In Vitro selection of groundnut cell lines fromCercosporidium personatum culture filtrates and regeneration of resistant plants through cell culture

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence ofCercosporidium personatum pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25–100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced byC. personatum. Selected cell lines were used for plant regeneration on regeneration media containingC. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R₂ generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants throughin vitro selection.     

Loss of resistance to Colletotrichum trifolii in in vitro regenerated alfalfa

Alfalfa plants were regenerated from callus cultures of three source plants that differed in resistance to anthracnose, caused by Colletotrichum trifolii. All regenerant plants were evaluated for variation in resistance to disease caused by races 1 and 2 of the pathogen. Of eighty-two plants that were regenerated and evaluated, no plants responded differently to inoculation with race 1 of C. trifolii, but two plants (2.4%) differed in resistance when inoculated with race 2. The source plant of these regenerants was resistant to races 1 and 2 of the pathogen but the regenerants were resistant to race 1 and susceptible to race 2. No variants to race 1 were detected. The susceptible response of the variant plants to race 2 was confirmed by cytological analysis and was consistent with the response of nonregenerant susceptible plants. These plants represent a near-isogenic plant model for studying the molecular biology of resistance and susceptibility to anthracnose of alfalfa.     

Isolation and characterization of isonicotinic acid hydrazide-resistant mutants of Nicotiana tabacum

Summary Twenty stable variant lines resistant to isonicotinic acid hydrazide (INH), an inhibitor of the conversion of glycine to serine in the glycolate pathway, were isolated in cell cultures initiated from allodihaploid Nicotiana tabacum. Plants were regenerated from 13 of these lines and explants were tested for resistance. For some lines virtually all of the regenerated plants scored as resistant; for others a mixed population of sensitive and resistant plants were obtained. One or more plants from 5 lines were fertile, presumably as a result of spontaneous diploidization of cells in the plant or culture. Callus initiated from the seed progeny of these plants was resistant to INH confirming the characteristic as a stable mutation. Seedlings from all INH-resistant plants were small and slow-growing, but the slow-growth trait could be separated from resistance in backcrosses of hybrids. In one case (line I21) crosses with sensitive lines show the resistant trait in that line to be dominant.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.     

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

The resistance of lettuce (Lactuca sativa L.) toNasonovia ribisnigri: Bionomics ofN. ribisnigri on near isogenic lettuce lines

The intrinsic rate of increase (r_m-values), mean relative growth rates and mortality ofNasonovia ribisnigri (Mosley) (Homoptera, Aphididae) on different lines of lettuce (Lactuca sativa L.) were determined. Near isogenic resistant and susceptible lines, plus their ancestors, were used. Bionomics ofN. ribisnigri on the resistant lines (NrNr) with the dominantNasonovia resistance gene (Nr-gene) differed clearly from the susceptible lines (nrnr). Mortality was high, no larvae reached adulthood, and no reproduction nor honeydew production was seen on the resistant lines. Transfer of aphids to susceptible plants after a period of 2 days on the resistant lines showed no signs of intoxication of aphids. Apparently there is no feeding on the resistant lines. It is not clear whether the aphids can not reach the phloem or do not accept it on the resistant line.     

Characterization and regeneration of salt- and water-stress mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Summary Protoplast-derived colonies of haploid N. plumbaginifolia leaves were used to select for resistance to NaCl, KCl and polyethylene glycol 6000 (PEG). Salt-and PEG-tolerant cell lines were isolated on the basis of growth in a culture medium containing inhibitory concentrations of either NaCl or KCl (200 mM) or PEG (25%). The frequency of resistant lines ranged from 10^-5 to 10^-6. One resistant line from each treatment was regenerated into plants. All resistant lines produced 10–25 times more proline than the wild type when grown on a non-selective medium. Similar values were also observed in the leaves of resistant progeny plants. In each mutant line, salt or PEG resistance was transmitted as a single dominant nuclear gene as shown by segregation ratios in progenies of crosses between resistant and wild-type plants. The latter observation demonstrates clearly the existence of a genetic basis for increased salt tolerance.     

Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens

Two pathogenesis-related (PR) protein genes consisting of a barley chitinase (chi-2) and a wheat lipid-transfer-protein (ltp) were introduced singly and in combination into carrot plants via Agrobacterium-mediated transformation using the phosphinothricin acetyl transferase (bar) gene as a selectable marker. Over 75% of regenerated plants were confirmed to be positive for the transgenes by PCR and RT-PCR and were resistant to the herbicide Liberty (0.2%, v/v). Northern analysis and immunoblotting confirmed the expression of the transgenes in about 70% of the plants, with variable expression levels among individual lines. Southern analysis revealed from one to three copies of each transgene. Transgenic plants were inoculated with two necrotrophic foliar fungal pathogens, Alternaria radicicola and Botrytis cinerea, and showed significantly higher resistance when both PR genes were expressed compared to single-gene transformants. The level of disease reduction in plants expressing both genes was 95% for Botrytis and 90% for Alternaria infection compared to 40–50% for single-gene transformants. The chi2 and ltp genes could be deployed in combination in other crop plants to significantly enhance resistance to necrotrophic fungal pathogens.     

Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz)

In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants.     

Identification of highly regenerative plants within sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines for molecular breeding

Summary Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration response, line FC607 also had the highest mean number (8.3±1.1) of shoots per explant. Individual plants within each line exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs.     

Selection of ethionine-resistant variants with increased accumulation of methionine from embryogenic protoplasts of the forage legume <Emphasis Type="Italic">Astragalus adsurgens</Emphasis>

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7–8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5–9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

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.     

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.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

Insect-resistant transgenic Pinus radiata

Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage.