Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Somaclonal variation in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) analyzed using polymorphic and sequenced AFLP markers

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.     

Culture-induced variation in plants of Coffea arabica cv. caturra rojo,regenerated by direct and indirect somatic embryogenesis

Amplified fragment-length polymorphism (AFLP) was used to evaluate the stability of DNA in regenerated plantlets of Coffea arabica obtained by direct (DSE) and indirect somatic embryogenesis (ISE). Cluster analysis using the unweighted pair-group method (UPGMA), showed no specific grouping pattern related to the type of embryogenesis. These results suggest that the somatic embryogenesis (SE) process has a mechanism for the selection of normal and competent cells. Bulked DNA from regenerated plants obtained by DSE and ISE, and from the mother plants, was used to characterize specific AFLP fragments associated with each SE process. Twenty-three primer combinations were tested. A total of 1446 bands were analyzed, with 11.4% being polymorphic and 84% being specific for regenerated plants. Furthermore, specific bands were detected for DSE, ISE, and the mother plants. These results indicate that the SE process induces rearrangements at the DNA level and demonstrates discrepancies between the mechanisms involved in each SE process. Coffea arabica breeding programs that involve DSE and ISE can use AFLP as an additional tool for assessing DNA stability.     

Assessment of genetic fidelity among <Emphasis Type="Italic">Agave tequilana</Emphasis> plants propagated asexually via rhizomes versus in vitro culture

Agave tequilana is a species of great economic importance for the Mexican society. Like most agaves, it can be propagated either sexually or asexually. However, plants originating from rhizomes are mainly used for commercial production. The aim of this study was to determine the genetic fidelity of plants that were obtained from the field and propagated from rhizomes and of those propagated by in vitro culture methods (somatic embryogenesis and proliferation of axillary buds). We used an inverse ISTR (sequence-tagged repeat) molecular marker based on retrotransposon sequences to detect variability among related individual plants. Cluster analysis showed that plants could be grouped according to the propagation method employed. Plants propagated by the same in vitro method were found to be distinct from those propagated in the field. Even within each method, plants were not genetically identical. Genomic changes were evidenced not only in plants subjected to somaclonal variation in vitro but were also evident in those propagated through the natural asexual process, despite the fact that they were considered true clones. The differences observed in plants confirm the existence of asexual genetic variability.     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Cloning and molecular characterisation of a potato SERK gene transcriptionally induced during initiation of somatic embryogenesis

Somatic embryogenesis offers great potential in plant propagation, long-term germplasm conservation, and as a suitable model system for deciphering early events during embryogenesis. The up-regulation and ectopic expression of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene has been shown to mark and enhance embryogenic competence in somatic cells of model plant species. We have cloned and characterised a SERK gene (StSERK1) from potato (Solanum tuberosum L.), an important crop plant. Sequence analysis of StSERK1 revealed high levels of similarity to other plant SERKs, as well as a conserved intron/exon structure which is unique to members of the SERK family. Furthermore, StSERK clustered most closely with SERK gene family members such as MtSERK1, CuSERK1, AtSERK1, and DcSERK, implicated in evoking somatic embryogenesis. Monitoring of SERK expression during progression of potato somatic embryogenesis revealed increased StSERK expression during the induction phase. Subsequently, during the embryo transition phases, StSERK expression was unchanged and did not vary among embryo-forming and inhibitory conditions. However, in isolated somatic embryos StSERK expression was again up-regulated. In other plant parts (leaves, true potato seeds, microtubers and flower buds), StSERK showed different levels of expression. Expression analysis suggests that the isolated StSERK could be a functional SERK orthologue. The possible role of SERK as a marker of pluripotency, rather than embryogenesis alone, is discussed.     

Retrotransposon-based markers from potato monoploids used in somatic hybridization.

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.     

