In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

禾谷镰刀菌粗毒素对小麦幼胚培养特性及再生R1、R2代植株的抗赤霉病性影响

研究了禾谷镰刀菌粗毒素对小麦幼胚培养特性的影响和再生R1、R2代植株的抗赤霉病性变异,结果表明,粗毒素对3个不同基因型幼胚的愈伤组织诱导、胚芽萌发及胚性愈伤组织形成和绿苗分化的影响有相同的趋势。①毒素浓度为6.0g/L时,愈伤组织生长受明显地抑制,但胚芽萌发生长受到促进,低于此浓度时,毒素对愈伤组织的生长有一定的促进作用;②毒素对胚性愈伤组织形成和苗分化均有较大的抑制作用,其中对前者的抑制作用最强;③适宜的粗毒素筛选浓度为1.5g/L~4.5g/L,在这一浓度范围内对愈伤组织形成无影响,而对胚性愈伤组织和苗分化有明显抑制,但又能使其保持有一定的分化能力。④经毒素筛选的抗耐毒素细胞系再生R1代植株中,抗病穗的比例明显提高,R2代再生植株的抗赤霉病性分离,并趋向于感病方的分离。     

In vitro Selection for Tolerance to Fusarium in F1 Microspore Populations of Wheat

Microspore populations of eight Fhybrids of winter wheat (Triticum aestivum L.) whose parents had different levels of resistance to Fusarium were screened in vitro, using phytotoxins of Fusarium as biochemical probe. Two selection methods were compared for the in vitro selection: either embryoids and calli were first initiated from anthers in toxin-free medium and then grown on medium with 0.3—0.9 %Fusarium toxin; or anthers were immediately cultured in modified liquid potato-2 medium in the presence of 1.5, 2.0 and 2.5ml Fusarium toxin per liter culture medium, a concentration which reduced the number of calli and embryoids to about 10 % compared to the toxin-free controls. Microspores from donor hybrids which were produced from very susceptible cultivars were killed by lower toxin concentrations than micro-spores from hybrids of less susceptible parents. From surviving calli and embryoids, originally initiated from 242,000 anthers in both procedures a total of 375 green lines could be regenerated. The results indicate that it is possible to enrich the fraction of regenerating microspores by those which contain the gene complex responsible for reduced susceptibility to Fusarium by the use of a pathotoxin.     

In vitro Selection for Fusaric Acid Resistant Barley Plants

Calli of two genotypes of barley,‘Dissa’and W 193, were used for selection of resistance against fusaric acid, a pathotoxin of Fusarium. Callus was initiated from 7- to 10 days old immature embryos. 1000 calli of the‘Dissa’and 500 of the W 193 genotypes were grown for 4 selection cycles on medium with 0.8 mM fusaric acid. In the first selection cycle, about 80 % of the calli were killed; after the 4 selection cycles, 8 to 11 % resistant calli were obtained and plants were regenerated. Resistant calli maintained on non-toxic medium showed retention of resistance ability after 3 months of sub-culturing. Plants could be regenerated from the surviving calli and testing by leaf bioassay revealed that many were resistant to the same toxin concentration employed for callus selection (100 %), while some were only resistant up to a concentration of 75 %.     

Studies on somaclonal variants for resistance to scab in bread wheat (Triticum aestivum L.) through in vitro selection for tolerance to deoxynivalenol

This study was conducted to develop an efficient in vitro selection system for scab resistance by using in vitro screening for tolerance to deoxynivalenol (DON). Immature embryos of two wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11, and their reciprocal F₁ hybrids were cultured on MS medium supplemented with 2,4-D 2 mg/l and 0.6 × 10^-4 M DON for callus induction. The responses of callus induction and plant regeneration to 0.6 × 10^-4 M DON differed significantly between resistant and susceptible varieties, according to observed scab resistance levels at the plant level in the field. The percentage of callus formation of resistant variety Sumai 3 on induction medium containing DON was higher than that of susceptible variety Mianyang 11. Regeneration of DON-tolerant calli on DON-containing differentiation medium differed significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was decreased 3-fold on DON-containing medium. By an inoculation test with conidiospores of Fusarium graminearum Schw, we obtained several resistant lines from progenies of regenerated plants from DON-tolerant calli. These somaclonal lines had lower disease scoring (reaction index, infected spikelets and disease incidence), shorter plants and better yield components than Sumai 3, a famous Chinese resistant variety. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfer of disease resistance within the genus Brassica through asymmetric somatic hybridization

