Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet)

Summary Genetic analysis was conducted on the qualitative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pennisetum glaucum (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23 BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (e₁) and yellow stripe (ys). Tissue culture regenerant populations (R₀) and progeny populations (R₁) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R₀, R₁, and R₂ progeny of the genetic marker line. In a population of 1,911 plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expression of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flowering time. The incidence of chlorophyll deficiency increased with time in culture, 51 % of the progeny regenerated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also observed in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmentation and a total of 6% showed chlorophyll variation in the first generation (S₀). The presence of qualitative variation in controls suggests that the inherent variation present in the original explant was expressed and perpetuated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R₁) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic.     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

Assessment of somaclonal variation in <Emphasis Type="Italic">Dieffenbachia</Emphasis> plants regenerated through indirect shoot organogenesis

The occurrence of somaclonal variation among regenerants derived through indirect shoot organogenesis from leaf explants of three Dieffenbachia cultivars Camouflage, Camille and Star Bright was evaluated. Three types of somaclonal variants (SV1, SV2, and SV3) were identified from regenerated plants of cv. Camouflage, one type from cv. Camille, but none from cv. Star Bright. The three variants had novel and distinct foliar variegation patterns compared to cv. Camouflage parental plants. Additionally, SV1 was taller with a larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 did not produce basal shoots (single stem) but basal shoot numbers between SV1 and parental plants were similar ranging from three to four. The variant type identified from regenerated cv. Camille had lanceolate leaves compared to the oblong leaves of the parent. This variant type also grew taller and had a larger canopy than parental plants. The rates of somaclonal variation were up to 40.4% among regenerated cv. Camouflage plants and 2.6% for regenerated cv. Camille. The duration of callus culture had no effect on somaclonal variation rates of cv. Camouflage as the rates between plants regenerated from 8 months to 16 months of callus culture were similar. The phenotypes of the identified variants were stable as verified by their progenies after cutting propagation. This study demonstrated the potential for new cultivar development by selecting callus-derived somaclonal variants of Dieffenbachia.     

Using AFLP markers for species differentiation and assessment of genetic variability of in vitro-cultured Papaver bracteatum (section Oxytona)

Summary Amplified fragment length polymorphism (AFLP) markers were employed to deteet genetic variation among species of Papever (section Oxytona) and assess genetic fidelity between in vitro cell lines of Papaver bracteatum and mature plants derived from the propagation of their callus cultures. Regenerated plants exhibited morphological and phytochemical characteristics dissimilar to those of their source material. Thebaine, the dominant alkaloid produced by Papaver bracteatum, was not detected in capsules from mature regenerated accessions, indicating that there may have been a loss of genetic uniformity. Instead, the dominant alkaloid produced by the regenerated plant was shown to be isothebaine (by TLC and GC/MS), a metabolic characteristic of P. pseudo-orientale. A Neighbor-Joining tree constructed from AFLP fingerprints distinetly separates the three species of Oxytona while firmly grouping the in vitro-cultured plants with P. pseudo-orientale. Additionally phytochemical data and chromosome counts indicate that the seed used to initiate cultures was of hybrid origin and ihat the loss in genetic uniformity was not due to somaclonal variation occurring during the in vitro culture process. AFLP fingerprinting was therefore able to differentiate Oxytona species and invesgigate allopolyploidy in closely related papaver species.     

Isozyme Variability in Plants Regenerated from Calli of Cereus peruvianus (Cactaceae)

Morphological and isozyme variation was observed among plants regenerated from callus cultures of Cereus peruvianus. Different morphological types of shoots (68%) were observed in 4-year-old regenerated plants, while no distinct morphological variants were observed in plants grown from germinated seeds. Isozyme patterns of 633 plants regenerated from calli and of 261 plants grown from germinated seeds showed no variation in isocitrate dehydrogenase isozyme, and the differential sorbitol dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and peroxidase isozyme patterns observed in regenerated plants were attributed to nonallelic variation. Allelic variation was detected at three isoesterase loci. The proportion of polymorphic loci for both populations was 13.6% and the deviation from Hardy–Weinberg equilibrium for the Est-1 and Est-7 loci observed in somaclones was attributed to the manner in which the regenerant population was established. The high values for genetic identity among regenerant and seed-grown plant populations are in accordance with the low levels of interpopulation genetic divergence. In somaclones of C. peruvianus, morphological divergence was achieved within a short time but was not associated with any isozyme changes and also was not accompanied by biochemical genetic divergence.     

