Responses of cells and protoplasts of Coffea arabica genotypes to partially purified culture filtrates produced by Colletotrichum kahawae

Hypocotyl-derived calli of genotypes and segregating populations of Coffea arabica, differing in susceptibility to Colletotrichum kahawae, were used to produce cell suspensions and protoplasts which were exposed to partially purified culture filtrates (PPCFs) prepared from the pathogen. The growth and viability of PPCF-treated cells and protoplasts were measured using packed cell volume, fluorescein diacetate staining and a colorimetric assay involving the tetrazolium salt MTT. Differential responses of cells and protoplasts were influenced by genotype, time of exposure and PPCF concentration. Protoplasts of resistant genotypes responded differentially from more susceptible genotypes as early as 4 h after challenge with the phytotoxin, suggesting that they were more sensitive than cell suspensions to the treatments. Protoplasts exposed to PPCFs from C. kahawae may therefore be used to screen and select genotypes resistant to, or tolerant of, coffee berry disease. Received: 10 April 1996 / Revision received: 25 August 1996 / Accepted: 15 September 1996     

In vitro selection of resistant rice plants against rice blast caused by Pyricularia oryzae via tissue culture technique

Abstract

A step by step protocol for resistant calli selection via a tissue culture technique under stress of Pyricularia oryzae culture filtrates was followed. Rice embryos dissected apart from the endosperm of susceptible rice seeds (Giza 176 and Riho) to P. oryzae produced embryonic calli on media containing various growth regulators of 2,4-D at concentrations of 0, 1, 1.5 and 2 mg/L and/or benzyl amino purine (BAP) at 0, 0.5, 1 and 1.5 mg/L when incubated under complete dark conditions for three weeks. Embryonic explants only produced shoots on media containing BAP. Selection of resistant calli was carried out in vitro under the challenging stress of increasing concentration of the pathogen P. oryzae culture filtrate (CF) from “0” up to 100%. The selection protocol has two directions. The first is step-by-step selection from lower to higher selective (CF) concentrations. The second is the exchangeable continuous cycles with and without the same selective (CF) concentration until the end of the selection regime to avoid calli adaptation to (CF). The regenerated calli to plantlets occurred under (CF) stress showed resistance and susceptibility when exposed to the pathogen infection under greenhouse conditions. The results reveal that the resistance in regenerated rice plantlets to P. oryzae pathogen segregated as 1 resistant: 2 moderate resistant: 1 susceptible giving the predication that the resistance in rice to P. oryzae may be controlled by one pair of genes. The in vitro selective regime via tissue cultures is advisable for the selection of novel disease resistant plants because of its time saving, space, money, it is easily applied and has a bio-safe approach.     

Pollen genotype selection for a simply inherited qualitative factor determining resistance to chlorsulfuron in maize

Pollen genotype selection for genes expressed in both the haploid and diploid phases of the plant life cycle can lead to correlated responses detectable in the sporophyte. A pollen selection was conducted in two genetic backgrounds of maize (Zea mays L.) for chlorsulfuron resistance, conferred by the XA17 allele. Plants of two backcross (BC) lines segregating 1 (heterozygote, resistant) : 1 (homozygote, susceptible) for chlorsulfuron resistance were used as pollen donor. Selection treatment was applied during microspore development, and tassels were cut about 10 days before anthesis and placed into a liquid medium with or without 40 mg l^–1 of chlorsulfuron. Pollen was used to fertilize an unrelated susceptible genotype (tester). The resulting testcrosses (TC) were evaluated in the greenhouse by spraying seedlings with chlorsulfuron at 23 g ha^–1. Non-selected TC progenies derived from heterozygous BC plants showed a proportion of resistant and susceptible plants close to the expected 1:1 ratio, while non-selected TC progenies derived from homozygous BC plants showed susceptible plants only. Selected TC progenies derived from heterozygous BC plants showed a frequency of resistant plants ranging from 89% to 100%. BC plants homozygous for the susceptible allele, subjected to selection treatment, gave poor seed set, and no resistant plants were found in their TC progenies. Resistant TC plants obtained through pollen genotype selection were selfed, and the proportion of resistant plants was close to 3:1 in all selfed families, in accordance with the hypothesis that all of them inherited the XA17 allele through selection. In this study, pollen genotype selection was extremely effective, and its effect persisted in the second sporophytic generation. Received: 19 November 1999 / Accepted: 30 April 2000     