Summary Asymmetric somatic hybrid plants between Brassica napus L. (oilseed rape genome AACC) and a transgenic line of Brassica nigra L. Koch (black mustard genome BB) were tested for their resistance against rapeseed pathogens Phoma lingam (black leg disease) and Plasmodiophora brassicae (club root disease). The transgenic B. nigra line used (hygromycin-resistant, donor) is highly resistant to both fungi, whereas B. napus (recipient) is highly susceptible. The asymmetric somatic hybrids were produced using the donor-recipient fusion method (with X-irradiation of donor protoplasts) reported by Zelcer et al. (1978) for the production of cybrids. Using hygromycin-B for selection, a total of 332 hybrid calli were obtained. Regenerants, resistant or susceptible to both diseases, were selected. Many hybrids expressed resistance to only one pathogen. Dot blot experiments showed that the asymmetric hybrid plants contained varying amounts of the donor genomic DNA. Furthermore, a correlation was detected between the radiation dose and the degree of donor DNA elimination.     

In Vitro Response to Fusarium Elicitor and Toxic Substances in Crosses between Resistant and Susceptible Carnation Cultivars

To assess the correlation between in vivo resistance to F. oxysporum f. sp. dianthi and in vitro behaviour, calli from several resistant and susceptible Mediterranean carnation cultivars and F₁ progenies of crosses were tested both for the ability to grow on a medium containing crude culture filtrate of F. oxysporum and to produce phytoalexins following treatment with cell wall components (elicitor) of the same fungus. The results show no significant correlation between in vivo resistance and in vitro tolerance to culture filtrate of the fungus, while there was a good correlation in die case of phytoalexin production. Moreover, the character phytoalexin production behaved as a dominant in the crosses between a resistant and a susceptible cultivar.     

The use of interspecific mixtures of Lupinus species to simulate the effects of different levels of Phomopsis Leptostromiformis resistance in L. angustifolius

Summary Mixtures of different proportions of two lupin species, one susceptible and one resistant to Phomopsis leptostromiformis were successfully used to generate different levels of fungal infection in the field. However there was not a good correlation between levels of Phomopsis infection and lupinosis toxin, possibly because of unusually high levels of toxicity encountered during 1978. The significance of this work in terms of breeding a lupin resistant to P. leptostromiformis is discussed.     

EMS离体诱变和抗大花萱草叶枯病突变体的筛选

利用甲基磺酸乙酯(EMS)诱变大花萱草‘红运’愈伤组织和分化芽,目的是获得抗叶枯病愈伤组织和分化芽突变体。结果表明：0.50%~0.75% EMS处理愈伤组织60 min;0.75%~1.00% EMS处理分化芽60min,分别获得“半致死剂量”效应。对存活的愈伤组织进行毒素浓度递增的多步筛选(10% 30 d—20% 30 d);分化芽80%毒素21d的一步筛选。经定向筛选,初步获得抗叶枯病分化芽突变体103株,有待进一步进行抗病鉴定。愈伤组织未能获得抗病突变体再生植株。     

Inheritance of resistance to angular leaf spot in Nicotiana tabacum L. cultivars burley 21 and kentucky 14

