Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid     

Inheritance of somatic embryogenesis in red clover (Trifolium pratense L.)

 Red clover genotypes capable of regenerating plantlets in vitro from non-meristematic tissue-derived callus are rare. Selection for genotypes capable of somatic embryogenesis identified a clone comprised of a group of plantlets regenerated from a hypocotyl-derived callus culture on L2-based media and another group of plantlets originating from crown divisions of the epicotyl-derived plant. The callus-derived plants of this clone were highly regenerative when reintroduced to callus culture, but the epicotyl-derived plants produced nonregenerative callus cultures. F₁, F₂ and BC₁ populations were evaluated to determine the mode of inheritance of the regeneration trait. Reciprocal crosses did not differ, indicating a lack of maternal effects. Results were compatible with genetic control of regeneration by two complementary genes. We propose the genotype Rn1-Rn2- for regenerative plants. Three petiole segment explants were sufficient to evaluate regenerative ability in seedlings. Regenerative ability was often associated with abnormal leaf morphology in a few to several leaves. Received: 17 February 1998 / Accepted: 7 April 1998     

Cytological Analysis of Seedling Roots, Transformed Root Cultures and Roots Regenerated from Callus of Swainsona galegifolia (Andr.) R. Br.

The stability of chromosome number was investigated in culturesof roots from Swainsona galegifolia. Roots from germinated seedsor plants grown in vitro when cultured in liquid medium howed90% or more cells with the diploid number of 2n = 32. The remainingcells showed aneuploidy mostly below 32. The stability of chromosomenumbers was not affected by transformation with Agrobacteriumrhizogenes although when roots were transformed with A. rhizogenesLB 9042 the range of chromosome numbers in the few aneuploidcells present was higher than in roots for which strain A4 wasused. In contrast, roots regenerated from callus had only 15%of cells with 2n = 32 and showed a modal number of 18. Six rootcultures established from individual roots regenerated fromcallus showed a wide variation in number (8–83). Fivecultures had a modal number around 18, the sixth, a modal numberof 39 which is above the diploid number. The implication ofthe results for the production of secondary metabolites fromroot culture is discussed. Key words: Agrobacterium rhizogenes, callus cultures, chromosome number, root cultures, Swainsona galegifolia     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

Inheritance of Chloramphenicol Resistance, a Trait Selected in Cell Cultures of Nicotiana Sylvestris. Speg. and Comes

Three cell lines with improved resistance to growth inhibitionby chloramphenicol were selected from cell cultures of Nicotianasylvestris. Resistance was retained in callus cultures of twoout of three plants regenerated from one of the lines, but notin cultures of plants regenerated from the other two lines.Sexual progeny of the two resistant plants were either sensitiveor showed slow segregation for chloramphenicol resistance. Incallus from only two of the seedlings was inheritance of chloramphenicolresistance clearly demonstrated. Nicotiana sylvestris, cell culture, choramphenicol resistance     

Cloning and Mapping of Telomere-Associated Sequences from Rice

We have isolated three telomere-associated sequences from riceusing cassette-ligation-mediated polymerase chain reaction (PCR).Each of the obtained clones hybridized to the terminal of oneor several rice chromosome arms. The telomeres recognized bythe clones displayed a high level of polymorphism between tworice varieties, Nipponbare (a japonica variety) and Kasalath(an indica variety). Variability in the chromosome termini wasalso detected among individual F₂ progeny plants, which werederived from a cross between the two rice varieties. One clonecontaining telomere-associated sequences was located to oneend of chromosome 5, and another clone to one end of chromosome11. For another clone, non-allelic segregation of polymorphichybridization bands was observed between japonica and indicarice; this clone was mapped to one end of chromosome 12 in japonicaand to one end of chromosome 11 in indica rice. This indicatesan exchange of termini between nonhomologous chromosomes.     

Experimentally Synthesized Plant Chimeras I. In vitro Recovery of Nicotiana tabacum L. Chimeras from Mixed Callus Cultures

Experiments were conducted to develop techniques for synthesizingchimeras between plants of known genotype by utilizing in vitrotechniques Chimeral calli composed of green and albino tobaccocells were obtained by initiating callus tissue from mixturesof albino and green cotyledons, hypocotyls, callus culturesand cell suspensions The most effective mixing of genotypesoccurred when callus was derived from mixed filtered cell suspensionsUpon shoot regeneration, chimeral calli yielded 1317 non-chimeraland four chimeral plants Chimeras may have arisen as a resultof experimental procedures or possibly from spontaneous chromosomalabnormalities since leaves of some albino control plants occasionallyproduced small green islands of cells Explanations for the recoveryof a high percentage of non-chimeral shoots are presented Tobacco, callus cultures, cell suspensions, tissue culture, shoot apical meristems, somatic-crossing over     

