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.     

Selection of regenerable maize callus cultures resistant to 5-methyl-DL-tryptophan,S-2-aminoethyl-L-cysteine and high levels of L-lysine plus L-threonine

Tissues resistant to lethal levels of equimolar L-lysine plus L-threonine (LT), 5-methyl-DL-tryptophan (5MT, a tryptophan analog), or S-2-aminoethyl-L-cysteine (AEC, a lysine analog) were selected from maize callus capable of plant regeneration (H99 and W77-R3019 genotypes).Resistance to LT resulted from resistant calli having a 19 times greater level of free threonine than wild type tissues. The resistance was expressed in roots of whole plants; threonine levels were two to nine times greater in leaves and kernels of resistant plants than in wild type plants. Slightly greater levels of isoleucine, lysine and methionine were also noted, particularly in the kernel. Genetic studies with individual resistant plants did not always produce inheritance ratios typical of simple Mendelian inheritance, but by the third generation after plant regeneration a trend towards homozygosity was apparent and the data suggests that LT resistance is inherited as a single dominant nuclear gene.Resistance to 5MT resulted from resistant calli having a 133 to 161 times greater level of free tryptophan than wild type tissues. Also, phenylalanine was 22 to 30 times as great and histidine, tyrosine and valine were about two times as great as in wild type tissues. Resistance was expressed in roots of whole plants, and tryptophan levels were at least 2000 times greater in resistant than in wild type plants. Phenylalanine was also 32 times greater. All regenerant plants resistant to 5MT were both male and female sterile.Resistance to AFC was caused by decreased AEC uptake by the callus tissue and was not due to increased levels of free lysine. Plants were not regenerated from this callus.     

Selection of downy mildew resistant somaclones,from a susceptible B line of pearl millet

Plants regenerated from seed-derived callus of a PNMS 6B line of pearl millet (Pennisetum glaucum (L.) R. Br.) were evaluated for their resistance induced by somaclonal variation for downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroter. Among the 201 lines regenerated, only 3 lines consistently proved highly resistant (free from disease incidence) for up to 5 generations; whereas, 17 lines were resistant (disease incidence ranging from 1 to 9%). Resistance was confirmed by testing the plants under both laboratory and field conditions. The plants were evaluated for their agronomic traits. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Lactuca universal hybridizer, and its use in creation of fertile interspecific somatic hybrids

A Lactuca sativa cv. Ardente line heterozygous for a gene encoding resistance to kanamycin, a positive and dominant trait, was crossed with cv. Girelle, which is heterozygous for a recessive albinism marker. The resulting seeds yielded 25% albino seedlings, of which 50% were also resistant to kanamycin. Such plantlets (KR, a) grown in vitro were used for preparation of universal hybridizer protoplasts, since green buds that can develop on kanamycin containing-medium should result from fusion with any wild-type protoplast. To test the practicability of this selection scheme, we fused L. sativa KR, a protoplasts with protoplasts derived from various wild Lactuca as well as various other related species. Protoplast-derived cell colonies were selected for resistance to kanamycin at the regeneration stage. Green buds were regenerated after fusion with protoplasts of L. tatarica and of L. perennis. So far, 9 interspecific hybrid plants have been characterized morphologically. In addition, random amplified polymorphic DNA (RAPD) analysis with selected primers confirmed that these plants are indeed interspecific hybrids. Some plants are female-fertile and production of backcross progenies with L. sativa is in progress. Since many desirable traits such as resistances to viruses, bacteria and fungi (Bremia lactucae) have been characterized in wild Lactuca species, the use of somatic hybridization in breeding programmes now appears a practical possibility.     

Identification of the aph IV gene from protoplast-derived maize plants by a simple nonradioactive DNA hybridization method

Summary Protoplast-derived, transformed maize plants were evaluated by Southern analysis for the presence of the aph IV gene which codes for resistance to the antibiotic, hygromycin B. This gene was used as a selectable marker for the transformation of maize protoplasts. Southern analysis was performed with fluorescein-labeled probe DNA. A new method for labeling molecular weight markers with fluorescein-N⁶ is presented. The nonradioactive Southern analysis method is compared to the radioactive method and the results show that the nonradioactive method is as sensitive as the radioactive method.     