Summary The genetics of resistance to angular leaf spot caused by Pseudomonas syringae pv. tabaci in Nicotiana tabacum cultivars Burley 21 and Kentucky 14 was investigated by studying disease reactions to three isolates of parental, F₁, F₂ and backcross generations derived from crosses between the resistant cultivars and the susceptible cultivar Judy's Pride. Studies were conducted in the greenhouse and in field plant beds. Chi-square values were computed to determine whether the observed ratios for disease reactions deviated from expected Mendelian ratios for a single, dominant gene controlling resistance to angular leaf spot in tobacco. Based on the resistance of the F₁ and the backcross generation to the resistant parent (BC-R), a 3 resistant: 1 susceptible segregation ratio in the F₂, and a 1 resistant: 1 susceptible segregation ratio in the backcross generation to the susceptible parent (BC-S), it was concluded that resistance to three isolates of Pseudomonas syringae pv. tabaci is governed by a single, dominant gene.     

Histological and inheritance studies of partial resistance in the Brassica napus–Albugo candida host-pathogen interaction

Traditional and doubled haploid (DH) genotypes of oilseed Brassica spp. resistant, partially resistant, moderately susceptible, and susceptible to Albugo candida were compared for phenotypic development of host‐pathogen interaction and histology of host‐pathogen interaction. The partially resistant genotype showed pinhead‐size pustules, mainly on the upper surface of cotyledonary leaves. Relatively less mycelium was observed in the partially resistant genotype compared with the susceptible genotype. In resistant B. napus genotypes, there was neither pustule development nor any mycelial growth. In the moderately susceptible genotype, the pustules were similar to those in the partially resistant genotype in being of pinhead‐size and occasionally coalescing. However, ample mycelial growth in the mesophyll tissue in the moderately susceptible genotype was similar to that in the susceptible control B. rapa cv. ‘Torch’. The susceptible genotype B. rapa cv. ‘Torch’ also showed large coalescing pustules. In the non‐host B. juncea cv. ‘Commercial Brown’, no pustules were formed although some mycelial growth was observed beneath the epidermal cell layer and in the mesophyll cell layer of the cotyledonary leaf tissue. For inheritance studies, two partially resistant B. napus genotypes were crossed with a resistant B. napus genotype. Various generations viz., F1, F1(reciprocal), F₂, and DHs produced from the crosses were inoculated with a zoospore suspension of race 7v of A. candida. The partially resistant phenotype appeared to be controlled by a single recessive gene designated as wpr with variable expression. The simple inheritance of partial resistance has implications for disease resistance breeding against white rust, as this type of resistance can be easily incorporated into elite breeding lines through conventional and DH breeding methods.     

Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenoloxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco

Summary When subjected to micropropagation by tissue culture, the two reputed cultivars of date palm (Phoenix dactylifera L.); Bou-Sthammi noire, resistant to Bayoud disease and Bou-Feggous, of high fruit quality, give rise to three types of calli, called white and root-forming callus, hyperhydric and degenerating callus and friable and embryogenic callus. All explant sources, calli and germinated embryos were analysed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for acid soluble protein composition. Phenol-oxidizing enzymes; peroxidase and polyphenoloxidase, were also, evaluated and the isoforms separated by polyacrylamide gel electrophoresis. When compared with the explant and germinated embryos, embryogenic calli of the two date palm cultivars could be identified by a concentrated polypeptide of molecular weight 27 500 and polypeptides of molecular weights 70 000 and 11 500. Hyperhydric and degenerating callus contained the polypeptide exhibiting the molecular weight 32 000. Embryogenic calli showed high levels of soluble, ionically and covalently bound peroxidases. The soluble acidic isoperoxidase of R _f 0.60, revealed in these calli and germinated embryos could be a marker of the two tissues. White and root-forming calli of Bou-Feggous cultivar were typified by soluble acidic isoperoxidases with high mobility (R _f 0.75) and anodic ionically wall-bound polyphenoloxidases similar to those of the explant sources. Polyphemoloxidase activities detected in calli and embryos were very low when compared with those of explants. Used as an early test to screen embryogenic calli of date palm, acid soluble proteins, peroxidase and polyphenoloxidase data could lead to introduce lightening and economy in the tissue culture technique.     

