Pear mutagenesis: In vitro treatment with gamma-rays and field selection for vegetative form traits

In vitro shoots of four pear (Pyrus communis L.) cultivars, 'Conference', 'Doyenné d'Hiver', 'Passe Crassane' and 'Bartlett', were irradiated with gamma rays (3.5 Gy). Around 1000 microcuttings of each cultivar were treated. After three subcultures, microcuttings from both the irradiated shoots and additional non-irradiated shoots were rooted; plants obtained were used to establish a survey orchard in Ostellato (Ferrara), Italy. For the first 2 years in the field, growth and survival were observed, but no data were collected. Subsequently, field surveys were conducted to characterize the population and identify mutants for vegetative traits. Trees that showed phenotypic evidence of vegetative growth characteristics more desirable than those shown by the overall population and by control plants were selected and measured for three years. Traits used for selection were small tree size, wide branch angle and short internodes. Frequencies of variants with compact habit varied with cultivars from 0.5% to 2.7% of irradiated trees. Effect of mutagenic treatment and efficacy of the selection methods are discussed.     

In vitro selection of strawberry (Fragaria × ananassa Duch.) clones tolerant to salt stress

The method of in vitro selection for increased salt tolerance at the seed germination and early growth phase of strawberry seedlings is proposed. Clone Pau/27 was selected on medium containing 200 mM of NaCl from population obtained by free pollination of cv. ‘Paula2019;. Subsequently, on the same medium 18 salt tolerant clones were attained from the population of seeds collected from self pollinated Pau/27 plants. In the next step we examined the influence of a mild salt stress (75 mM of NaCl) on vegetative growth parameters of selected clones and two cvs.; ‘Paula2019; and ‘Senga Sengana’. All materials in the study on the basis of calculated indexes were divided into four groups differing in reaction to salt. First group (clustered together cv. ‘Paula’, Pau/27 and three second generation clones: Pau/27/11, Pau/27/24, Pau/27/30) represents sensitive genotypes. Second group, including ‘Senga Sengana’, Pau/27/06, Pau/27/10, Pau/27/12, Pau/27/13, Pau/27/15, Pau/27/18, Pau/27/20, Pau/27/21, Pau/27/26, Pau/27/27, Pau/27/31 and Pau/27/32 was designated tolerant. Third group contains only one highly tolerant clone Pau/27/08. The last group comprises two highly sensitive clones (Pau/27/01 and Pau/27/03). This revised version was published online in August 2006 with corrections to the Cover Date.     

Induction of mutations for heat tolerance in potato by using in vitro culture and radiation

Heat tolerant mutants were obtained in two commercial potato cultivars, `Kufri Jyoti' and `Kufri Chandramukhi' through in vitro mutagenesis of in vitro propagated plantlets. Gamma-irradiated (20 and 40 Gy) shoots were micropropagated for three cycles (M₁V₃). A large number of the micropropagated shoots produced microtubers at 28 ^°C. Microtubers induced at high temperature had distorted shape but showed normal germination in field. Under stress conditions of high temperature, the frequency of chlorophyll variants increased in the gamma irradiation-derived material, however, nearly 40% of the plants had normal leaf tissue, whereas control plants showed completely damaged leaves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Salt-tolerance in Brassica juncea L. I. In vitro selection,agronomic evaluation and genetic stability

Summary In vitro selection of salt tolerant plants of Brassica juncea L. (Indian mustard) cv. Prakash has been accomplished by screening highly morphogenic cotyledon explant cultures on high NaCl media. Out of a total of 2,620 cotyledons cultured on high salt medium, 3 survived, showed sustained growth and regenerated shoots. They were multiplied by axillary bud culture on NaCl free medium. The salt-selected shoots retained salt tolerance following 3 month of growth and multiplication on control medium. While two of these somaclones flowered and set seeds, third one grew slowly, had abnormal leaf morphology and was sterile. The seed of the two fertile plants were sown in the field to raise R₁ segregating generation. Data were recorded for field, other agronomic components and oil content. The somaclonal lines, both selected salt-tolerant and non-selected, showed tremendous amount of variation for all the characters studied. One of the two tolerant somaclones invariably showed reduced height, longer reproductive phase and higher 1000 seed weight. Based on the agronomic performance of R₁ plants of these somaclones, some plants were selected and their progeny were evaluated for agronomic performance under standard field conditions and salt-tolerance in the greenhouse using sand pot culture method. Most of the lines bred true for their specific characteristics. In the greenhouse, selected salt-tolerant somaclones (SR-2 and SR-3) performed better for plant growth, yield and other agronomic traits at higher salt treatments, indicating thereby that salt-tolerance trait selected in vitro was expressed in the whole plants and is genetically stable and transmitted onto the progeny. The two tolerant lines, however, differed in their salt-tolerance during vegetative and reproductive phases as indicated by their salt-tolerance and stress susceptibility indices. The mechanism of salt-tolerance is not clear and needs to be further investigated.     