In vitro selection for methomyl resistance in CMS-T maize

Summary Many plants resistant to methomyl (Lannate), an insecticide which selectively damages maize with the Texas (T) type of cytoplasmic male sterility (CMS-T), were obtained by in vitro selection and also without selection. The selection procedure used 0.6–0.7mM methomyl and callus from CMS-T versions of several field and sweet corn genotypes (W182BN, Wf9, P39, MDM1, SW1 and hybrids of SW1, IL766A1, IL766A2, and 442 with W182BN-N). Addition of 1 mM methomyl to the regeneration medium greatly reduced recovery of methomyl-sensitive escapes. Resistance was linked with reversion to male fertility and maternally inherited. Most progeny of resistant plants exhibited stable maternally inherited resistance for two generations in field tests. First-generation progeny of seven culture-derived plants segregated for resistance and sensitivity; this suggests that ears of these seven regenerants were cytoplasmically chimeral. Resistance to methomyl was associated with resistance to T toxin from Helminthosporium maydis race T and with changes in mitochondrial physiology. Prolonged culture (14–16 months versus 6–8 months) increased the frequency of resistance among both selected and non-selected regenerants. Little or no resistance was found among regenerants from certain genotypes. Selection with methomyl may be useful for production of improved sweet corn lines and as a source of mitochondrial mutants. This system is also convenient for studies of the effects of nuclear background and of culture and selection systems on the generation of cytoplasmic mutants.     

Characterization of resistance to clofentezine in populations of European red mite from orchards in Ontario

Resistance to clofentezine was identified in four populations of the European red mite, Panonychus ulmi (Koch), from apple orchards in Ontario after ca. 5 years use. Resistance was expressed at high levels (> 2000-fold at the LC 50) to clofentezine in a population selected in the laboratory. This population was resistant to hexythiazox and to the organotin compounds cyhexatin and fenbutatin-oxide. However, a population selected with fenbutatin-oxide did not show cross resistance to clofentezine. The clofentezine-resistant population was not resistant to pyridaben, propargite or dicofol. The synergists, piperonyl butoxide and DEF, were ineffective in overcoming the resistance. Resistance to clofentezine declined rapidly in a mixed population (most resistance was lost in fewer than three generations). There were several fitness factors (fewer eggs/female, longer egg stage, longer development time for males) associated with the resistant population which may have contributed to the loss of resistant phenotypes. However, in a second test in which selection was removed from a resistant population, resistance persisted for at least 10 generations. Observations on field populations indicated that resistance persisted for at least two seasons. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dominant pleiotropy controls enzymes co-segregating with paraquat resistance in Conyza bonariensis

Summary The genetics of paraquat-resistance in Conyza bonariensis was studied. Reciprocal crosses were prepared between resistant and sensitive individuals. The enzymes of the pathway that detoxifies superoxide to innocuous oxygen species involved in resistance were evaluated in the F₁ and F₂ generations. All F₁ plants were as resistant as the resistant parent, irrespective of parental sex, demonstrating dominance and excluding maternal inheritance. The activities of superoxide-dismutase, ascorbate-peroxidase and glutathione-reductase in the F₁ were constitutively as high as in the resistant parent. Resistance in the F₂ generation was distributed in a 31 ratio (resistant to sensitive). Leaves from F₂ plants were removed for a resistance assay and enzyme immuno-assays of single plants were performed. The high levels of superoxide-dismutase and glutathione-reductase, the two enzymes for which antibodies were available, were similar in resistant individuals to the levels in the resistant parent; the levels were low in the susceptible individuals. These results indicate either a very tight linkage, or more probably, that one dominant nuclear gene controls resistance by pleiotropically controlling the levels of enzymes of the activeoxygen detoxification pathway.     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

Response of wheatgrasses and wheat × wheatgrass hybrids to barley yellow dwarf virus

Summary Resistance to barley yellow dwarf virus (BYDV), manifested by low enzyme-linked immunosorbent assay (ELISA) values in plants exposed to viruliferous aphids, was identified in several wheatgrasses (Agropyron spp.). ELISA results were similar for root and leaf extracts of infested plants. No difference in reaction to BYDV was found between plants grown in the field and those in the growth chamber. Interspecific hybrids were generated using pollen from single resistant plants of Agropyron spp. to pollinate soft red winter wheat spikes. Resistance in hybrids appeared to be at the level of virus replication rather than at the level of vector inoculation. The hybrids varied in their reaction to BYDV. Expression of BYDV resistance in hybrids was influenced not only by wheat genotype and Agropyron species but, in some cases, reaction varied even among hybrids between the same wheat genotype and Agropyron plant. Implications of these results are discussed.Contribution from the Purdue Univ. Agric. Exp. Stn., West Lafayette, IN 47907, and USDA-ARS. The research was supported in part by Public Varieties of Indiana. Purdue Univ. Agric. Exp. Stn. Journal No. 11656     