Brassica coenospecies: a rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae

Development of leaf spot resistant mustard cultivars is a relevant objective in view of heavy crop losses caused by this pathogen. Thirty-eight species belonging to 9 genera, including cultivated and wild allies, of the genus Brassica were evaluated under epiphytotic conditions for two years. Inoculations were done on whole plants (in vivo) and on detached leaves (in vitro). Data on incubation period, number of lesions per leaf, lesion size and leaf area covered by lesions were recorded. Species which never produced disease symptoms throughout the growing period in pots and until 72 hours after inoculation in detached leaf assays during both years were treated as resistant, while those that produced symptoms were classified as moderately resistant, susceptible or highly susceptible depending upon incubation period, size of lesions and leaf area covered by disease symptoms. Eight species (Brassica desnottesii, Camelina sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides, and Erucastrum gallicum) were found completely resistant, whereas others were classified as moderately resistant (12), susceptible (11) or highly susceptible (9). Since resistance is unavailable within the cultivated species, these 8 resistant wild species could be used as donor parents for introgressing resistance to leaf spot disease in Indian mustard. This revised version was published online in August 2006 with corrections to the Cover Date.     

Contribution of host-pathogen interactions to the expression of the blackleg disease of spring rape (Brassica napus L.) caused by Leptosphaeria maculans (Desm.) Ces. et de not

Variation in the responses of selected genotypes of spring rape to infection by a wide range of isolates of Leptosphaeria maculans was analyzed in this study.Differences in severity of disease damage to seedlings between host and pathogen genotypes were highly significant. Interactions between host and pathogen were also highly significant. The generally resistant cultivar Wesreo was susceptible to a number of isolates whereas the normally susceptible cultivars Zollern-gold and Ceska were resistant to some isolates. Overall, however, Wesreo was resistant to a far greater number of isolates than were the susceptible genotypes.Significant differences in severity of disease damage were also observed between groups of isolates from different sites. All host genotypes were resistant to a substantially greater proportion of isolates from certain sites than from others. Interactions were again detected in that the ranking of sites on the basis of the proportion of isolates causing severe damage varied between host genotypes.The significance of host-pathogen interactions in the B. napus-L. maculans relationship is discussed with special reference to current views on the nature of horizontal resistance.     

Assessment of Gossypium sturtianum and G. australe as potential sources of Fusarium wilt resistance to cotton

When challenged with Fusarium oxysporum f. sp. vasinfectum (Fov) from vegetative compatibility groups (VCGs) 01111 and 01112 in glasshouse tests, Gossypium australe Mueller and Gossypium sturtianum Willis accessions showed a variety of disease responses ranging from highly resistant to highly susceptible. Under high disease pressure G. sturtianum accession Gos-5275 was significantly more resistant than the commercial G. hirsutum cultivars that are designated standards for Fusarium resistance by Australian cotton breeders. Under low disease pressure G. sturtianum accession Gos-5250 was more susceptible than a highly susceptible commercial cultivar. A series of glasshouse tests was performed at two locations (Indooroopilly, QLD. and Canberra, ACT), and under low and high disease pressure. In these tests, a hexaploid cross (Gos-5271) generated from a Fusarium-resistant G. sturtianum (Gos-5275) and a Fusarium-susceptible G. hirsutum L. (CPI-138969) was significantly more resistant to Fusarium wilt than its G. hirsutum parent. Thus G. sturtianum, with a diploid genome and a range of responses to Fov challenge, has the potential to provide the basis for the elucidation of the genetic basis of resistance to Fusarium wilt in cotton species. In addition, resistant accessions of G. sturtianum are identified as a potential source of Fusarium wilt resistance genes for cotton breeding. In the glasshouse tests used to assess the resistance of various Gossypium accessions to Fusarium wilt disease, the scoring of vascular browning was found to give a more reliable indication of disease severity than the scoring of foliar symptoms. This revised version was published online in August 2006 with corrections to the Cover Date.     

