Long-term storage of Narcissus anthers and pollen in liquid nitrogen

Summary Three methods for the long-term storage of narcissus pollen were compared; anthers in glass vials held in a desiccator with calcium chloride at 2°C, and polypropylene straws containing either anthers or naked pollen immersed in liquid nitrogen. Pollen from all storage treatments showed 15–16% germination in vitro after 3 days, compared with 27.4% for fresh pollen. Seed set per pod using pollen stored for 3 days was comparable to that of fresh pollen. However, after 351 days, pollen from anthers at 2°C exhibited only 0.1% germination and failed to set seed whereas no further change in germination rate was recorded for pollen from the two liquid nitrogen treatments and seed set was still equivalent to fresh pollen.     

In vitro and in vivo genetic parameters and character associations in potato

Relative behaviour of genetic parameters and character associations for in vitro and in vivo systems was studied by evaluating 22 potato genotypes for seven morpho-agronomic characters under six in vitro and four in vivo conditions. There was considerable similarity between in vitro and in vivo systems with regard to pattern of coefficient of variation (both phenotypic and genotypic), heritability (broad sense), genetic advance (as% of mean) and correlation coefficients (both phenotypic and genotypic) between characters. The magnitude of various genetic parameters was higher under in vitro than in vivo conditions, particularly for tuber yield and its components. Treatment-to-treatment variation in genetic parameters and character associations was higher under in vivo than in vitro conditions. In vitro selection for agronomic characters in potato may be possible. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monohaploid plants from anthers of a dihaploid genotype of Solanum tuberosum L.

Summary Monohaploid Solanum tuberosum plants were produced from the anthers of a dihaploid genotype. From another dihaploid genotype plants containing 36 chromosomes were obtained. For plantlet production anthers containing pollen at the uninucleate microspore stage were inoculated on a Linsmaier and Skoog-based medium supplemented with 1 mg/1 indole-3-acetic acid and 1 mg/1 benzyl aminopurine.Anthers from donor plants grown either in the summer or in the winter responded similarly on a range of media. Anther response in the form of callus induction and root formation was at least partly dependent on the genotype of the donor dihaploid plant.     

Pollen grain germination inside the anthers of two chasmogamous angiosperms: Almond (Prunus amygdalus L. Batsch) and apple (Malus pumila Mill.)

Summary Pollen grain germination inside anthers has so far been known in only eleven chasmogamous angiospermous species. The discovery of this phenomenon in some varieties of Prunus amygdalus (almond) and Malus pumila (apple) is therefore significant. The anomaly appears to be genetically controlled, the gene expression occurring under specific environmental conditions.     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

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.     

Characterisation of transgenic oilseed rape expressing pea lectin in anthers for improved resistance to pollen beetle

In order to evaluate pea lectin as a resistance factor against the pollen beetle (Meligethes aeneus), transgenic oilseed rape (Brassica napus) has been produced wherein the pea lectin gene expression is driven by a pollen-specific promoter. The aim of the present study was to identify and characterise non-segregating transgenic and non-transgenic lines to be compared in various future tests for benefits and risks associated with such transgenic crop plants. Three doubled haploid (DH) populations (1436, 1440 and 1451) expected to include lectin-producing lines and two DH populations (1443 and 1449) expected to be free of lectin were produced. All five populations originated from different transformation events in the cultivar Westar. The relative amounts of DNA from the marker gene cassette were quantified by conventional as well as real-time PCR analyses and lectin concentrations were estimated by western blot analysis. Two populations with high lectin concentrations, 1436 and 1451, contained higher amounts of the marker DNA and thus more lectin gene copies as compared with 1440 which had lower concentrations of the lectin. As expected all DH lines from 1443 and 1449 were free of lectin. Maximum pea lectin concentration obtained corresponded to 3% of total soluble protein in the anthers. There were significant differences between the populations with respect to bud and flowering stage phenology as well as seed yields, but the differences were not related to their transgenic status. All in all there were 171 lines tested for phenology and transgenic status, out of which 89 with similar phenology were subjected to tests for lectin concentration and seed yield, and 20 of these lines were retested in the next generation. Finally two lines with high lectin concentrations and one with an intermediate level along with two matching non-transgenic lines were selected for future benefit/risk experiments.     

Interspecific hybridization in Cucumis L. II. The crossability of species,an investigation of in vivo pollen tube growth and seed set

Summary The crossability of 12 Cucumis species of African and Asiatic origin was studied in a diallel cross, in order to find ways to realise the cross between the common cucumber (C. sativus L.) and its wild relatives which carry resistances against diseases and pests.Self-pollinations and cross-pollinations within species gave normal pollen tube growth and seet set. The different accessions within a species, as a rule reacted alike in interspecific crosses. In crosses between African species different crossing patterns were found, viz. bilateral congruity, bilateral incongruity and unilateral incongruity. Within C. sativus all accessions intercrossed freely, except one, which displayed unilateral incongruity.Good seed was harvested from several crosses and in some cases embryo culture was needed for further development of seeds. No good seeds were obtained from any cross between a species of the African group and C. sativus L.     

Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum)

Summary Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25° C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l glutamine and solidified by gelrite (4g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15 E m^–2 S^–1) at 26°C at 16h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28°C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regenerated and fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.     

In vitro culture variation of wheat and rye caused by genes affecting plant growth habit in vivo

Summary The influence of genes affecting the plant growth habit in wheat (Rht8 and Ppd1) and rye (ct1 and ct2) on tissue culture response was studied using immature embryos. Whereas the semi-dwarfing gene Rht8 seems to promote only a minor effect, the day-length sensitive allele ppd1 determined a major increase in callus growth and regeneration ability. With regards to their tissue culture efficiency, the four alleles studied could be ranked as follows: ppd1>Rht8>rht8>Ppd1.In contrast to wheat, the GA insensitive semi-dwarfing genes of rye (ct1 and ct2) appear not to influence in vitro response.     

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.     

Mutation breeding of Chrysanthemum morifolium Ram. using in vivo and in vitro adventitious bud techniques

Summary During experiments, which are being carried out to study the factors which control the process of adventitious bud formation in vivo on detached leaves of Chrysanthemum morifolium Ram, adventitious shoots were produced from leaves, irradiated with 500 rad of X-rays. The most important but disadvantageous result was that the majority of the adventitious shoots proved to be of a chimeral nature and obviously developed from more than one cell.An in vitro adventitious bud technique was developed using different types of explants. Pedicel segments regenerated the highest number of adventitious shoots and, moreover, they developed faster as compared to explants of young flower heads or leaves. The mutants produced by irradiating the various explants were almost exclusively of a solid (non-chimeral) nature. In addition, histological observations suggest that single epidermal cells are involved in the initiation of the adventitious shoot apices.The optimum dose for mutant production is approximately 800 rad X-rays. Rather often, more than one phenotypically identical mutant was found, which was always derived from the same explant. They could for instance originate from a multi-apical meristem formed by a single mutated cell.     

In vitro interspecific fertilization,embryo development and formation of hybrid seedlings between Gossypium hirsutum and G. arboreum

Summary In vitro hybridization between Gossypium hirsutum, and G. arboreum was carried out. Hybrid seedlings were obtained after successive use of the following five kinds of media: 1) pollen grain germination medium, 2) double-fertilization medium, 3) embryo development medium, 4) seedlings formation medium, 5) green seedlings growth medium. The factors affecting in vitro interspecific fertilization, embryogenesis and seedling formation were studied. The key factors were temperature and relative humidity (RH). The optimum RH for in vitro interspecific fertilization was 65%, and a suitable temperature range was 26–30°C. Pollen grain germination ratio decreased rapidly at a RH of 80%. When the temperature and the RH were higher than 32°C and 80%, respectively, the fertilization rate decreased to zero.Effects of petals and calyces of the maternal flowers on in vitro interspecific fertilization and ovule growth were identified. Even though the petals or calyces were excised, hybrid plantlets were also obtained after media were improved and a shake culture was used. In addition, the process of double-fertilization and embryo development were studied cytologically. The developmental characteristics of the hybrid embryos derived from in vitro interspecific fertilization were a later occurrence of the double-fertilization process and a much lesser division of endosperm cells. But the embryo development was not affected by these characteristics, and young hybrid embryos can develop to their cotyledon stage finally on artificial media.     

In vitro culture of tissues,cells and protoplasts of Trifolium alexandrinum L. (Egyptian clover)

Summary Callus induction derived from root, hypocotyl and cotyledon explants were investigated for four cultivars of Trifolium alexandrinum L. The Murashige & Skoog (1962) medium containing 2.0 mg/L NAA and 0.5 mg/L BAP was able to induce callus from different explants. A wide range of culture media were tested to determine the morphogenetic potential of different callus types derived from different explant types. Shoot morphogenetic development was observed with the cultivars Sakha 4 and Giza 10. Cell suspension cultures were established from hypocotyl derived callus. Methods for isolation and culture of protoplasts from cotyledons are described.     

In vitro propagation of Japanese garden iris,Iris ensata Thunb.

Summary Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.     

Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.     

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.     

Screening immature bulbs of daffodil (Narcissus L.) crosses for resistance to basal rot disease caused by Fusarium oxysporum f. sp. narcissi

Summary Immature bulbs were used in two screening trials to identify daffodil genotypes resistant to basal rot disease caused by Fusarium oxysporum f. sp. narcissi. Progenies, having at least one parent with some resistance, were grown in compost infested with chlamydospores of the fungal pathogen, and survivors were recovered at final harvest. Survival rates were very low when one-year old bulbs were used; the greater survival of two-and three-year old bulbs indicates that resistance increased with bulb age. Selection pressure was influenced by inoculum concentration in the compost. Continuous variation of percentage bulb survival between progenies suggests a polygenic mode of inheritance; there was no evidence of maternal inheritance. Parental general combining ability (GCA) was highly significant and accounted for much of the difference in bulb survival between progenies, but non-additive parental effects were also apparent. GCA rankings in the two trials were similar and reflected the resistance of parental cultivars in the field. A three-dimensional graphing procedure was devised to depict goodness of fit of progeny data to the additive model.     

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.     

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.