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Pearl Millet (Pennisetum americanum)

Immature embryos of Pennisetum americanum (pearl millet), culturedin the presence of 2,4-dichlorophenoxy acetic acid (2,4-D) produceda pale-yellow and compact callus tissue by proliferation ofthe scutellum. Teased pieces of the compact callus were placedin a liquid medium on a gyrotory shaker to establish suspensioncultures. The cultures were composed of large, elongated andhigly vacuolated cells, and a population of richly cytoplasmiccells. The latter, here termed embryogenic cells, containednumerous plastids with starch, and occurred in tight groupsof four or more cells, and occasionally as single cells. Structuresresembling various stages of embryogenic development were foundin the suspension cultures. When the cultures were plated ina 2,4-D-free agar medium containing abscisic acid, embryoidswith the typical organization of cereal embryos were produced.The embryoids ‘germinated’ in vitro to give riseto plantlets, which were successfully transferred to soil. Theregenerated plants showed the normal diploid chromosome numberof 14. Embryoids apparently arose from single embryogenic cells,either directly or after the formation of a proembryonal massof cells. embryogenesis, pearl millet, Pennisetum americanum, regeneration, suspension culture     

Callus Induction and Plant Regeneration from Barley Mature Embryos

Callus cultures were induced starting from excised mature embryosin spring barley, Hordeum vulgare cv Maxima On a medium containinga high level of auxin, a first primary callus was induced whichwas friable, unorganized and capable of direct plant regenerationin the tested conditions This callus type was characterizedby fast growth and high variability in chromosome number Subsequently,a secondary callus type arose from the primary calli subculturedon the same medium in the light This callus type was white andcompact and consisted predominantly of diploid cells When transferredto hormone-free medium it gave rise to green shoots Completerooting of the shoots was achieved on half-strength basal mediumfollowed by exposure to higher light intensity Regenerated plantletscould then be transferred directly into soil without sufferingany loss in vitality Although showing different degrees in morphologicalvariability, they all maintained the diploid chromosome number Hordeum vulgare L, spring barley, morphogenic calli, organogenesis     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

Plant regeneration in zigzag clover (Trifolium medium L.)

A study was conducted to regenerate plants from explant tissue and from callus culture of zigzag clover (Trifolium medium). Petiole segments from two strains of zigzag clover were cultured on L2 or in SL2 media. Shoots were regenerated via organogenesis from petiole segments of both strains of zigzag clover. Direct shoot regeneration was noticed as early as eight days after the initiation of cultures. Shoots were also regenerated via somatic embryogenesis from petiole-derived calli of the two strains on L2 and SL2 media. Regenerated plants have normal morphological characteristics.     

Agrobacterium rhizogenes Mediated Transformation of the Forage Legumes Medicago sativa and Onobrychis viciifolia

Three cultivars of M. sativa and one cultivar of O. viciifoliawere evaluated for their response to inoculation with A. rhizogenesstrain A4T (containing pRiA4b). A cultivar-dependent responsewas observed in M. sativa with 94%, 25%, and 4% of infectedstem explants producing transformed roots in the cultivars Vertus,Regen-S, and Rangelander, respectively. In O. viciifolia cv.Hampshire Giant, an explant-dependent response was observedwith 78% and 50% of seedling cotyledon and hypocotyl explantsresponding, respectively. Leaf explants failed to produce transformedroots. Transformed roots showed plagiotropic and negativelygeotropic growth on hormone-free agar MS medium. Productionof transgenic shoots from O. viciifolia root cultures occurredspontaneously. Recovery of transgenic plants from M. salivacv. Rangelander was achieved by transfer of callus (inducedon UM medium containing 2·0mg dm^–3 2,4-D and 0·25mg dm^–3 kinetin) to MS medium containing 0·5 ingdm^–3 BAP and 0·05 mg dm^–3 NAA. Cultured rootsof both species synthesized opines (agropine and mannopine).Extensive morphological variation was observed in plants ofM. sativa (clone Al) and O. viciifolia (clone A4Tl) establishedin the glasshouse. DNA sequences homologous to TL-DNA and TR-DNAwere present in root clones and regenerated plants. Key words: Agrobacterium rhizogenes, Medicago sativa, Onobrychis viciifolia, transformed roots, transgenic plants     

Peroxidase Isoenzyme Patterns in Cell Cultures derived from Cotyledon, Stem, Leaf and Fruit from Grapevine (Vitis vinifera cv. Monastrell)