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F₁, from which F₂ and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F₁ plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F₂ populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F₁ hybrids and used commercially to prevent downy mildew at the cotyledon stage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Leaf and neck blast resistance reaction in tropical rice lines under green house condition

Rice blast (Magnaporthe oryzae) is the most destructive and epidemic disease of rice. Use of host resistance is the best alternative for disease management. The leaf and neck blast resistance of 182 rice breeding lines were assessed for leaf and neck blast and classified relative to a susceptible check-Masuli and resistant check-Laxmi, from greenhouse experiment in 2005 and 2006. The test plants were inoculated with 10⁵ conidial suspension/ml of M. oryzae at 21 days old seedlings for the leaf blast, and at neck base for the neck blast. Among them, for leaf blast, 77 rice lines were resistant, 43 were moderately resistant, 39 were moderately susceptible and 23 were susceptible. While among the selected 31 rice lines evaluated for neck blast, 4 lines were resistant, 4 were moderately resistant, 16 were moderately susceptible and 7 were susceptible. Leaf and neck infection was significant and positively correlated (r = 0.30, P = 0.05). The rice lines, Barkhe 1032, Barkhe 1034, Barkhe 1035, Barkhe 1036, Barkhe 2014, Barkhe 2024, Barkhe 3019, Super 3004 were resistant to leaf blast and Barkhe 1006, Barkhe 1032, Barkhe 1035, Barkhe 3004 were resistant to neck blast. The rice lines with identification # 11, 69, 137, 168, 182 from Masuli × MT4 parentage, and Barkhe 3017 were susceptible to both leaf and neck blast. Progenies of Irradiated Pusa Basmati (IPB), Kalinga III/IR64 (KIII/IR64), and Masuli/IR64 were resistant to both leaf and neck blast. However, most progenies from Masuli/MT4 showed susceptible reaction to both leaf and neck blast. Thus, rice lines form the IPB, KIII/IR64 and Masuli/IR64 will be promising resistant sources for rice blast in breeding programme.     

Fungitoxic effect of scopolin and related coumarins on Sclerotinia sclerotiorum. A way to overcome sunflower head rot

Summary The content of coumarins, as probable phytoalexins, was analysed in four sunflower genotypes that ranged in responses to head rot from highly susceptible to highly resistant. Low levels of all coumarins (scopolin, scopoletin and ayapin) were detected in the three most susceptible genotypes irrespective of time after inoculation. However, in the resistant genotype there was a clear time-dependent disease-induced increase of all coumarins that reached a maximum after 10–14 days. Detailed comparison of the most susceptible and the resistant genotype showed that in the resistant but not the susceptible, scopoletin peroxidase activity increased during the course of the experiment. Results confirmed a clear negative correlation between coumarin content and disease symptoms and in particular for scopolin. Furthermore we show for the first time that scopolin is inhibitory to Sclerotinia at similar doses to scopoletin. As scopolin is known to be less phytotoxic than ayapin and scopoletin, its accumulation may well confer head rot resistance with minimal plant damage and might be one of the bases for resistance to Sclerotinia.     

稻瘟病毒素对水稻根冠活细胞的影响

利用稻瘟病的菌丝悬浮液提取稻瘟病菌毒素,以毒素浓度、毒素处理根尖的时间及不同品种进行复因子试验,结果表明.毒素处理的不同水稻品种间根冠细胞死亡率差异极显著;而不同毒素浓度处理及不同处理时间的根冠细胞死亡率差异不显著.对另外13个水稻品种在毒素抗性方面的研究结果表明,稻瘟病菌毒素对水稻根冠活细胞有较强的毒性作用,感病品种经毒素处理后细胞死亡率极显著的高抗病品种,细胞死亡率与稻穗颈瘟发病率呈极显著正相关,相关系数为0.91.这为快速鉴定水稻对稻瘟病的抗性提供了一个新方法.     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.