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.     

Agronomic evaluation of tissue-culture-derived soybean plants

Summary Genetic alterations of regenerated plants based on the tissue culture process (somaclonal variation) have become common for many plant species including soybean [Glycine max (L.) Merr.]. The objective of this study was to test for the presence of tissue-culture-derived genetic variation in eight agronomic traits in homozygous progeny regenerated by organogenesis using the commercially important cultivar Asgrow A3127. A total of 86 lines derived by repeated self-pollination of nine regenerated plants was grown in two locations for 2 years. When compared to the unregenerated parent, statistically significant variation (P<0.05) was found for maturity, lodging, height, seed protein and oil, but not for seed quality, seed weight, or seed yield. All of the variation noted was beneficial and did not involve decreased yield. Since the differences were not large, the results indicate that the tissue culture process is not necessarily detrimental to plant performance, which is an important consideration since tissue culture techniques are used in many genetic engineering methods.     

Improved protoplast culture and stability of cytoplasmic traits in plants regenerated from leaf protoplasts of cauliflower (Brassica oteracea ssp.botrytis)

Nearly 1000 plants have been regenerated from leaf protoplasts of two cauliflower (Brassica oleracea ssp.botrytis) alloplasmic inbred lines. One line (7642A) carried the Ogura (R1) cms cytoplasm derived from radish; the other line (7642B) carried a normalBrassica cytoplasm and was the fertile maintainer for the cms line. The majority of regenerated plants displayed normal vegetative morphology; they formed normal cauliflower heads and retained the floral characteristics of seed-grown plants from which they were derived. We found no change in either male sterility or in the low temperature-induced chlorosis associated with the 7642A line. Mitochondrial DNA analysis by hybridization with five cloned mtDNA probes revealed no apparent alteration in 75 regenerated plants of both lines. These results indicate that cytoplasmic traits inBrassica oleracea are stable after one cycle of in vitro culture and regeneration.     

General features of chromosome substitutions in Triticum aestivum x T. timopheevii hybrids

Summary Tissue culture of the Zea mays inbred line A188 resulted in the regeneration of plants having a high level of phenotypic variation compared to seed-grown control plants. To determine how such variation was induced and whether this could be related to specific in vitro culture methods, callus cultures were established and maintained on different, commonly used culture media. Plants were regenerated and the genomic DNA of callus cultures and regenerants analysed for RFLP differences. The results show that regardless of the gene probe used, callus formation resulted in significant deviations from the DNA pattern normally found in seed-grown control plants. Alterations in gene copy number also occurred. As differentiation and organogenesis began, the level of DNA variation fell, and most of the regenerated plants showed a genetic similarity to the controls; those with RFLP differences were the somaclonal variants.     

Detection of androclonal variation in anther-cultured rice lines using rapds

Summary Random amplified polymorphic DNA analysis was used to determine the occurrence and extent of variation in rice (Oryza sativa L.) plants regenerated from anther culture. Androclonal variation in morphologically uniform progenies was detected using 40 10-mer oligonueleotide arbitrary primers. Among 27 plants from nine anther culture-derived lines, variation was detected in three plants from two lines by two primers, namely UBC 160 and UBC 209. Primer UBC 160 amplified a polymorphic band on one of the three progenies from DH-34, while UBC 209 detected polymorphisms on two out of three progenies from line DH-58. Apart from these, the amplification produets were monomorphic across all the regenerants from anther culture-derived plants. Out of 40 tested primers, no difference in the banding pattern was observed in three seed-derived plants. The significance of possible androclonal variation at the DNA level in rice doubled haploid breeding and genetic mapping is discussed.     

Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Genetic instability during embryogenic cloning of celery

Genetically marked tissues of celery (Apium graveolens) were employed to contrast genetic and chromosomal stability in serially bulk-transferred callus and regenerated plants. After six months in culture, 84% of the callus cells were karologically indistinguishable from normal, while the remainder exhibited chromosome loss and/or fusion. All of 50 clones derived from this tissue expressed the control phenotype with respect to heterozygous isozyme markers. Of 95 plants regenerated from the same tissue, 94 were phenotypically indistinguishable from the original explant donor, and cytogenetic analyses revealed the presence in 4.3% of an accessory chromosome, while the remainder were normal diploids. Analysis of the selfed progeny of these regenerated plants revealed the presence of a new recessive mutation causing abnormal leaf morphology at a frequency of 1.8%. Only one of 40 cells in 12-month-old callus tissue was karyologically indistinguishable from normal, the remainder consisting primarily of hypodiploids. The observation that all 50 clones were phenotypically heterozygous was statistically inconsistent with the hypothesis that hypodiploidy was associated with random complete chromosome loss. The culture had, at this point, lost the ability to regenerate. It is speculated that embryogenic cloning of celery may be suitable under certain circumstances for direct field establishment, but that levels of new genetic variation are sufficiently high to preclude its use for seed production.     

Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.     

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

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

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Genetic analysis of variation in micropropagated plants of Melia azedarach L.

Summary Plants were regenerated by shoot multiplication from four clones of Melia azedarach L. during 12 mo. of subculturing. One hundred and one of these plants were examined by randomly amplified polymorphic DNA analysis. All regenerated plants showed at least one polymorphism. However, no chromosome number alterations were observed. The pattern of variation obtained by principal coordinated analysis showed a random distribution of variation among regenerated plants and their controls, indicating that genetic alterations were not cumulative during in vitro culture. Similar results were found using Shannon's index, which revealed that 50% of the observed diversity resided among plants coming from the same subculture generation. This high intraclonal variation does not provide a clear scenario for predicting the amount of culture time required to preserve genetic fidelity in commercially micropropagated M. azedarach plants. Our work suggests that other mechanisms, such as chimerism, contribute to intraclonal heterogeneity in vitro.     

Stability of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) plants regenerated via somatic embryos,axillary bud proliferated shoots,microtubers and true potato seeds: a comparative phenotypic,cytogenetic and molecular assessment

The stability, both genetic and phenotypic, of potato (Solanum tuberosum L.) cultivar Desiree plants derived from alternative propagation methodologies has been compared. Plants obtained through three clonal propagation routes—axillary-bud-proliferation, microtuberisation and a novel somatic embryogenesis system, and through true potato seeds (TPS) produced by selfing were evaluated at three levels: gross phenotype and minituber yield, changes in ploidy (measured by flow cytometry) and by molecular marker analysis [measured using AFLP (amplified fragment length polymorphism)]. The clonally propagated plants exhibited no phenotypic variation while the TPS-derived plants showed obvious phenotypic segregation. Significant differences were observed with respect to minituber yield while average plant height, at the time of harvesting, was not significantly different among plants propagated through four different routes. None of the plant types varied with respect to gross genome constitution as assessed by flow cytometry. However, a very low level of AFLP marker profile variation was seen amongst the somatic embryo (3 out of 451 bands) and microtuber (2 out of 451 bands) derived plants. Intriguingly, only AFLP markers generated using methylation sensitive restriction enzymes were found to show polymorphism. No polymorphism was observed in plants regenerated through axillary-bud-proliferation. The low level of molecular variation observed could be significant on a genome-wide scale, and is discussed in the context of possible methylation changes occurring during the process of somatic embryogenesis.     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Agronomic evaluation of inbred lines derived from tissue cultures of maize

Summary Tissue culture-induced variation has been proposed as a novel source of variation for crop improvement. In maize (Zea mays L.), chromosome aberrations and qualitative genetic variants have been induced during in vitro culture. The proportion of regenerated plants carrying such variants has been shown to increase with culture age. The objective of this research was to evaluate the relationship between culture age and somaclonal variation for several agronomic traits. Six sib-pollinated ears of S₀ (F₂) plants in four OH43 ms/A188 populations each provided control seed and embryos for culture initiation. S₂ lines derived from control seed and from plants regenerated 4 and 8 months after culture initiation were grouped according to their source ear and grown in 6 separate trials. A total of 305 tissue culture-derived and 48 control lines were evaluated as lines per se and in a testcross at each of three locations. Tissue culturederived lines and their testcrosses generally had lower grain yield and moisture. Since grain yield and moisture were not positively correlated in any trial, the highest yielding lines could be selected without increasing grain moisture. Grain yield and plant height tended to decrease with culture age. Although tissue culture-derived lines were, on average, inferior, the highest yielding line per se in three of six trials and the top-ranked line in five of six trials for yield and moisture were derived from tissue culture. The results indicate that tissue culture may generate variation for agronomic traits. Some of the variation, particularly the trend towards earlier maturity, could be useful. However, this method may require screening large populations because of the tendency to generate a large proportion of inferior lines.Contribution from Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108. Minnesota Agric. Exp. Stn. Scientific Journal Series Paper No. 15,172