橡胶树体细胞胚发生过程中DNA甲基化分析

为探讨巴西橡胶树(Hevea brasiliensis)自根幼态无性系与供体间差异产生的原因,应用甲基化敏感扩增多态性扩增技术,对巴西橡胶树体细胞胚发生过程中基因组DNA 胞嘧啶甲基化程度和模式进行了分析。结果表明,在巴西橡胶树体细胞胚发生过程中不同阶段的DNA 甲基化程度不同,以花药的DNA 甲基化程度最高,体细胞胚的DNA 甲基化水平最低。在体细胞胚发生过程中出现了I、Ⅱ和Ⅲ 3 种类型的甲基化多态性带型的改变,包括他们的出现与消失。因此,橡胶树体细胞胚发生过程中可能通过DNA 甲基化甲基化和去甲基化来调控基因的表达。     

DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

DNA-methylation profiles of leaf tissues of Rosa hybrida cv. Carefree Beauty collected from in vivo-grown greenhouse plants, in vitro-grown proliferating shoots at different passages, regenerants of embryogenic callus, regenerants of organogenic callus, as well as calli from undifferentiated callus (UC), embryogenic callus, and organogenic callus were investigated using an amplified fragment-length polymorphism (AFLP)-based detection technique. Three types of AFLP bands were recovered. Type I bands were observed with both isoschizomers Msp and HpaII, while type II and type III bands were observed only with MspI and HpaII, respectively. Sequence analysis of the three types of AFLP bands revealed that a nonmethylated MspI/HpaII-recognition site 5-CCGG-3 resulted in a type I band, while an inner 5-methylcytosine generated most type II and type III bands. About 40% of inner and 20% of outer cytosines in 5-CCGG-3 sequences were fully methylated, and only a few hemimethylated outer cytosines were observed. Changes in types of AFLP bands among different tissues were frequently observed, including appearance and disappearance of type I, II, and III AFLP bands, as well as exchanges between either type I and type II or type I and type III AFLP bands. Methylation alterations of outer cytosines in 5-CCGG-3 sequences triggered appearance and disappearance of type I and II AFLP bands. Methylation changes of both outer and inner cytosines resulted in either removal or generation of type III AFLP bands. Methylation alteration of an inner cytosine was responsible for exchange between type I and type II, while hemimethylation of an outer cytosine accounted for exchange between type I and type III AFLP bands. During UC induction, a significant DNA-methylation alteration was detected in both inner and outer cytosines. Variations in methylation profiles significantly differed between somatic embryogenesis and in vitro organogenesis. Demethylation of outer cytosines occurred at a high frequency during somatic embryogenesis, and most altered AFLP bands in embryogenic callus were passed on to its regenerants. However, most methylation-altered AFLP bands during organogenesis were recovered in shoot regenerants derived via organogenic callus. Seven tissue-specific bands were isolated, cloned, and sequenced. Blast search revealed that two of these might be derived from functional genes.Mingliang Xu and Xiangqian Li contributed equally to this paper     

Analysis of genetic diversity through AFLP,SAMPL, ISSR and RAPD markers in <Emphasis Type="Italic">Tribulus terrestris</Emphasis>, a medicinal herb

Tribulus terrestris is well known for its medicinal importance in curing urino-genital disorders. Amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers were used for the first time for the detection of genetic polymorphism in this medicinal herb from samples collected from various geographical regions of India. Six assays each of AFLP and SAMPL markers and 21 each of ISSR and RAPD markers were utilized. AFLP yielded 500 scorable amplified products, of which 82.9% were polymorphic. SAMPL primers amplified 488 bands, 462 being polymorphic (94.7%). The range of amplified bands was 66 [(TC)₈G + M-CAG] to 98 [(CA)₆AG + M-CAC] and the percentage polymorphism, 89.9 [from (CT)₄C (AC)₄A + M-CTG] to 100 [from (GACA)₄ + M-CTA]. The ISSR primers amplified 239 bands of 0.4–2.5 kb, 73.6% showed polymorphism. The amplified products ranged from 5 to 16 and the percentage polymorphism 40–100. RAPD assays produced 276 bands, of which 163 were polymorphic (59%). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.9 for all the four marker systems. The dendrograms and PCA plots derived from the binary data matrices of the four marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. The relative efficiency of the four molecular marker systems calculated on the basis of multiplex ratio, marker index and average heterozygosity revealed SAMPL to be the best. Distinct DNA fingerprinting profile, unique to every geographical region could be obtained with all the four molecular marker systems. Clustering can be a good indicator for clear separation of genotypes from different regions in well-defined groups that are supported by high bootstrap values.     

