Molecular analysis of genetic stability in micropropagated apple rootstock MM106

Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic stability of 10 micropropagated plants regenerated through axillary buds of clonal apple (Malus pumila Mill.) rootstock MM106. Eleven random decamer primers were successfully used to analyse genomic DNA from mother plants and in vitro plant material. A total of 129 scorable fragments were amplified with an average of 11.73 bands per primer. Among them, 99 were monomorphic and 30 were polymorphic with 23.2% polymorphism. Among these 30, 12 were found monomorphic across seven plants and parent. Three plants could be regarded as off-types. Our results show that RAPD markers could be used to detect the genetic similarities and dissimilarities in micropropagated material.     

High multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.)

RAPD and microsatellite markers were used to determine the genetic fidelity of micropropagated plants from the three varieties of tea plant derived from explants of field grown mother bush as well as in vitro germinated seedlings. Rate of shoot multiplication was better from nodal explants than from shoot tips. A maximum of 32–33 shoots was observed in cotyledonary node in 1/2 MS medium with BAP (6 mg/l), GA₃ (1 mg/l) and IBA (0.5 mg/l). 90% of the in vitro derived microshoots were micrografted into rootstocks. The micropropagated plantlets showed both cytological and genetical stability. SSR primers showed complete stability among the regenerants. The results convinced that plants derived from axillary as well as adventitious mode of propagation can be genetically true to type. This cost effective technique would help in fast clonal propagation at a commercial scale.     

中国石蒜组培苗体细胞无性系变异的ISSR和SRAP检测

以中国石蒜离体培养再生的幼苗为试材,首次采用ISSR和SRAP分子标记对再生苗的遗传稳定性进行了检测,以探讨体细胞无性系变异情况及成因.结果表明:所选用的14条ISSR引物共产生191条带,其中只有1条为多态性条带,并且该条带表现为新增条带;SRAP标记所选用的15对引物共扩增出219条带,其中3条为多态性条带,表现为共有条带缺失.综合2种分子标记检测结果来看,再生植株表现出的体细胞变异率极低(0.97％).现有数据同时也表明,已建立的离体培养体系在保存中国石蒜种质资源和遗传改良上是稳定、可行的.     

Determination of essential oil contents and micropropagation of Gaultheria fragrantissima,an endangered woody aromatic plant of India

Summary

Leaves of mature of Gaultheria fragrantissima Wall. plants (Indian wintergreen) were collected from various locations in the Eastern Himalayan region on the Indo-China border and were analysed by steam distillation and gas chromatography to identify an elite line that contained 1.79% (v/v) essential oils, 98% of which was methyl salicylate. Subsequently, a highly reproducible micropropagation protocol using adult shoot tips from this elite genotype was developed in order to conserve this highly-valued, endangered, woody oil-bearing aromatic shrub in India. Among several plant growth regulator (PGR) combinations tested for in vitro multiplication, up to 35 shoots per explant could be induced within 14 weeks of culture on woody plant medium (WPM) fortified only with 0.22 mg l^–1 thidiazuron (TDZ). These shoots could elongate on WPM containing 1.0 mg l^–1 kinetin. Rooted plantlets were acclimatised ex vivo, with 70% success. Random amplified polymorphic DNA (RAPD) analysis indicated the genetic uniformity of both the micropropagated plantlets and the donor plants. This is the first report on in vitro micropropagation of G. fragrantissima.     

Arbuscular mycorrhizal inoculation of micropropagated persimmon plantlets

Summary

Growth, development and nutrient status of micropropagated persimmon (Diospyros kaki) ‘Rojo Brillante’ in response to inoculation with two species of arbuscular mycorrhizal (AM) fungi were studied under nursery conditions. The species of AM fungi were Glomus intraradices and G. mosseae. Shoot growth depression and low root colonization percentage were observed to G. mosseae inoculation. Shoot and root growth enhancements were observed for plants colonised by G. intraradices. Inoculated plantlets with G. intraradices and high level of controlled-release fertilizer signi®cantly decreased shoot height in relation to treatment with low fertilization level. Furthermore, G. intraradices had unique effects on the mineral status of the persimmon plantlets. N and Ca were significantly increased and decreased, respectively, within the shoot tissue of plants colonised by G. intraradices. Early inoculation with G. intraradices appears to favour growth of micropropagated persimmon plantlets.     