In vitro selection and regeneration of carnation (Dianthus caryophyllus L.) plants resistant to culture filtrate of Fusarium oxysporum f.sp. dianthi

Callus cultures derived from internodal segments of two cultivars of carnation susceptible to Fusarium oxysporum f.sp. dianthi were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant lines were selected by culturing calli on growth medium containing various concentrations of the culture filtrate of F. oxysporum f.sp. dianthi. Resistant calli obtained after two cycles (25 days/cycle) of selection were used for plant regeneration. About 32% of the plants regenerated from the resistant calli had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

In vitro detection of Cornus florida callus insensitive to toxic metabolites of Discula destructiva

Summary Dogwood anthracnose, caused by the fungus Discula destructiva Redlin, is a severe disease of flowering dogwood (Cornus florida L.) and Pacific dogwood (C. nuttallii Aud.). Disease control is inadequate in nurseries and landscapes and absent in the forest, and resistant cultivars are not commercially available. The ability to select tissues insensitive to culture filtrates from D. destructiva in vitro offers a novel and important approach for the selection of dogwood genotypes that are resistant to or tolerant of this devastating fungus. Embryo-derived dogwood callus cultures were established on Murashige and Skoog medium amended with benzyladenine (BA) and either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA). Selection for insensitivity to D. destructiva metabolites was done by placement of individual cultures on media amended with progressively higher concentrations of a partially purified culture filtrate (PPCF) containing lowmolecular-weight compounds. Following this selection process, cultures were challenged in a dose-response format with PPCF to determine whether the sensitivity of the callus to the culture filtrate had changed. During the selection period, the fresh weight of callus grown on medium containing 2,4-D and amended with PPCF was always less than that of callus grown on medium amended with the same concentration of potato-dextrose broth (PDB, negative control). Fresh weight of callus was greater on medium containing NAA amended with PPCF than on medium with the same concentration of PDB. Callus selected in the presence of NAA showed decreased sensitivity to toxic metabolites at higher concentrations of culture filtrate. The in vitro system described may assist in the identification of disease-resistant germplasm important to the long-term survival of flowering dogwood.     

Effects of Alteria alternata f.sp. lycopersici toxins at different levels of tomato plant cell development

Since the host-specific toxins of Alternaria alternata f. sp. lycopersici play an important role in pathogenesis, they potentially could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Prerequisite for this is that sensitivity to the Alternaria alternata f.sp. lycopersici pathotoxins is manifest at the cellular level. To gain insight into cellular effects of AAL-toxins and into the mechanisms of plant insensitivity to AAL-toxins, effects of AAL-toxins on leaves, leaf discs, roots, calli, suspension cells, minicalli and protoplasts of susceptible and resistant tomato genotypes were studied. In leaves of susceptible genotypes, toxins cause severe necrosis, while in leaves of resistant genotypes necrosis was never observed. Inhibition effects of toxins were observed at all other levels in susceptible and resistant genotypes: toxins inhibited shoot induction on leaf discs, root growth and growth of calli, suspension cells and protoplasts. This indicates a cellular site for AAL-toxins. Differences in sensitivity to AAL-toxins between susceptible and resistant genotypes were observed in leaves and roots, but were not observed during shoot induction on leaf discs, in calli, suspension cells and protoplasts. However, differences in sensitivity to AAL-toxins in roots were at least 20 times less than in leaves. Therefore insensitivity seems related to a higher level of tomato plant differentiation and is most pronounced in leaves.     

<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.     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

