Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

Selection for Fusarium wilt disease resistance from regenerants derived from leaf callus of strawberry

Resistant lines of strawberry to the fungal wilt disease caused by Fusarium oxysporum f. sp. fragariae were selected strawberry plants regenerated from leaf-derived callus tissues. Regenerants were transplanted to a field heavily infested with this pathogen, and normally growing plants were selected as the putative resistant lines. Daughter plants produced vegetatively through runner formation of the lines were similarly tested in the pathogen-infested field over an additional three generations. Finally, two resistant lines were obtained from a total of 1,225 regenerants. The stable propagation of disease resistance in these lines was confirmed by directly inoculating the daughter plants with the pathogen and planting in a pathogen-infested soil. All of the control plants were efficiently infected and died within one month. The isolated plant lines grew and developed runners even after direct inoculation and produced daughter plants in this soil. Thus, the present study demonstrated the existence of somaclonal variation for disease resistance against a soil-borne fngal pathogen.     

Transformation of Medicago by Agrobacterium mediated gene transfer

Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence of the structural gene of the kanamycin resistant determinant in total DNA isolated from several independent transformants. All data presented clearly demonstrate the transfer, stable maintenance and functional expression of the kanamycin resistance marker in Medicago varia cells which retain their morphogenic property.Abbreviations Km kanamycin - KmR kanamycin resistant - Cb carbenicillin - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine - T-DNA transferred DNA into plants     

Multiplication of tobacco mosaic virus in tobacco callus tissues and in vitro selection for viral disease resistance

Tobacco mosaic virus-resistant tobacco was selected in vitro using callus tissues induced from axillary buds of systemically infected tobacco plants. Callus lines in which the virus was continuously multiplying were first isolated and redifferentiated into shoots. By the procedure, non-diseased, healthy shoots were successfully isolated from diseased shoots, which showed typical mosaic symptoms of the virus, and regenerated into intact plants.These regenerated plants showed resistance to virus inoculation, and selfed progeny of virus-resistant regenerants segregated the resistance and susceptibility according to the Mendelian system.     

Somaclonal variation and resistance to Verticillium wilt in lucerne,Medicago sativa L., plants regenerated from callus

Somaclonal variation in disease reaction type to infection by the vascular wilt pathogen Verticillium albo-atrum Reinke & Berth was assessed in a population of lucerne plants regenerated from callus lines obtained from a susceptible cultivar. Disease severity in the regenerant population was reduced by comparison with parental controls. Seed progeny and plants recovered via a second tissue culture cycle reverted to mainly susceptible reaction types. In a further experiment a low molecular weight toxic fraction from culture filtrates of the fungus was incorporated into the callus medium prior to regeneration. Toxin treatment reduced the regenerative capacity of callus, and there was little evidence for a higher frequency of wilt resistant plants in populations selected at low toxin concentrations. The results suggest that somaclonal variation as an alternative breeding strategy for disease resistance in lucerne offers no advantages over conventional recurrent selection.     

Biotechniques for improving acid aluminum tolerance in alfalfa

Alfalfa (Medicago sativa L.), cultivars ARC, Regen Y and Saranac were selected in vitro in a recently developed acid/aluminum toxic media. The new media produced higher initiation rates and higher fresh callus weights than those obtainable with the media described by Meredith and Connor for the selection of aluminum resistant variants in Nicotiana plumbaginofolia. Both rescue and direct initiation yielded adequate amounts of healthy callus for the initiation of embryogenesis. The toxic effect of the acid/aluminum media is expressed in both the percent of explants initiating callus and in the fresh-weights obtained during initiation and two subsequent subcultures.     

In Vitro Selection Against Ascochyta Blight of Chickpea (Cicer arietinum L)

Callus cultures established on MS medium containing 2.0 mg l^-1 2, 4-D were inoculated on the regeneration medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3%, v/v) of culture filtrate (CF) of Ascochyta rabiei infesting chickpea. Out of 486 callus pieces and 270 regenerants obtained from immature embryo derived callus screened, 50 callus lines and 74 regenerants were found resistant. Further, these resistant callus lines and regenerants were subjected to stability test by growing them on a medium containing 3% CF. Seventeen callus lines and 28 regenerants of the selected lines showed normal growth on the selection medium. The regenerated plants were tested in pots under artificial epiphytotic conditions where they showed normal growth behaviour and high degree of resistance.     

Hyphal growth of Phytophtora cinnamomi on pine callus tissue

A procedure was developed which demonstrates the expression of differential resistance in pine callus tissues to the fungal pathogen Phytophthora cinnamomi Rands. Callus tissues were maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D and inoculated with hyphae of P. cinnamomi at 26°C in the dark. The number of intracellular hyphae was used as an index of resistance. Loblolly and loblolly × shortleaf pine hybrids were determined to be more resistant to infection and invasion by the fungus than were shortleaf and Virginia pine.Abbreviations (AL) loblolly pine—Alabama - (PL) South Carolina - (AS) shortleaf pine—Alabama - (CS) Georgia - (AV) Virginia pine—Alabama - (H1) loblolly × shortleaf pine hybrids—14–42 × 6-I-43 - (H2) I-523 × 6-D-8     

Regeneration of amphidiploid plants from tissue cultures of Allium wakegi

Callus was induced from the bulb of Allium wakegi Araki on MS semisolid medium supplemented with several growth regulating substances. The calli were subcultured every 40 days. At the time of every subculture the callus was subdivided to be used for chromosome studies, plant regeneration, or continuous callus multiplication. The chromosome constitution of cells in callus and regenerated plants varied over the culture period, and at the 3rd subculture amphidiploid plants were obtained. They appeared even more frequently than amphihaploid plants in the 4th subculture. Hypoamphihaploid regenerants appeared as stumpy shoots but none of these shoots proceeded further to form a normal plant. By Giemsa C-banded karyotype, the chromosome constitution of amphidiploid plants was found to result from exact doubling of the chromosome sets of amphihaploid common species. Amphidiploid plants show better viability and growth than common plants. The possibility and the expectation of new crop plants to be developed from amphidiploid plants will be discussed.     

Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)

Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.Abbreviations BAP benzylaminopurine - KIN kinetin - NAA naphthaleneacetic acid - PAR photosynthetically active radiation Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2     

Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-II gene and a glyphosate resistance plant-derived 5-enolpyruvylshikimate-3-phosphate synthase gene was used to transform flax hypocotyl tissues. Transformed shoots could be regenerated from the inoculated tissue and were proven to be transgenic by the combination of leaf callus assays, nopaline assays and progeny tests. Co-segregation was observed in the progeny for kanamycin and glyphosate resistance.     

Agrobacterium mediated transfer of chlorsulfuron resistance to commercial flax cultivars

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-ll gene and a mutant acetolactate synthase gene (conferring resistance to the herbicide chlorsulfuron) from Arabidopsis was used to transform flax (Linum usitatissimum) hypocotyl tissue. Transgenic regenerants were recovered from the inoculated tissue and were tested for expression of the foreign genes by leaf callus assays on kanamycin and on chlorsulfuron. Transgenic plants were grown to maturity; selfed progeny were similarly tested to determine segregation pattern for the novel genes, and some were grown in chlorsulfuron containing soil. Lines from two major commercial cultivars express chlorsulfuron resistance in greenhouse tests.     

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.     

Selection, biological properties and fitness of resistance-breaking strains of Tomato spotted wilt virus in pepper

In glasshouse tests, infective sap from plants infected with 17 different isolates of Tomato spotted wilt virus (TSWV) from four Australian states was inoculated to three Capsicum chinense accessions (PI 152225, PI 159236 and C00943) carrying single genes that confer hypersensitive resistance to TSWV. The normal response to inoculation was development of necrotic (hypersensitive) local lesions in inoculated leaves without systemic invasion, but 3/1386 infected plants also developed systemic susceptible reactions in addition to hypersensitive ones. Similarly when two isolates were inoculated to C. chinense backcross progeny plants, 1/72 developed systemic susceptible reactions in addition to localised hypersensitive ones. Using cultures from the four plants with susceptible reactions and following three to five further cycles of serial subculture in TSWV‐resistant C. chinense plants, four isolates were obtained that gave systemic susceptible type reactions in the three TSWV‐resistant accessions, and in TSWV‐resistant cultivated pepper (C. annuum). When three of these isolates were inoculated to tomato (Lycopersicon esculentum) breeding lines with single gene resistance to TSWV, resistance was not overcome. Similarly, none of the four isolates overcame partial resistance to TSWV in Lactuca virosa. When TSWV isolates were inoculated to tomato breeding lines carrying partial resistance from L. chilense, systemic infection developed which was sometimes followed by ‘recovery’. After four successive cycles of serial passage in susceptible cultivated pepper of a mixed culture of a resistance‐breaking isolate with the non resistance‐breaking isolate from which it came, the resistance‐breaking isolate remained competitive as both were still found. However, when the same resistance‐ breaking isolate was cultured alone, evidence of partial reversion to wild‐type behaviour was eventually obtained after five but not four cycles of long term serial subculture in susceptible pepper, as by then the culture had become a mixture of both types of strain. This work suggests that resistance‐breaking strains of TSWV that overcome single gene hypersensitive resistance in pepper are relatively stable. The findings have important implications for situations where resistant pepper cultivars are deployed widely in the field without taking other control measures as part of an integrated TSWV management strategy.     

Effect of dilution rate on the biological nitrogen fixation in thePhaseolus vulgaris-Rhizobium phaseoli symbiosis

Summary Continuous culture was used to produceRhizobium phaseoli cells at different dilution rates.Phaseolus vulgaris seeds in sterile Leonard jars were then inoculated with the cells produced, and the system was left to interact for six weeks. Dry weight of the plants and number and weight of the nodules were measured. These data were compared to the response obtained with cells produced in batch culture. An increase in plant biomass and nodule weight and number were observed at higher dilution rates.     

