP displayed no congruence vis-a-vis RAPD and ISSR data. However, strong correlation was observed between RAPD and ISSR marker systems. This paper reports the start of molecular biology programme targeting nuclear genome of safflower, a major world oilseed crop about whose genetics very little is known.

Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae)

Abstract The most important commercial species of coffee, Coffea arabica, which produces 73% of the worlds coffee crop and almost all of the coffee in Latin America, is the only tetraploid (allotetraploid, 2n=4x=44) species known in the genus. High-frequency somatic embryogenesis, plant regeneration and plant recovery were achieved from leaf explants of a mature, elite plant of C. arabica cv. Cauvery (S-4347) using a two-step culture method. To assess the genetic integrity of the nuclear, mitochondrial and chloroplast genomes among the hardened regenerants, we employed multiple DNA markers (RFLP, RAPD, ISSR) for sampling various regions of the genome. Although the nuclear and mitochondrial genomes of the mother plant and five ramets derived from the mother ortet were similar in organization, this was not so in the somatic embryo-derived plants where both nuclear and mitochondrial genomes changed in different, characteristic ways and produced novel genome organizations. A total of 480 genetic loci, based on the data obtained from a total of 16 nuclear, mitochondrial and chloroplast gene probes, in combination with nine restriction enzyme digests, 38 RAPD and 17 SSR primers, were scored in 27 somatic embryo-derived plants and the single control. Among these, 44 loci were observed to be polymorphic. A relatively low level of polymorphism (4.36%) was found in the nuclear genome, while polymorphism in the mitochondrial genome (41%) was much higher. No polymorphism was detected in the chloroplast genome. The polymorphism in the mitochondrial genome was found in only 4 plants. Such selective polymorphism was not true for the nuclear genome. Thus, this in-depth and comprehensive study demonstrates, for the first time, the presence of subtle genetic variability and novel genome organizations in the commercially well-established somatic embryogenesis-derived plants of this important coffee species.

GISH technology in plant genome research

Genomic in situ hybridization, a modification of fluorescence in situ hybridization technology, is one of the most exciting and versatile research tools to be developed in recent years. It has enabled plant chromosome and genome research to progress at phenomenal rate and its potential is far from exhausted. Genomic in situ hybridization has applications in characterization of genomes and chromosomes in hybrid polyploids, hybrid plants, partial allopolyploids, polyhaploids and recombinant breeding lines; and in the localization, and detection of amount of introgressed alien chromatin. Genomic in situ hybridization has also given new insights into somaclonal variation, the origin of B chromosomes, control of chromosome pairing and other aspects of chromosome evolution. This review highlights some of these areas and provides source material for the reader who seeks more information on a specific field.

Genetic variation vis-a-vis molecular polymorphism in groundnut, Arachis hypogaea L.

Recent studies on the genus Arachis using molecular markers have revealed very little demonstrable polymorphism in the cultivated groundnut, A. hypogaea. This has led to the hasty generalization that the groundnut lacks genetic variation. However, this is in complete contradiction to the results of other lines of investigations into the origin and evolution of A. hypogaea. Further, a characterization of the world collection for various traits also shows significant levels of variation for almost all genetic traits. The literature review in this article suggests that the lack of genetic variation was inferred because of an inadequacy in the material studied, and the range of techniques used to study molecular polymorphism. A comprehensive and rigorous examination of the material available in the groundnut world collection, either by improving current techniques, or by using such advanced techniques as SSRs and AFLP could well reveal polymorphism at the molecular level.

DNA amounts and chromatin compactness in Vicia

Abstract2C DNA amounts and areas of chromatin were determined with a M 86 Vickers microdensitometer in 56 species of Vicia (x=5, 6, 7), exhibiting large differences in chromosome size. There were significant differences between the species both in DNA content and chromatin area. The nuclear DNA amounts range from 3.85 to 27.07 pg. DNA distribution appears discontinuous; species cluster into distinct groups and the average nuclear DNA amount separating each successive pair is approximately the same (2.23 pg). The compaction of DNA in interphase nuclei increases with increasing DNA amount, which is, at least partly, due to a disproportionate increase in the heterochromatin relative to the euchromatin component of DNA. Comparisons of DNA readings at various stages of the cell cycle show that the DNA amounts are underestimated by microdensitometry in nuclei with high DNA density. Estimation of relative DNA content and area of individual chromosomes were made in twelve species. The results show that changes in DNA content within chromosomes affect the degree of metaphase coiling in an orderly fashion.

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

Abstract 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.

Quantitative DNA variation between and within chromosome complements of Vigna species (Fabaceae).

Cytogenetical investigations, so far, on the organisation and evolution of the genomes of Vigna species have proved difficult due to small chromosome size, large chromosome number and uniformity in chromosome shape and size within and between the complements. In this investigation the nature and extent of DNA variation between thirteen diploid and one polyploid species have been estimated. The DNA variation between diploid species was small and species clustered around a mean value of 2.7 pg. The polyploid species had a greater DNA value of 4.95 pg. No significant variation in 2C DNA content was found between accessions of V. radiata. A comparison of the distribution of DNA among the chromosomes within complements has shown that the excess DNA acquired in evolution was distributed evenly in all chromosomes despite significant differences in chromosome size. The relative changes in chromatin area and DNA density which accompany evolutionary DNA variation was also compared.

AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.