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.     

Assessment of genetic variation among thornless blackberries (Rubus spp.) using random amplified polymorphic DNA

Summary

To assess genetic relatedness in thornless blackberry (Rubus spp.), 11 different blackberry cultivars were screened using random amplified polymorphic DNA (RAPD) markers. The blackberries selected represented four different thornless backgrounds. Genetic similarity was estimated using 140 random primers, and the cluster analysis conducted using the RAPD data grouped the cultivars into three distinct clades. Ninety-eight primers produced 113 cultivar-specific RAPD fragments capable of identifying each cultivar. In addition, reproducible polymorphism using two primers was observed within the ‘Evergreen’ (R. laciniatus) clade that consisted of the pure thornless blackberry ‘Everthornless’, the chimeral ‘Thornless Evergreen’, and their thorny progenitor ‘Evergreen’. All three plants are believed to be identical, except for a single mutational event that caused the phenotypic change from thorny to thornless. The R. laciniatus RAPD marker data provide information that may eventually be useful to identify the gene(s) responsible for thornlessness in that species.     

Vegetative and yield component characteristics of micropropagated muscadine grape (Vitis rotundifolia Michx.)

SUMMARY

Evaluations were made of the field performance of micropropagated (tissue cultured axillary buds) versus conventionally rooted softwood cuttings of muscadine grape, Vitis rotundifolia Michx. Trunk cross-sectional area did not differ between treatments during three years of field evaluation following planting. There were no differences in leaf area and dry weight or shoot number in the two propagation types; leaf morphology was normal with no apparent juvenile characteristics. Yield components including flower number per shoot and inflorescence, fruit number, total fruit weight and yield efficiency were greater in micropropagated plants during the second year, the first year of cropping; differences diminished and yield components were not different in year three. Performance of tissue cultured plants was therefore as good as, or surpassed, conventionally propagated plants during early vine establishment.     

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.     

Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

Summary

An efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.     

Development of sex-associated RAPD markers in wild Pistacia species

Summary

Marker assisted selection may greatly facilitate pistachio rootstock breeding as well as cultivar improvement, because of the long juvenile period of Pistacia species. Early diagnosis of seedling sex type would assist breeding and nurserymanagement in these dioecious species. We searched for RAPD markers linked to sex in P. atlantica, P. terebinthus and P. eurycarpa, the main wild species in Turkey that are used as rootstocks for P. vera. For this purpose, leaf samples were collected from male and female individual trees from each species and sex-pooled DNA samples were prepared by mixing the DNA of ten male and ten female individuals, to screen for sex associated RAPD bands. A total of 472 primers have been screened so far and two bands, amplified by primers BC156 and BC360, appeared to be sex assocaited in P. eurycarpa. The bands were tested in 30 male and 37 female individuals. Band BC156 (1300) was present in all, except one, female trees and was absent from all the male trees. Band BC360 (500) was amplified in 31 out of 37 females and was absent from all the males. In P. atlantica, one primer, OPAK09, amplified a femaleassociated band (850 bp), that was present in all 46 female individuals tested and absent in all the 38 male trees tested. It is likely that these markers are linked to sex-determining loci. The sex determination mechanism has not been characterized in Pistacia and segregating populations from controlled crosses are required to elucidate such mechanism and also to measure the genetic distance of our markers from the putative sex loci.     