In vitro selection of wheat callus tolerant to high levels of salt and plant regeneration

Summary Embryogenic calli isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The effect of NaCl on the selected and unselected cell lines has been investigated. The results indicated that the relative growth rate of callus decreased as the concentration of NaCl increased in both callus lines. The selected callus line gave a higher growth weight in the presence of NaCl in the medium and was highly significant as compared with unselected callus line across medium protocols in all wheat cultivars. The dry weight of both kinds of callus lines of all wheat cultivars increased markedly with increasing NaCl concentration in most cases. The Na⁺ and Cl^- contents of both callus lines were increased with increasing salinity levels while K⁺ content was decreased. The selected callus line of each cultivar at the same salinity level produced significant amounts of Na⁺, K⁺ and Cl^- higher than the unselected callus line in most salinity levels. However, the unselected callus lines of the cultivars Giza-157 and Sakha-90 at the same salinity level produced significant amounts of K⁺ higher than the selected callus line in most salinity levels. The proline content of both kinds of callus lines for all wheat cultivars was increased with increasing salinity level. However, the selected callus line gave a significantly higher proline content than the unselected callus line in all wheat cultivars at the same Salinity level. Results from the in vitro selection for NaCl tolerance showed that the stepwise method of increasing NaCl in the medium was more effective for plant regeneration than other methods.     

In vitro selection for oleic and linoleic acid content in segregating populations of microspore derived embryos of Brassica napus

Microspore derived embryos (MDEs) in Brassica napuscontain large amounts of storage lipids which show a genotype specific fatty acid composition (FAC). One cotyledon of regenerating emblyos can be dissected at an early stage during the in vitro culture and used for fatty acid analysis. Thus, in breeding programmes to modify oil quality, only MDEs having the desired FAC need to be regenerated to plantlets and transferred to the greenhouse. In the present study the applicability of this method for the selection of a high oleic acid content and a low linoleic acid content in the seed oil has been tested by crossing a Brassica napus mutant line having a high oleic acid (C18:1) content in the seed oil (75%) with a wild type doubled haploid line with 62% C18:1 in the seed oil. Microspore culture was applied to the F1 plants. In total 59 MDEs were obtained, from which 31 were cultured with and 28 without 15μM abscisic acid for 3 weeksin vitro. One cotyledon was dissected under aspetic conditions and used for fatty acid analysis. The remaining part of the embryos were further regenerated to plantlets and transferred to the greenhouse to obtain seeds after self pollination. Seeds harvested from the doubled haploid lines in the greenhouse were used for fatty acid analysis and also for growing in the field. The abscisic acid treatment of the MDEs generally improved the correlations for linoleic and oleic acid between the MDEs and the seeds harvested in the greenhouse and the field. The correlations ranged from 0.68** to 0.81**.This indicates that selection for high oleic acid can be started already during an early stage of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Application of cell and tissue culture and in vitro selection for disease resistance breeding — a review

Summary Somaclonal variation, i.e. the variation induced by cell and tissue culture, offers an opportunity to broaden the genetic variation of crops. As a result of somaclonal variation a wide range of plant characteristics can be altered. However, the selection of agronomically important traits, e.g. disease resistance, has many limitations. The efficiency of selection can be increased by the application of in vitro selection procedures. Selection strategies that may be applied to obtain disease resistant somaclonal variants are described. Their merits and limitations, in relation to the efficiency of the procedures, the frequency of disease resistant variants and the genetics of the resistance obtained, are discussed.     

In vitro androgenesis of wheat: from fundamentals to practical application

Wheat anther cultures have a history of almost 30 years and are nowemployed efficiently in many countries of the world for the developmentof doubled haploid lines for breeding. The present paper discusses keyquestions of the elaboration and perfection of the method: cytologicalaspects of in vitro androgenesis, the conditions required for theembryogenic development of microspores and the applicability of anthercultures in the Martonvásár wheat breeding programme.     