Development of sequence characterized DNA markers linked to leaf rust (<Emphasis Type="Italic">Hemileia vastatrix</Emphasis>) resistance in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Coffee leaf rust due to Hemileia vastatrix is one of the most serious diseases in Arabica coffee (Coffea arabica). A resistance gene (S_H3) has been transferred from C. liberica into C. arabica. The present work aimed at developing sequence-characterized genetic markers for leaf rust resistance. Linkage between markers and leaf rust resistance was tested by analysing two segregating populations, one F₂ population of 101 individuals and one backcross (BC₂) population of 43 individuals, derived from a cross between a susceptible and a SH3-introgressed resistant genotype. A total of ten sequence-characterized genetic markers closely associated with the S_H3 leaf rust resistance gene were generated. These included simple sequence repeats (SSR) markers, sequence-characterised amplified regions (SCAR) markers resulting from the conversion of amplified fragment length polymorphism (AFLP) markers previously identified and SCAR markers derived from end-sequences of bacterial artificial chromosome (BAC) clones. Those BAC clones were identified by screening of C. arabica genomic BAC library using a cloned AFLP-marker as probe. The markers we developed are easy and inexpensive to run, requiring one PCR step followed by gel separation. While three markers were linked in repulsion with the S_H3 gene, seven markers were clustered in coupling around the S_H3 gene. Notably, two markers appeared to co-segregate perfectly with the S_H3 gene in the two plant populations analyzed. These markers are suitable for marker-assisted selection for leaf rust resistance and to facilitate pyramiding of the S_H3 gene with other leaf rust resistance genes.     

Growth and Enzyme Activity in Roots and Calli of Resistant and Susceptible Allium Lines as Influenced by Sterile Culture Filtrates of Phoma terrestris

Growth and activities of peroxidases, chitinases and glucanases were studied in order to evaluate the response of calli and roots of pink root-susceptible Allium cepa cvs. Valcatorce and T-412 and resistant A. fistulosum cv. Nogiwa Negi, to sterile culture filtrates of Phoma terrestris. Untreated calli and roots of A. fistulosum exhibited higher activity of peroxidases and glucanases than that of Valcatorce and T-412. Enzyme activities and growth of roots and calli were not affected in filtrate-treated A. fistulosum. The growth of calli and roots of A. cepa cultivars decreased significantly after exposure to P. terrestris filtrates while the peroxidase and glucanase activities increased. Peroxidase and glucanase activities were also enhanced in roots of Valcatorce bulbs grown in P. terrestris-inoculated soil as compared to healthy control plants. We conclude that a high constitutive activity of glucanases and perhaps chitinases might account for the resistance of A. fistulosum. The differential reaction (with respect to root growth) of pink root-susceptible and resistant materials to culture filtrates indicates that this in vitro-system might be useful for the screening of onion breeding lines.     

Selection of resistance breaking strains of tomato spotted wilt tospovirus

In glasshouse tests, sap from plants infected with 15 different isolates of tomato spotted wilt tospovirus (TSWV) from three Australian states was inoculated to nine genotypes of tomato carrying TSWV resistance gene Sw-5 or one of its alleles. A further two resistant tomato genotypes were inoculated with four isolates each. The normal response in resistant genotypes was development of necrotic local lesions in inoculated leaves without systemic invasion, but 22/752 plants also developed systemic reactions in addition to local hypersensitive ones. Using cultures from two of these systemically infected plants and following four cycles of subculture in TSWV resistant tomato plants, two isolates were obtained that gave susceptible type systemic reactions but no necrotic spots in inoculated leaves of resistant tomatoes. When these two isolates, Da_WA-1d and To_TAS-1d, were maintained by repeated subculture for 10 successive cycles in Nicotiana glutinosa or a susceptible tomato genotype, they still induced susceptible type systemic reactions when inoculated to resistant tomato plants. They were therefore stable resistance breaking isolates as regards overcoming gene Sw-5. When resistance-breaking isolate Da_WA^-1ld multiplied together with original isolate Da_WA-l in susceptible tomato, it was fully competitive with the original isolate. However, when Da_WA-ld and To_TAS-ld were inoculated to TSWV resistant Lycopersicon peruvianum lines PI 128660R and PI 128660S and to TSWV resistant Capsicum chinense lines PI 152225, PI 159236 and AVRDC CO0943, they failed to overcome the resistance, producing only necrotic local lesions without systemic infection. Thus, although the ease of selection, stability and competitive ability of resistance breaking isolates of TSWV is cause for concern, L. peruvianum and C. chinense lines are available which are effective against them. The effectiveness of the resistance to TSWV in nine tomato genotypes was examined in a field experiment. Spread was substantial in the susceptible control genotype infecting 42% of plants. Resistance was ineffective in cv. Bronze Rebel, 26% of plants developing infection. In contrast, it held up well in the other eight resistant genotypes with only 1–3 or no plants of each becoming infected. Accumulated numbers of Thrips tabaci, Frankliniella occidentalis and F. schultzei were closely correlated with TSWV spread.     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

A Comparison of the Disease Reactions of Stems and Detached Leaves of Soil and in vitro Grown Plants and Regenerants of Oilseed Rape to Leptosphaeria maculans and Protocols for Selection for Novel Disease Resistance

Protocols for selecting plant tissues of winter oilseed rape (Brassica napus subsp. oleifera) with resistance to Leptosphaeria maculans by either stem or leaf inoculation of both soil and in vitro grown plant material are described. The stem inoculation procedure gave good correlation (r = 0. 92) between the 50 day stem disease scores of eight out of nine cultivars of soil grown winter oilseed rape inoculated with isolate 41A4 of L. maculans and the N. A. B. esistance ratings or resistance data from field trials. The exception was the cultivar Liradonna. Inoculation of stems of five cultivars with isolates 41A4, 433 and 478 indicated a range of isolate virulence 478 > 41A4 > 433. This was the inverse of that observed in leaf inoculations. Application of the stem inoculation procedure to in vitro shoot cultures allowed differentiation of resistant and susceptible cultivars, including the cultivar Liradonna, after 20 days incubation at 20°C. The protocol was also applicable to plantlets regenerated from thin cell layer explants grown in vitro. Inoculations with isolate 433 allowed the differentiation of resistant, intermediately resistant and susceptible leaf material of soil grown plants, when leaf discs from young leaves were incubated on water agar supplemented with BAP (1 × 10^?5 M) at 25°C for 10 days. Intermediately resistant leaves were resistant after 10 days and susceptible after 15 days of incubation. Leaves of shoot cultures grown in vitro were more susceptible than the corresponding soil grown material. However, inoculation of old leaves with isolate 41A4 (an isolate of less virulence on leaves than 433) distinguished the cultivars after 15 days of incubation. These protocols allow the accurate assessment of resistance to L. maculans at the stem or leaf level and are of use in traditional as well as in vitro selection programmes.     

Variation ofHelminthosporium resistance and biochemical and cytological characteristics in somaclonal generations of barley

SC₂ and SC₃ progenies of nineteenin vitro regenerated barley plants (SC₁) from resistant calli selected against purified culture filtrate ofHelminthosporium sativum and one parent ‘Dissa’ genotype were studied for stability of resistance and protein, soluble protein, maltose and saccharose contents. Cytological studies were also carried out on the SC₃ generation. Stability of resistance toHelminthosporium sativum was found in 50% of the somaclonal lines. Significant variation among different somaclonal lines and among different callus lines from which the plants were regenerated were found for yield, disease score and biochemical characters assessed except saccharose content in the somaclonal lines. Significant increase and decrease over the donor parent for most of the characters were obtained. Cytological abnormalities such as multilobed nuclei, multinucleate cells, abnormal anaphase and mixoploidy were also observed.     

Evaluation of in vitro selection regimes for a mitochondrial trait (methomyl resistance) in cms-T maize

Several factors affecting the success of selection in plant populations were examined for their relevance to in vitro selection. Three in vitro selection schemes and two growth assessment procedures were evaluated for effectiveness in selecting for a mitochondrial trait in maize: resistance to the insecticidal compound methomyl. Regenerable maize callus was derived from immature embryos of the three-way hybrid P39/IL766A2 x W182BN containing Texas male sterile cytoplasm (cms-T). Either low, gradually increasing, or high selection pressures were used to grow callus over a period of 3–5 months. There was no significant difference in recovery of resistant plants using these 3 methods. Growth of callus on medium containing methomyl was assessed by increase in fresh weight during the final month of selection or by increase in number of callus pieces over the course of selection. These quantitative measures of growth were unreliable indicators for gain in resistance within the callus population. A procedure for recovery of methomyl resistant and male-fertile cms-T plants is suggested.     

Marker-assisted selection provides arabica coffee with genes from other <Emphasis Type="Italic">Coffea</Emphasis> species targeting on multiple resistance to rust and coffee berry disease

Selecting superior genotypes is facilitated by marker-assisted selection (MAS), which is particularly suitable for transferring disease resistance alleles because it nullifies environmental effects and allows selection of resistant individuals in the absence of the pathogen or race, enabling preventive breeding. Molecular markers linked to two major genes (S_H3 and S_H?), conferring resistance to coffee rust, and those linked to the Ck-1 gene, conferring resistance to coffee berry disease (CBD), have previously been identified. These markers were validated and used in a progeny of crosses between Indian selections with Coffea arabica cultivars. Eleven resistant individuals homozygous for S_H3 were identified by MAS. Of these, seven carry S_H? from Híbrido de Timor and the gene introduced from Coffea liberica (S_H3). S_H? was characterized as derived from Coffea canephora. Thus, it was possible to identify C. arabica genotypes carrying important genes for rust resistance introgressed from other coffee species. MAS also allowed identification of sources of CBD resistance for use in preventive breeding for resistance to this serious disease. Using two validated molecular markers, two coffee plants carrying Ck-1 were identified: the UFV 328-60 genotype (F₂) was resistant and homozygous based on both molecular markers but exhibited no markers related to S_H3 and S_H?, and the UFV 317-12 genotype (F₁) was resistant and homozygous but resistant and heterozygous based on CBD-Sat207 and CBD-Sat235, respectively. Along with possessing Ck-1, the latter carries S_H?. Overall, plants carrying different genes for resistance to rust and CBD were identified. These plants are important sources for gene pyramiding in breeding programs aimed at multiple and durable resistance.     

Linkage of a RAPD marker with powdery mildew resistance <Emphasis Type="Italic">er-1</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

The aim of this study was to investigate the inheritance of powdery mildew disease and to tag it with a DNA marker to utilize for the marker-assisted selection (MAS) breeding program. The powdery mildew resistant genotype Fallon ^er and susceptible genotype 11760-3 ^ER were selected from 177 genotypes by heavy infestation of germplasm with Erysiphe pisi through artificial inoculation The F₁ plants of the cross Fallon/11760-3 indicated the dominance of the susceptible allele, while F₂ plants segregated in 3: 1 ratio (susceptible: resistant) that fit for goodness of fitness by χ² (P > 0.07), indicating monogenic recessive inheritance for powdery mildew resistance in Pisum sativum. A novel RAPD marker OPB18 (5′-CCACAGCAGT-3′) was linked to the er-1 gene with 83% probability with a LOD score of 4.13, and was located at a distance of 11.2 cM from the er-1 gene.     

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     

Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots

The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The A. rhizogenes strain A4RS proved to be the most virulent. High transformation efficiencies (70%) were obtained using a 2-week co-cultivation period at a temperature of 15–18°C. Using a p35S-gusA-int construct inserted in the pBIN19 binary plasmid, we could estimate that 35% of transformed roots were GUS positive (co-transformed). Using the GUS assay as visual marker, 40% composite plants bearing a branched co-transformed rootstock could be obtained after only 12 weeks without selection with herbicides or antibiotics. Transgenic coffee roots obtained with A. rhizogenes did not exhibit the ‘hairy’ disturbed phenotype and were morphologically similar to normal roots. PCR analyses demonstrated that all co-transformed roots were positive for the expected rolB and gusA genes. Transformed and non-transformed root systems from both susceptible and resistant varieties were inoculated with Meloidogyne exigua nematode individuals. Inoculation of composite plants from the Caturra susceptible variety resulted in the normal development of nematode larvae. Numbers of extracted nematodes demonstrated that transformed roots retain the resistance/sensibility phenotype of varieties from which they are derived. These results suggest that composite plants constitute a powerful tool for studying nematode resistance genes.     

Selection ofMycosphaerella fijiensis-resistant cell lines from micro-cross sections of banana and plantain

Anin vitro selection system using microcross sections of banana and plantain cultivars belonging to AAA and AAB genomic groups were used to produce plants resistant against the Black Sigatoka disease. The fungus resistant plantlets were obtained in a double selection system. This involved in a first step the use of a fungal crude filtrate and in the second step the purified host-specific toxin 2,4,8-trihydroxytetralone extracted from the fungusMycosphaerella fijiensis (M. fijiensis), the causal agent of Black Sigatoka disease. Resistant plantlets obtained from the double selection system were inoculated with conidia ofM. fijiensis in a growth chamber to reproduce Black Sigatoka symptoms. Compared to non-treated control plantlets, which were highly susceptible to the fungus, 10.7–19.3% toxin-resistant plantlets which arose from tissues that went through the double selection system were resistant againstM. fijiensis. This technique of using micro-cross sections for selection on fungal toxins seems to be amenable to differentMusa genotypes for the production of fungus-resistant plants.F. A. Schulz died 11. 3. 1995