Further Investigation of the Specificity of Interactions Between Wild Lactuca spp. and Bremia lactucae Isolates from Lactuca serriola

Callus cultures derived from isogenic lines of the tomato cultivars Moneymaker and Craigella, resistant or susceptible to F. oxysporum f. sp. lycopersici, were inoculated with Fusarium oxysporum f. sp. lycopersici race 1. Fungal growth was restricted on callus derived from resistant plants, after inoculation with a conidial suspension, whereas callus derived from susceptible plants was totally overgrown by the fungus within 7 days. The concentration of the phytoalexin rishitin was significantly higher in the callus culture derived from a resistant tomato line compared with the callus culture from a susceptible line, 2 and 3 days after inoculation with mycelium. The results of the experiments were compared with experiments with whole plants. Rishitin production as well as growth of the fungus was comparable with responses in plant-fungus interaction. Therefore callus culture may be useful in studying the interaction between tomato plants and race 1 of F. oxysporum f. sp. lycopersici.     

Scopolin, a Biochemical Marker for Resistance to Thielaviopsis basicola in Callus and Crown-Gall Tissue Cultures of Tobacco

Scopolin concentration increased in tissue cultures where Thielaviopsis basicola growth ceased (primary and established callus cultures of the resistant tobacco cultivar Ky 170) while it decreased in tissue cultures where fungal growth persisted (primary and established callus cultures of the susceptible tobacco cultivar Ky 151 and crown-gall cultures of Ky 170 and Ky 151). The concentration of chlorogenic acid, scopoletin, and two other unknown soluble phenols varied after inoculation and no, correlation with tissue culture resistance could be established. Incorporation of Agrobacterium tumefaciens T-DNA in the genome of both cultivars induced a drastic reduction of scopolin in inoculated tissue cultures. T-DNA incorporation had less, influence on uninoculated tissues. Scopolin at concentrations found in tissue cultures was not toxic to the fungus.     

Studies on the nature of resistance in tomato plants to Verticillium albo-atrum

Extracts of healthy resistant and of healthy susceptible plants of tomato had the same effect on growth of Verticillium albo-atrum in vitro. Tracheal saps from resistant and from susceptible plants showed no difference in their effect on spore germination and mycelial growth of V. albo-atrum. Cuttings from resistant plants, inoculated with V. albo-atrum and fed with low concentrations of casamino acids, or glucose, at first wilted more than controls but soon recovered. Continuous treatment with dilute ethanol solutions for 2 weeks induced marked wilting in inoculated cuttings of resistant plants: treatment for shorter periods caused less severe symptoms, from which cuttings recovered slowly. Metabolic inhibitors did not break resistance of cuttings, but the pathogen survived longer in cuttings treated with sodium diethyldithiocarbamate, salicylaldoxime or 8-hydroxyquinoline than in controls. When one end of segments of stems of resistant plants was inoculated with the pathogen, and 48 h later the uninoculated end was placed near a colony of V. albo-atrum on agar, growth of the fungus colony towards the stem segment was sometimes inhibited. There was no such inhibition when segments from susceptible plants were used. Both tracheal sap and diffusates from segments of inoculated resistant plants supported less growth of germ tubes of V. albo-atrum than sap and diffusates from uninoculated plants. These differences were not obtained with the susceptible variety and production of fungitoxic substances in resistant plants after infection is inferred.     

Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens

Summary To determine the best combination for potential use in transformation of Pisum sativum L., 13 genotypes were inoculated with wild-type Agrobacterium tumefaciens strains A281, C58 and Ach5. A281 appeared to be the most virulent strain, as determined by size and number of tumours, followed by C58 and Ach5. Genotypes differed considerably in their response to inoculation and genotype x strain interaction was evident. Genotypes also responded differently to in vivo or in vitro inoculation. Axenic calli from tumours could be grown on hormone-free medium and the presence of the specific opines for each strain in the callus indicated successful transfer and expression of T-DNA. Southern blot analysis of DNA from callus of A281-inoculated material showed that both T_R and T_L T-DNA had been incorporated into the pea genome.NRCC No. 30265     

Optimum tissue culture conditions for selection of resistance to Phytophtora cinnamomi in pine callus tissue

The present experimentation compared the best nutrient medium, temperature, and growth hormones for callus induction and growth of various pine species from different seed sources with their effect on growth of Phytophthora cinnamomi. Callus tissues maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D at 26°C in the dark optimized the expression of differential resistance when inoculated with hyphae of P. cinnamomi. High concentration of 2,4-D (5×10^–5M) inhibited growth of P. cinnamomi.Abbreviations AL loblolly pine-Alabama - PL South Carolina - AS shortleaf pine-Alabama - CS Georgia - AV Virginia pine-Alabama