Methods for evaluating fruit characters and incorporation in a final index in the Norwegian pear breeding programme

Summary In the Norwegian pear breeding programme selection is based on evaluation of 20 fruit samples together with field observations of precocity, productivity and resistance to biotic and abiotic factors. Fruit weight is recorded in grams, and then transformed to a scale of 20 gram classes. Appearance, shelf life and flavour are recorded on a 0 to 9 scale, and observations of shelf life and flavour are made at 4 defined intervals following ripening at 20 °C. The final index is made by summarizing the scores of fruit size and appearance, mean score of internal breakdown, mean score of flavour, maximum score of flavour and scores of precocity, productivity and resistance. All parameters, except mean flavour, are given a weighting of one, mean fruit flavour is given a weighting of 2. Data for 160 seedlings evaluated in 1992 is presented.     

In vitro screening of strawberry plants for cold resistance

Formation of embryo autonomy of strawberry, plant regeneration fro membryo components, plant freezing conditions in vitro and the possibility to differentiate objectively genotypes by freezing them in vitro and in vivo were studied to create strawberry screening technology in vitro for cold resistance. It was established that autonomy of strawberry embryos manifests itself not earlier than on 14–16^th day after pollination and full autonomy is reached on 20–22^nd day. Plants regenerated from 26 days old embryos grew most intensively. At the highest rate strawberry plants regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons x on the medium with 1.0 BA and 0.5 NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was -8 to 12 ^°C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo, provided hardening proceeded not less than 21 days. The correlation between cold resistance in vitro and in vivo reached 0.93. Domination of cold resistance manifested itself in strawberry seedlings from various crossing combinations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of gamma and ultraviolet irradiation on adventitious regeneration from in vitro cultured pear leaves

Summary Irradiation of in vitro explants and subsequent adventitious regeneration has been tested for 4 commercially important varieties of pear (Pyrus communis) with the aim to create mutants with a reduced susceptibility to fire-blight (Erwinia amylovora). The effect of gamma and ultraviolet irradiation of leaves on adventitious regeneration ability has been studied. The LD50 (50% decrease of regeneration) after gamma irradiation was genotype-dependent and was between 20 and 50 grays. The curves of regeneration showed a threshold dose underneath which none or a very slight decrease was registered. The decrease might result from cumulative events. After an ultraviolet irradiation as low as 62.5 J/m², the leaves became crumbly and rolled up, and their metabolism seemed to be altered. The LD50 was about 125 J/m² for all varieties and the decrease of regeneration was linear. Histological investigations showed leaves with flattened epidermal cells after ultraviolet irradiation and slack spongy parenchyma after gamma irradiation.Abbreviations Gy grays - J joules - LD lethal dose     

Haploidy breeding and mutagenesis for drought tolerance in wheat

Several intraspecific crosses between known drought tolerant wheat varieties and stable high yielding recombinants were made with the objective to develop improved cultivars for the moisture stressed rainfed areas of Pakistan. Five of these crosses were selected for further creation of useful mutations through the application of low doses of gamma rays and development of doubled haploids through anther culture. Anther culture response of the selected irradiated F₁ generations was studied on liquid and solid induction media. The highest number of calli among almost all crosses was produced on Potato-2induction medium. All the crosses varied greatly in response to callus induction and maximum calli (75%) were obtained from Lyl-73/vee’s’ cross. Similarly, genotypic differences were found for green vs. albino regenerants. The highest number of green plantlets (12.1%) was recorded for Lu-26/3062. From the developed doubled haploid population 25 DH-mutants were initially selected and nine lines were finally included in multi-locational field tests. Two DH-mutants (i.e. DHML-50 and DHML-9) have potential for better grain yield, earliness, disease resistance and moisture stress tolerance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic progress after four cycles of recurrent selection for yield and grain traits in common bean

The objective of this study was to evaluate the genetic progress after four cycles of recurrent selection in common bean. The base segregating population was obtained from 10 parents, and derived the S_0:1 and S_0:2 families that were evaluated. The S_0:3 families with higher grain yield and grain color, like the standard carioca were selected, and were intercrossed to generate the population of the following cycle. This process was repeated for four cycles. The best families were evaluated in each cycle by many generations and locations, and the five best lines of each cycle were identified. The 20 lines thus obtained were evaluated in two growing seasons, sown in July and November 2002. The grain yield (kg/ha) and grain type (scale of scores) were evaluated. Genetic progress was confirmed for both traits. The mean annual gain with selection for the grain type was 10.5% and 5.7% for grain yield, with no evidence of variability reduction in the population. These results show that recurrent selection is a good alternative for improving common bean quantitative traits.     