A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

Detection of protoplast-derived DNA tetraploid Lisianthus (Eustoma grandiflorum) plants by leaf and flower characteristics and by flow cytometry

Plants of lisianthus (Eustoma grandiflorum (Griesbach)Schinners=Lisianthus russellianus Hook.) were regenerated from protoplasts and grown in pots until flowering. Vegetative and floral characteristics were measured and compared with parent plants. Larger leaves and petals and longer guard cells, sepals and filaments were recorded from protoplast-derived plants suggestive of polyploidy. The nuclear DNA contents of protoplast-derived and parental plants were determined by flow cytometry. Protoplast-derived plants were confirmed as DNA tetraploid by flow cytometry with a DNA index of 1.95. Their nuclear DNA content was measured as 6.33±0.04 pg DNA per 2C nucleus compared with 3.26±0.10 pg DNA per 2C nucleus from parental plants. Polyploidisation induced during protoplast regeneration offers an alternative to that of colchicine treatment.     

Resistance of rape (Brassica napus) to attack by the cabbage aphid (Brevicoryne brassicae L.)

Resistance to Brevicoryne brassicae L. attacks in a New Zealand variety of forage rape resulted from a combination of host non–preference and antibiosis. Using clonal plant material obtained from cuttings, only half the numbers of immigrant alate cabbage aphids that settled to reproduce on the susceptible rape were to be found on the resistant rape. The reproduction rate of these alates was about 12 % slower on the resistant plants than on the susceptibles and the young took about 13% longer to mature. Antibiosis then shortened the reproductive life of the apterae by one-third, reduced their fecundity by nearly 50 % and caused 40% mortality in their progeny. The over-all effect of this was considerable and under conditions specified in the text could result in the population on the resistant plants being about one-eighth that on the susceptible plants in under 1 month. The resistant plants lost their resistance on flowering and became normally susceptible to cabbage aphid attack. With maturity, however, the B. brassicae-resistant plants became slightly resistant to Myzus persicae.     

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.     

Inherited resistance of sugar beet to aphid colonization

Results of glasshouse experiments have confirmed that inbred lines of sugar beet differ in each of three types of resistance to Myzus persicae Sulz. and Aphis fabae Scop., namely: resistance to settling, resistance to multiplication, and tolerance. Resistance to multiplication was not invariably associated with resistance to settling, although plants of some lines showed both forms of resistance. Plants that were resistant to settling of alatae were not always resistant to apterae of the same species, and there was not a close relationship between resistance to M. persicae and to A. fabae. The mechanisms involved in resistance to aphids in sugar beet are not understood. Progenies of plants, selected for resistance to aphids from inbred lines, were often more resistant than progenies of unselected plants. Inheritance of each type of resistance is probably polygenic. The potential value of the different kinds of resistance, in reducing direct feeding damage and controlling the spread of virus yellows in the field, is discussed. The ultimate breeding objective is to produce commercial varieties in which appropriate kinds of resistance to aphids are combined with resistance to virus yellows. The use of such varieties would reduce the need to control aphids in the field by applications of chemicals.     

Symbiotic properties of 5-methyltryptophan-resistant mutants of Bradyrhizobium japonicum

The effect that resistance to 5-methyltryptophan (MT) has on the symbiotic properties of B. japonicum was examined in a survey of fourteen clones. Resistance to MT often involves a mutational alteration in the regulation of tryptophan biosynthesis.Resistant clones (MT^R) were isolated from agar plates containing MT. In the selection process care was taken to avoid pigmented clones that are likely to accumulate large amounts of indole compounds or show increased tryptophan catabolism. Wild-type control clones (WT_c) were isolated from plates containing no selective agent. In greenhouse studies. Tracy-M soybean plants were inoculated with the two types of clones. After six weeks, plants which were inoculated with the MT resistant clones showed a much greater range of symbiotic effectiveness than did plants that received the control clones.While most MT-resistant clones were poor symbionts or unchanged in their symbiotic performance, one clone was obtained that had significantly improved symbiotic properties. The procedure may offer a way of selecting for clones with improved symbiotic performance. These results also indicate a link between tryptophan biosynthesis and symbiotic effectiveness.     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Resistance of bacteria from cooling waters to bactericides

Summary Bacteria from water cooling systems developed resistance to three different bactericides i.e. quarternary ammonium compound (QAC), isothiazolone and thiocarbamate. Resistance was induced by exposing isolates to increasing sublethal concentrations for a period of 10 weeks.Bacillus subtilis became resistant to 1000 mg l^–1 QAC. Cross-resistance was also detected, e.g. isothiazolone induced resistance to QAC and thiocarbamate.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine