Analysis of Specific RAPD and ISSR Fragments in Maize (Zea mays L.) Somaclones and Development of SCAR Markers on Their Basis

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

Analysis of specific RAPD- and ISSR-fragments in somaclonal maize (Zea mays L.) and development of SCAR markers based on them

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

The effects of growth regulators on somaclonal variation in rye (Secale cereale L.) and selection of somaclonal variants with increased agronomic traits

The aim of this research was to characterize somaclonal variation in populations derived from embryos cultured on two types of induction medium (supplemented with either 2,4-D or dicamba), as well as to select and characterize several somaclonal lines. The sexual progenies of 40 R(0)regenerants - A somaclones (derived on the medium with 2,4-D) and B somaclones (derived on the medium with dicamba) - were analysed according to the following traits: plant height, total number of tillers, number of productive tillers, spike length, number of spikelets per spike, spike compactness, number of normally developed grains per spike, weight of grains per spike, and the weight of 1000 grains. The results for twenty-two R(1)plants surpassed the variability range for the control. The transmission of positive changes to the next generation was proved in the case of 8 originally chosen R(1) plants: 7 plants selected from the A somaclones and one plant from the B somaclones. Five out of the eight created somaclonal lines proved to be stable somaclonal variants. The absolute rate of the efficiency of positive somaclonal changes was calculated as 0.64%.     

A comparative analysis of the development and quality of nursery plants derived from somatic embryogenesis and from seedlings for large-scale propagation of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Plants of Coffea arabica L. derived via somatic embryogenesis, namely, somaclones, were evaluated with C. arabica seedlings grown in the nursery. At the time of their transfer to the nursery, somaclones of C. arabica cvs. Caturra and Costa Rica 95 (Catimor) were smaller and less vigorous than seedlings of the same cultivars. Following an initial slow growth for a period of 10 weeks, somaclones began to grow faster than seedlings until both groups of plants were equal in size at 21 weeks (entire duration of growth in the nursery). Comparisons of aerial and root systems of 30-cm long somaclones and seedlings of two cultivars revealed that plants of somaclones were more vigorous than seedlings, based on the higher number of leaves (13–16 vs. 9), larger leaf area (1060–1280 vs. 730–890 cm²), and greater dry weight of aerial organs (8.5–12 vs. 7.0–7.5 g). For cv. Caturra, the root dry weight of somaclones was significantly greater than that of seedlings (2.7 vs. 1.9 g) and was attributable to the large diameter roots (>0.5 mm). Analysis of 176,000 F₁ hybrid somaclones revealed that these exhibited more heterogeneous growth than did the seedlings derived from zygotic embryos; moreover, there was a genotype effect. Almost 9–20% of somaclones required an additional 3–4 months of growth in the nursery, and 8–12% were culled for other undesirable horticultural attributes. Only 0.10–0.23% of somaclones displayed variant phenotypes. The observed somaclone vigor in the nursery was carried over to field performance as these plants were more precocious than seedlings and yielded coffee beans 1 year earlier than seedlings.     

Organ-specific and ploidy-dependent somaclonal variation; a new tool in breeding

The organ-specific somaclonal variation means the differences between the variability of somaclones originated from different somatic tissue of plant. Significant differences in some agronomical characters were achieved among somaclones of seed and plumule meristem origin. The ploidy-dependent somaclonal variation means the differences between the variability of somaclones originated from different ploidy-level tissue. Increased variation among regenerated plants was postulated by origin from cultured cells of reduced ploidy level. The comparison of somaclonal variation in the progenies of diploid plants regenerated from callus of haploid and diploid origin supported the ploidy dependent theory. The pollenhaploid somaclone method (PHS-method) was developed and tested for utilization somaclonal variation in rice breeding. The PHS-method comprises the two well-known and widely applied in vitro methods which are the androgenesis (another culture) and genetic instability of cultured haploid somatic cells (callus cultures). Developmental varieties produced by this breeding sheme are under certification in Hungary.     

In vitro selection in resistance breeding of strawberry (Fragaria x ananassa duch.)

Genetic resistance to pathogenetic soil-borne fungus Verticillium dahliae Kleb. was examined in two strawberry somaclones. Strawberry somaclones were obtained in sterile culture from runner tips of cultivars 'Merton Dawn' and 'Selva'. In vitro selection was performed with the use of homogenate of liquid cultures of Verticillium dahliae. Microplants of both somaclones were inoculated at stage of 4. Leaves. Disease symptoms were observed at 15., 30., 45., 60. and 75. days post inoculation. Extent of leaf chlorosis was rated on a scale of 0-4. Under the controlled in vitro culture conditions a different response to infection by this pathogenic fungus was observed. After 75. days post inoculation the contribution of necrotic plants in somaclone of 'Merton Dawn' reached the value of 76%, whereas in somaclone of 'Selva' this value reached 86%. In control somaclones of 'Merton Dawn' and 'Selva' the contribution of necrotic plants after 75. days post mock-inoculation with sterile distilled water reached the considerably lower value of 13%. These results revealed that somaclone of 'Merton Dawn' was more genetically resistant to infection by V. dahliae than somaclone of 'Selva'. The observed response to in vitro infection caused by Verticillium dahliae in examined somaclones was similar in comparison with original cultivars. Furthermore, somaclonal variation induced in tissue cultured strawberry was sufficient to select variants that showed enhanced genetic resistance to Verticillium wilt caused by V. dahliae. In vitro selection can be efficiently used as an alternative program to conventional resistance breeding in strawberry.     

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.     

Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants.     

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.     

In vitro induction, screening and detection of high essential oil yielding somaclones in turmeric (Curcuma longa L.)

Leaf of turmeric contains an essential oil used extensively in perfumery, pharmaceuticals and aromatherapy. Five somaclones were induced in turmeric on MS media with varying amounts of plant growth regulators. All somaclones were subsequently transferred to the field. Essential oil was extracted from leaves of in vitro and ex vitro grown plants and subjected to quantitative and qualitative evaluation. A positive correlation was established between the leaf oil content and oil constituent of in vitro grown and field transferred somaclones. Somaclones (C2, C4, C5) containing 0.16–0.18 % oil in vitro retained normal oil content (0.48–0.5 %) in the field. Similarly in vitro grown somaclones C3 and C7 with 0.36 and 0.25 % oil content retained proportionately increased oil yields of 1 % and 0.76 under ex vitro condition. GC–MS analysis of the oil revealed similar spectrum of constituents both among in vitro and ex vitro grown plants with alpha-phellandrene as major one. Thus the novel method of in vitro screening could be applied for rapid identification of high essential oil yielding turmeric genotypes thereby reducing labour, cost and time required in conventional ex vitro screening of somaclones.     

Microsatellite DNA somaclonal variation in micropropagated trembling aspen (Populus tremuloides)

Microsatellite DNA markers of ten simple sequence repeat (SSR) loci were used to examine somaclonal variation in randomly selected micropropagated plantlets derived from three different Populus tremuloides donor trees (genotypes). The plantlets were obtained from tissue cultures of dormant vegetative buds, and those derived from the same donor tree, grown in the greenhouse, did not exhibit any sign of visible morphological variation. No microsatellite DNA variation was observed among 13 somaclones of one tree and 4 somaclones of another tree at eight of the ten SSR loci. However, despite the small number of micropropagated progeny per tree sampled, microsatellite DNA variation was detected among the plantlets derived from the same donor trees at two SSR loci. The primer pair for the SSR locus PTR5 revealed somaclonal variation in 1 out of the 13 plantlets obtained from one genotype, while the primer pair for the PTR2 SSR locus revealed somaclonal variation in one out of the four plantlets obtained from another genotype. The variation at the PTR2 locus resulted in the appearance of a new allele of increased size, possibly due to an addition of the repeat units, while the variation at the PTR5 locus resulted in the appearance of third allele, presumably due to the presence of a single extra chromosome or duplication of a chromosomal segment. These results demonstrate that the genetic fidelity of micropropagated plants of P. tremuloides cannot always be assured and somaclonal variation can occur even when tissues of well organized vegetative buds are used for tissue cultures; that somaclonal variation cannot always be detected at the gross morphological level; and that microsatellite DNA markers provide useful and sensitive markers for determining the clonal fidelity and somaclonal variation in P. tremuloides.     

Genetic Variation in Potato Cv. Record: Evidence from In Vitro 'Regeneration Ability'

Regeneration ability in vitro was studied in 170 individualtubers putatively derived from several or many parent plantsof the potato cv. Record. Of these, 120 were sprouted and thesprouts used to establish in vitro shoot cultures for leaf discproduction. The other 50 were grown in a glasshouse for theproduction of leaf discs. The reliable regeneration of somaclonesfrom leaf disc calluses was successful from only 11 parentaltubers. In ten of these, somaclones were derived from in vitroshoot cultures, and from a glasshouse-grown plant in the other.Four parental tubers gave the majority of somaclones, and one,R149, produced 85% of all somaclones at 15 months from initiationof leaf disc cultures. This differential regeneration abilitymay be due to genetic differences between tubers in this potatocultivar as it was found to be maintained in subsequent tubergenerations. The results are discussed in terms of seed potatoproduction and in vitro genetic conservation of vegetativelypropagated species. Potato, Solanum tuberosum cv. Record, regeneration ability, leaf disc culture, somaclonal variation     

RAPD Analysis of Maize Somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 17 decanucleotide primers used each allowed amplification of 2–17 fragments ranging 200–2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64–74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.     

RAPD-analysis of corn somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 15 out of 17 decanucleotide primers used each allowed amplification of 2-17 fragments ranging 200-2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64-74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.     

Progeny tests of barley,wheat, and potato regenerated from cell cultures after in vitro selection for disease resistance

Summary Because plant cells cultured in vitro express genetic variability and since they can be regenerated into functional plants, procedures have been designed to use this system for the production of plants with new important agronomic characteristics, particularly for disease resistance. For barley, wheat, and potato somaclones have been found that were less susceptible to a toxin of Helminthosporium, fusaric acid, Fusarium coeruleum, F. sulphureum, or Phytophthora infestans, when screened in the first in-vitro-derived generation. Here the progeny of such somaclones is evaluated after natural and artificial infection, using greenhouse-grown or field material. The progenies of the same somaclones did not express detectable differences, which indicated that no heterozygous mutations occurred. Most lines and clones differed in their level of susceptibility to the pathogen compared to the level of the starting material, but these data were in no instance significant. It is discussed here whether this lack of significance is due to a lack of genetic differences or whether the test procedures are in adequate for detecting and securing the slight, probably quantitative, alterations.     

Heritable variability induced by the in vitro culture and genetic improvement of cultivated plants

Abstract

Genetic variability is found among plants derived from in vitro cultures of somatic cells. A number of different factors, such as the pre-existing genetic variation developed in vivo during tissue differentiation, the variation induced during the in vitro culture and also the selection for specific genotypes during plant regeneration, are considered as possible causes of the phenomenon.

The nature of the genetic changes induced in somaclones (variation in chromosome number, gross and cryptic chromosomal rearrangements, transposition of genetic elements, gene amplification and somatic gene rearrangements) is also discussed.     

Development and Evaluation of a Laboratory-Based Technique for Screening Somaclonal Regenerants of Watercress (Rorippanasturtium-aquatium) for Resistance to Crook Root Disease (Spongospora subterranea f. sp. nasturtii)

A laboratory based method has been developed for infecting watercress with the crook root fungus ( Spongospora subterranea f.sp. nasturtii ). This utilised plastic trays of nutrient solution on which watercress shoots, supported by floats, were grown for a pre-inoculation period of 14 days before an inoculum consisting of 1 g of crook roots was added. Seven watercress someclones derived from plantlets regenerated from callus of a parental commercial control line (clone A) were tested onthree separate occasions. No variation within the parental control plants was seen but significant variation both between and within the individual somaclones over successive tests was observed. Two somaclones showed increased resistance to crook root disease compared with parental control plants.     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Selection for virus resistance in tomato exposed to tissue culture procedures

Protocols elaborated with the objective of achieving valuable material for selection procedure of variants with virusresistance traits in tomato genotypes are presented. Preliminary results are demonstrated in the domain of testing for variability in somaclones obtained through indirect adventitous organogenesis initiated on leaf explants of cultivated tomato (Lycopersicon esculentum Mill.). Somaclones were grown in greenhouse conditions and variation of their symptoms upon infection with tomato mosaic (ToMV) or cucumber mosaic (CMV) respectively was observed. Tests for resistance to the local isolates of the above cited viruses were performed using enzyme linked immunosorbent assay and back inoculation onto diagnostic plants. Screening data are presented. Desirable variants were selected from cultivars ‘Moneymaker’, ‘Potentat’ and ‘Rutgers’. Some of the ‘Moneymaker’ somaclones exhibited increased tolerance to cucumber mosaic virus, a few seemed to be even fully resistant though most were susceptible as donor plants. The most favourable somaclonal lines are actually further tested and monitored for changes in horticultural characteristics. The described procedure of searching for resistance trait in specific pathogen-free (SPF) plants regenerated from infected tissue looks promising and thus can serve as aid in attaining appropriate objectives of breeding programme. Additionaly experiments were initiated to obtain somaclones from cultivars ‘Beta’, ‘Krakus’ and Stevens Rodade hybrid via regeneration of isolated protoplasts. To this end the callus stage was obtained from all donors.