Development of a regeneration protocol through indirect organogenesis in prickly pear cactus (Opuntia ficus-indica (L.) Mill)

An indirect organogenesis regeneration protocol for Opuntia ficus-indica (L.) Mill var “Blanco sin Espinas” is described. One centimeter square cladode explants sections from previously micropropagated prickly pear plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 20 different combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyladenine (BA). The best calli induction and regeneration response were observed when 2.26 μM 2,4-D and 2.21 μM BA combination was applied to the nopal explants. Regenerating calli was capable of forming new buds when transferred to MS basal medium supplemented with 0.5 μM BA (proliferation medium). Shoot elongation and rooting were achieved on MS medium without plant growth regulators. Excellent acclimatization to greenhouse conditions was observed for all transferred plantlets. By this procedure no morphological differences were observed between the regenerated and mother plants. This protocol may be also utilized to carry out plant regeneration after genetic transformation, in order to develop transformed plants without the presence of chimeric zones.     

Further Work on Plant Injection for Diagnostic and Curative Purposes

Summary

Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-relationship among31 acid citrus species and cultivars, including sour oranges (six accessions); ‘Yuzu’ (four accessions) andits relatives (21 accessions). Out of the 60 decamer primers screened, 27 were selected which produced 108 markers; 76 of which were polymorphic. Species or cultivar-specific RAPD markers were also found. A dendrogram based on genetic distance implied that sour oranges were very distinct from ‘Yuzu’ and its relatives. ‘Yuzu’ accessions were very closely linked to each other, however; for the other specimens genetic polymorphism could easily be detected by RAPDs and the genetic variation between accessions was quite high and revealed their different origins. In this study some RAPDs allowed the distinction of very close cultivars, for instance ‘Kabosu’ from ‘Aka kabosu’.     

Variation in essential oil components in regenerated lavender (Lavandula vera DC) plants

The variation in morphology and essential oil components in 63 regenerated plantlets of Lavandula vera was investigated. Ten regenerated plantlets appeared dwarf-like and exhibited reduced spike lengths and floral stalk lengths. Phenotypic variants were obtained using plant growth regulators at all concentration regimes, but 4.4×10⁻⁶ M of 6-benzylamino purine (BA) for shoot induction produced the highest frequency of dwarfism (30.8%). None of the regenerated plantlets produced as much essential oil as the original plant. However, three regenerated plantlets had a different fragrance from other regenerated plantlets and the original plant. In a comparative analysis, these three plantlets exhibited much higher levels of linalool and lavandulol than linalyl acetate and lavandulyl acetate. From these results, we suggest that the activity or quantity of acetyltransferases may be reduced in those plants. However, neither the shoot or root induction medium altered the essential oil compositions. These results suggest that somaclonal variation may be useful to produce variants with different fragrance in L. vera.     

Relationship between vegetative and rhizome characters and final rhizome yield in micropropagated ginger plants (Zingiber officinale Rosc.) over two generations

Correlation and path analysis for yield and yield contributing characters in two types of micropropagated ginger plants (plantlets directly regenerated from aerial stem explants and plantlets regenerated from aerial stem derived callus) were carried out over first and second generations in two varieties viz. var. ‘Jamaica’ and var. ‘Varada’. Irrespective of the regeneration method, the in vitro derived plants showed high positive correlation and maximum positive direct effect of circumference of cormlets, length of cormlets and number of cormlets with the rhizome yield in the first generation. But tiller number exhibited negative correlation and negative direct effect with the rhizome yield in the first generation. In the second generation of the aerial stem regenerated plants, tiller number, number of nodes per cormlets, circumference of cormlets, number of cormlets and plant height exhibited high positive correlation and maximum direct effect with rhizome yield. However, in the second generation also, the callus regenerated plants showed the same trend as in the first generation. Even though the tiller number showed positive significant correlation with rhizome yield, it showed negative direct effect with the yield.     