A procedure suitable for in vitro pollen selection inBrassica oleracea

Summary Experiments to determine whetherBrassica oleracea pollen could effect fertilisation following incubation in liquid culture medium are reported. Pollen was incubated in vitro, collected by centrifugation and used to pollinate compatible pistils. While retaining viability and the capacity to germinate such pollen was unable to penetrate the stigma papillae. However, incubated pollen produced functional tubes following pollination of styles from which the stigmas had been removed. These tubes grew through the style to the ovary and viable seed was obtained. The potential application of this procedure in pollen selection is discussed.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. I. Double-layer culture technique

Summary Calluses of spring and winter wheats (Triticum aestivum L.) were selected for Fusarium resistance in vitro, using the double-layer culture technique. Potato-dextrose agar medium in vials was inoculated with mycelia of Fusarium graminearum and F. culmorum. After one week, fungal cells were killed by autoclaving and the agar medium containing the thermostable toxic metabolites was overlayered with MS callus-growing medium. Later, wheat calluses were placed on the upper medium for 4–5 weeks, and from the surviving calluses plants were regenerated. R₂ seedling populations from self-fertilized R₁ plants of 4 varieties were tested for Fusarium resistance by artificial infections in the greenhouse, and 3% of the regenerated R₂ plants have been found to be more resistant than the original cultivars.     

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.     

Stability of bacterial leaf spot resistance in peach regenerants under in vitro,greenhouse and field conditions

Summary Phenotypic stability of bacterial leaf spot resistance in peach (Prunus persica (L.) Batsch) regenerants, either selected at the cellular level for insensitivity to a toxic culture filtrate of Xanthomonas campestris pv. pruni or screened at the whole plant level for resistance to X. campestris pv. pruni, was investigated. A detached-leaf bioassay was used to evaluate the original regenerants again after three years in the greenhouse and also after a two to three year cycle of tissue culture propagation. Peach trees derived through micropropagation from the original regenerants were also evaluated after one to three years growth in the field. Although leaf spot resistance was retained in some regenerants over time in the greenhouse, following in vitro propagation, and under field conditions, resistance was either lost or not expressed in others. Regenerants # 19-1 and #156-6, derived from embryo callus of bacterial spot susceptible Sunhigh, were significantly more resistant than Sunhigh. High levels of resistance were exhibited in greenhouse plants and field-grown trees of regenerant #122-1, derived from embryo callus of moderately resistant Redhaven. This research provides additional evidence that selecting or screening for somaclonal variants with disease resistance is a feasible approach to obtaining peach trees with increased levels of bacterial spot resistance.Abbreviations TC Tissue-Cultured - TF Toxic culture Filtrate     

Breeding tomato for pollen tolerance to low temperatures by gametophytic selection

Haploid selection for traits related to pollen cold tolerance in tomato was performed in segregating populations derived from a Lycopersicon esculentum × L. pennellii hybrid. BC₁ populations were obtained by combining normal and low temperature treatments on two stages of pollen development: pollen formation, and germination and pollen tube growth. F₁ hybrids were cultivated under low and normal temperatures and their pollen was used to pollinate L. esculentum plants at low and normal temperatures. The four BC₁ populations obtained were tested for the quality and quantity of pollen produced at low temperatures. The population obtained by cold treatment at both stages had a significantly improved pollen germination ability at low temperatures. The two other coldselected BC₁ populations showed no differences compared with the unselected BC population. A second cycle of pollen selection, corresponding to BC₂, was applied in order to test its persistence in the subsequent generations and the possibility to further improve the character. This second cycle showed no improvement although some plants retained the high pollen germination ability at low temperatures that was observed in the first cycle. Hence, gametophytic selection of some characters related with tomato pollen performance may be feasible, at least for the first cycle of selection.     

Genetic markers and the selection of quantitative traits in forage grasses

Summary Marker assisted selection is based upon the principle that if a gene (or block of genes) is linked to an easily identifiable genetic marker it may be more efficient to select in a breeding programme for the marker than for the trait itself. The recent developments in molecular marker technology has allowed several approaches to be applied to the forage grasses. The most effective methods involve the production of detailed genetic maps which can be used for determination of the location of Quantitative Trait Loci (QTLs). Application of these methods toLolium has lead to the identification of 10 QTL's affecting mainly phenological characteristics. Up to 80% of the variation in inflorescence production in the establishment year may be accounted for by one region of the genome.     

Inheritance and selection of some fruit characters of apple. II. The relation between leaf and fruit pH as a basis for preselection

Summary The leaf pH of two year old apple seedlings was found to be sufficiently related to the fruit pH, measured when the seedlings were six to seven years old, to serve as a basis for pre-selection. Discarding all seedlings with an above average leaf pH (40% of the total) eliminated the majority (74%) of seedling with low-acid cum sweet fruits. The method was not effective for reducing the proportion of seedlings with high-acid cum sour fruits.