The expression of peroxidase isoenzymes capable of oxidizing4-hydroxystilbenes was studied during the establishment of cellcultures derived from different tissues (cotyledon, stem, leafand fruit) of Vitis vinifera cv. Monastrell vines. This wascarried out in order to elucidate whether different tissuesof the same plant maintain persistent tissue-specific patternsof gene expression during in vitro culture or whether in vitrocultures are characterized by identical patterns of gene expressionirrespective of the tissue's origin. The results illustratedthat both the isozyme patterns and the substrate specificityof the peroxidase activity secreted to the medium are analogousfor the profile of acidic (Prx A) and basic (Prx B) peroxidaseisoenzymes, only quantitative differences being shown in theneutral peroxidase isoenzyme (Prx N) pattern. These resultssuggest that in vitro cultures of grapevines are characterizedby similar patterns of gene expression, no matter what theirtissue of origin.Copyright 1995, 1999 Academic Press Grapevine, vitis vinifera, cell cultures, 4-hydroxystilbene oxidizing peroxidase isoenzyme, substrate specificity     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

The Production of Plantlets from Tissue Cultures of Brussels Sprout (Brassica oleracea L. var gemmifera D.C.)

Shoots were induced on callus derived from sprout sections andpetiole slices of an inbred parent line of Brussels sprout (Brassicaoleracea L. var gemmifera D.C.). The shoots, when excised andtransferred to fresh medium, enlarged and formed roots. Theseplantlets could be transferred to soil or their number increasedby a multiplication process involving the production of newshoots from the dormant lateral buds. Some of the plantletsderived from sprout callus were grown to maturity in the fieldand their morphology and chromosome number compared to seedgrown plants. There were no significant differences in sproutsize and stem diameter but there were significant differencesin plant shape. None of the plants in the field experiment showedpolyploidy. Plants derived from callus possessed an enhanced ability toform callus and redifferentiate when sections from these plantswere placed back on to nutrient medium.     

Isozymes as biochemical and cytochemical markers in embryogenic callus cultures of maize (Zea mays L.)

Isozyme analyses were carried out on protein extracts of non-embryogenic and embryogenic callus fromZea mays L., using polyacrylamide gel electrophoresis. We examined the isozyme patterns of glutamate dehydrogenase, peroxidase and acid phosphatase for their utility as biochemical markers of maize embryogenic callus cultures. These isozyme systems were also used to examine possible correlations between isozymes and different stages of regeneration. The zymograms of peroxidase and glutamate dehydrogenase differed for non-embryogenic and embryogenic callus. Further, some isozymes were correlated with the morphological appearance of the tissue while others seemed to be involved with the duration of the culture period. Using the same enzyme assays on fresh tissue samples we were able to test the three enzymes as cytochemical markers in embryogenic cultures. Glutamate dehydrogenase proved to be most successful to discriminate embryogenic from non-embryogenic cells.     

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.     

Oxygen Supply Limits Nitrogenase Activity of Clover Nodules After Defoliation

The aim of this study was to test the effect of oxygen partialpressure as a possible limiting factor of nitrogen fixationfollowing defoliation. The response of nitrogenase activity(C₂H₂-reduction) of defoliated and undefoliated white and redclover plants (Trifolium repens L. and Trifolium pratense L.)to either 19 kPa oxygen or 55 kPa oxygen was investigated. Priorto defoliation, white clover plants were grown for five weeksin a growth chamber, and red clover plants for 7 or 11 weeksin a glasshouse. The results included measurements of ¹⁶N₂-uptake. Increasing oxygen partial pressure from 19 to 55 kPa severelyrestricted nitrogenase activity of undefoliated white cloverplants; however, 2 h after complete defoliation, the same treatmentcaused a significant increase. A fivefold increase in nitrogenaseactivity upon exposure to the elevated oxygen partial pressurewas found at the end of a 24 h period. This beneficial effectdecreased gradually from 1 to 5 d after defoliation. The responseof recently defoliated red clover plants to 55 kPa oxygen partialpressure was similar to that of white clover, independentlyof plant age. The gradual recovery of nitrogenase activity duringthree weeks of regrowth was associated with a simultaneous changein the response to increased oxygen partial pressure, leadingagain to the response of undefoliated plants. These data suggested that lack of oxygen at the site of nitrogenfixation, resulting from a dramatic increase in oxygen-diffusionresistance, is the main factor limiting nitrogenase activityfollowing defoliation. Trifolium repens L., Trifolium pratense L., white clover, red clover, defoliation, regrowth, nodules, nitrogen fixation, nitrogenase activity, oxygen limitation