Detection of somaclonal variants in somatic embryogenesis-regenerated plants of <Emphasis Type="Italic">Vitis vinifera</Emphasis> by flow cytometry and microsatellite markers

Flow cytometry and microsatellite analyses were used to evaluate the trueness-to-type of somatic embryogenesis-regenerated plants from six important Spanish grapevine (Vitis vinifera L.) cultivars. Tetraploid plants were regenerated through somatic embryogenesis from all of the cultivars tested with the exception of ‘Merenzao’. In addition, an octoploid plant was obtained in the cv. ‘Albariño’, and two mixoploids in ‘Torrontés’. The most probable origin of these ploidy variations is somaclonal variation. The cv. ‘Brancellao’ presented significantly more polyploids (28.57%) than any other cultivar, but it must be noted that 50% of the adult field-grown ‘Brancellao’ mother plants analysed were mixoploid. Hence, it is probable that these polyploids originated either from somaclonal variation or by separation of genotypically different cell layers through somatic embryogenesis. Microsatellite analysis of somatic embryogenesis-regenerated plants showed true-to-type varietal genotypes for all plants except six ‘Torrontés’ plants, which showed a mutant allele (231) instead of the normal one (237) at the locus VVMD5. There was not a clear relationship between the occurrence of the observed mutant regenerated plants and the callus induction media composition, the developmental stage of the inflorescences, the type of explant used for starting the cultures or the type of germination (precocious in differentiation medium or normal in germination medium) in any of the cultivars tested, except ‘Torrontés’. The mutant plants described herein have been transplanted to soil for future evaluation of putative phenotypic traits of interest. These mutants can be useful both for breeding programs and for functional genomic approaches aimed at increasing knowledge of the biology of grapevine.     

Assessment of ploidy stability of the somatic embryogenesis process in <Emphasis Type="Italic">Quercus suber</Emphasis> L. using flow cytometry

Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of true-to-type propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.     

AFLP analysis of variation in pecan somatic embryos

Before somatic embryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability of AFLP (amplified fragment length polymorphism) analysis for the assessment of genetic variability in somatic embryos of pecan [Carya illinoinensis (Wangenh.) C. Koch] and made comparisons between and within embryogenic culture lines. AFLP readily detected differences between culture lines, with 368 polymorphic loci identified. Individual culture lines generally produced somatic embryos with similar overall banding patterns. Embryos derived from the same culture line generally grouped together in a phenogram generated by UPGMA (unweighted pair-group method, arithmetic average) analysis. However, a few somatic embryos exhibited higher levels of polymorphism and failed to group with others regenerated from the same line. The relation between the detected within-line differences and their contribution to phenotypic variation is yet to be determined. Received: 9 September 1998 / Revision received: 12 November 1998 / Accepted: 8 December 1998     

A new image of plantain diversity assessed by SSR,AFLP and MSAP markers

Using both SSR and AFLP markers, the genetic diversity of 30 plantains constituting a representative sample of the phenotypic diversity was assessed. The results confirmed a very narrow genetic base of this cultivar group. SSR and AFLP data support the hypothesis that these cultivars may have arisen from vegetative multiplication of a single seed. MSAP were used to survey cytosine methylation status at CCGG sites in order to obtain an alternative source of diversity data. A higher degree of polymorphism was revealed allowing the classification of the samples into three clusters. No correlation was observed between the phenotypic classification and methylation diversity. Implications for breeding programs are discussed.     

Variation in potato microplant morphology in vitro and DNA methylation

Heterotrophic and autotrophic culture in agar and in polyurethane foam, the latter used as an alternative tissue support to agar, resulted in potato microplants with different in vitro morphologies. The microplants were visually characterised in terms of their relative developmental maturity, by comparing the respective leaf shapes in vitro with ontogenetic differences in leaf shape in glasshouse-grown potato plants. Cytosine methylation in the DNA of microplants of the different morphologies was determined using a method based on the AFLP technique but employing methylation-sensitive restriction enzymes (MSAP analysis) to test the hypothesis that DNA methylation could be used to characterise differences in microplant development in vitro. In three of the four treatments there was a good correlation between the visual assessment of relative morphological maturity and DNA base methylation levels. In these microplants there was increased DNA methylation in the leaves with mature leaf morphology represented by a decreased number of restriction fragments. The fourth in vitro morphology had the most juvenile leaf shape but did not have the predicted level of DNA methylation, having a relatively low number of restriction fragments. Subtraction analysis was used to discriminate the fragments that were unique to the juvenile and mature in vivo leaf morphologies. Comparison of the fragment patterns from the microplants with the latter reference profiles, confirmed the relationship with the total DNA methylation as detected by MSAP analysis, that is, the number of common fragments with the juvenile or mature in vivo leaf profiles, respectively. However, none of the fragment profiles, while sharing some common bands at random, was identical to any other; or to that of either the juvenile or mature in vivo leaf. The anomalous relationship of the microplants with most juvenile leaf shape and highest DNA methylation was confirmed. The measurement of DNA methylation in in vitro plants is discussed in the context of the development of a method to assess the quality of microplants produced by different in vitro protocols.     