Genetic variation and its relationship to root weight in the sweet potato as revealed by RAPD analysis

The aim of this work was to evaluate the genetic variations of the sweet potato by a random amplified polymorphic DNA (RAPD) assay with emphasis on correlations with morphological traits. Cuttings of superior and inferior lines derived from tissue culture-regenerated plants and asexually propagated plants of the Tainung 57 (TN57) and Tainung 66 (TN66) sweet potato were planted in the field. Three important agronomic traits, top weight, root weight, and root numbers, were measured 3 months after planting. The RAPD-PCR (polymerase chain reaction) technique was used for detecting genomic variations within and between varieties. Of the 160 primers tested in this study, 38 revealed clear and repeatable RAPD polymorphisms. Among the 38 primers, 8 showed consistent amplified band patterns among the plants with variations within and between varieties, while the others indicated polymorphisms within or between varieties. RAPD markers demonstrated a clear association with root weight. However, the presence and absence of these genetic markers did not correspond well with either the top weight or root number. Genetic instability revealed in the selection stress of superior and inferior plants was found to be independent of variety. In addition, both asexual propagated and tissue culture-induced somaclonal variations were observed in this study. Our results show that RAPD is a useful tool for detecting somaclonal variations from varietal and intra-varietal sweet potato for monitoring of DNA changes in somaclonal variants. The identification of regions which are associated with the character of root weight by RAPD markers enables us to use these markers as selection tools to improve root weight in sweet potato.     

Morphological and molecular analysis of genetic diversity in jackfruit

Summary

RAPD markers were used to estimate genetic diversity in 12 high-yielding jackfruit (Artocarpus heterophyllus Lamk.) accessions obtained from different locations in southern India. Marker data were compared with morphological data obtained over three successive seasons. PCR-amplifiable DNA was isolated using the CTAB method and 171 amplified fragments were obtained using 23 random primers. The genetic dissimilarity matrix was calculated based on Squared Euclidian Distances, which revealed a maximum genetic distance of 7.9% between a clone of ‘Mottavarica’ (‘M₀’), and ‘Chandrahalasu’ from distant locations, while the minimum genetic distance (5%) was between the genotypes (‘M₀’) and ‘Kerala’, indicating their similar geographical origin. Ward's method of cluster analysis grouped all individuals on the dendrogram into two major clusters according to their geographical location. The present study showed low-to-moderate genetic diversity among the 12 jackfruit accessions, which will assist in the identification and management of jackfruit germplasm for breeding purposes.     

Evaluation of genetic diversity among some Iranian wild asparagus populations using morphological characteristics and RAPD markers

An evaluation of genetic diversity in 39 wild asparagus populations was carried out using morphological and RAPD markers. A combination of morphological traits and random RAPD primers was used to examine the level of genetic variation and polymorphisms among the populations. A factor analysis using Ward's method on mean values of morphological characteristics indicated seven main factors resulting in four groups. Analysis of polymorphic bands using Jaccard's similarity coefficient indicated that genetic similarity ranged between 0.71 and 0.29. At a similarity level of 0.64, the populations were divided in three sub-clusters, containing 34, four and one populations, respectively. Significant regression associations were found between 21 morphological characteristics and 18 RAPD markers, revealing some informative markers associated with some traits. The highest R² was related to 18 RAPD markers associated with gender (53.5%) that among them BA-04₂₀₀₀ had a maximum R². The results showed that Iranian wild asparagus with its high levels of genetic variation could be considered as a valuable gene pool for future asparagus breeding programs. Furthermore, it could be inferred that morphological characteristics and RAPD markers are suitable tools to discriminate asparagus populations for the evaluation of genetic diversity.     

Development of SCAR markers to differentiate between mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.)

Summary

Mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.) are similar in fruit and tree morphology, and exhibit high cross- and graft-compatibility with each other. It is therefore difficult to differentiate mume and apricot cultivars on the basis of morphological and phenotypical characteristics. Molecular markers were developed to differentiate nine mume from ten apricot cultivars. Four dominant, random amplified polymorphic DNA (RAPD) markers that can discriminate between mume and apricot cultivars (designated OPA15₆₂₈, OPO10₅₅₀, OPO20₂₅₉, and OPU03₄₁₅) were identified from 21 decamer primers. Two RAPD markers (OPO10₅₅₀ and OPU03₄₁₅) were developed into dominant sequence-characterised amplified region (SCAR) markers (SCO10 and SCU03). These SCAR markers could differentiate between all mume and apricot cultivars.     

Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.