Molecular characterisation of resistance against potato wart races 1, 2, 6 and 18 in a tetraploid population of potato (Solanum tuberosum subsp. tuberosum)

Potato wart is caused by the obligate biotrophic fungus Synchytrium endobioticum, which is subject to quarantine regulations due to the production of long persisting spores in the soil and the lack of effective fungicides. The objective of this study was to identify quantitative trait loci (QTL) for resistance against potato wart races (R) 1, 2, 6 and 18 in a tetraploid potato population developed by crossing cv. Saturna (resistant to R1) with cv. Panda (resistant to R1, R2, R6, R18). A total of 92 progenies were used for phenotyping and genotyping. Resistance tests were performed for races 1 and 18 in 2 years and for races 2 and 6 in 1 year on 10 to 20 eyepieces per genotype. Based on amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, linkage maps were established for the female and male parent, respectively. Single marker analysis followed by a multiple regression analysis revealed initial marker–trait associations. The interval mapping routine of TetraploidMap was applied for QTL analysis. A major QTL for resistance against race 1 explaining between 46 % and 56 % of the phenotypic variation was identified near Sen1, a known resistance locus for potato wart race 1 on chromosome XI. Other resistance QTL were detected on chromosomes I (to R2), II (to R6, 18), VI (to R1, 2, 6, 18), VII (to R2, 6, 18), VIII (to R1, 2, 6, 18), X (to R2, 6, 18), XI (to R2, 6, 18) and on an unknown linkage group (to R18) explaining minor to moderate effects of the phenotypic variation. Resistance QTL against different potato wart races often overlapped, particularly concerning races 2, 6 and 18. Overall, this study gives a valuable insight into the complex inheritance of resistance against potato wart.     

Asexual genetic variability inAgavaceae determined with inverse sequence-tagged repeats and amplification fragment length polymorphism analysis

Agaves are succulent monocot plants rich in fibers, sugars and other important compounds. They are also valued as ornamental plants and for their ability to grow in poor soils. In the present study, inverse sequence-tagged repeats (ISTR) and amplified fragment length polymorphism (AFLP) analysis were used to study genetic diversity in differentAgavaceae plant samples. Comparison of the banding patterns between the mother plant and rhizome-derived daughter plants showed that genetic variability is generated during asexual reproduction in these species. Phylogenetic relationships amongAgave species were obtained using unweighted pair-group method, arithmetic average (UPGMA) analysis. Genetic diversity through asexual propagation allows for genetic selection and improvement within these asexually propagated plants.     

Field assessment of dihaploid Solatium tuberosum and S. brevidens somatic hybrids

Summary Following both chemical and electrical fusion of protoplasts of a dihaploid line of potato (Solanum tuberosum), (PDH40), with those of the wild species, Solanum brevidens, 11 and 40 somatic hybrid plants, respectively were obtained. Fifteen of these somatic hybrid genotypes and the two parents were studied further in a small field trial to assess field performance and phenotypic variability. In the UK, somatic hybrid plants are classified as genetically engineered organisms, and the UK Advisory Committee on Genetic Manipulation have imposed various restrictions on field experiments. Examination of the somatic hybrids in the field showed extensive phenotypic variability, and no two genotypes were identical. Some of the variation reflected changes in chromosome numbers, but other factors were also involved. Half the somatic hybrid genotypes produced tubers in the field, although the tubers were smaller and differed morphologically from those of PDH40. The results of the study suggest that the extent of somaclonal variation manifested in somatic hybrids is greater than that found in protoplast-derived plants of potato. The implications of this observation and the current regulations concerning field experiments of somatic hybrid plants in the UK are discussed.