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.     

Structure and variation of subterranean clover populations from Sicily,Italy

Summary A collection of subterranean clover lines singled out from populations of the species Trifolium brachycalycinum and T. subterraneum collected in Sicily, Italy, was examined at two localities for flowering time and at one locality for oestrogen content and seed yield. The structure and variation of the populations of the two species were compared. The relationship between flowering time of the populations and some environmental features of their collection sites was examined to assess whether the maturity requirements of the two species were similar and to frame selection models focused on developing varieties of appropriate maturity. Populations of T. subterraneum were, on average, more complex than those of T. brachycalycinum, being characterized by higher number of lines per population and greater intra-population variation for flowering time, oestrogen content and seed yield. Furthermore, populations of T. subterraneum were, on average, about 15 days earlier than sympatric populations of T. brachycalycinum. Both the greater variation and the relative earliness of T. subterraneum occurred irrespective of the environments of origin of the populations. Inferences are drawn on the adaptive advantages that such features confer to T. subterraneum. Mean flowering time of the populations increased on increasing annual rainfall and altitude of the collection sites. However, the changes in maturity appeared almost exclusively related to variations in rainfall in T. subterraneum, while in T. brachycalycinum the effect of altitude was greater and that of rainfall less marked than in the former species.     

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.     

Somatic in vitro culture response of Lolium perenne L.: genetic effects and correlations with anther culture

Summary Genotypic effects on callus induction and plant regeneration in callus, suspension and protoplast culture, and their correlations with both phenotypic and GCA-values for anther culture response, were studied using 21 genotypes of perennial ryegrass. Differences between genotypes accounted for approximately 40% of the total variation for callus induction and initial callus growth, and 59 and 83% of the variation in callus culture for regeneration percentage and percentage of green plants. Effects of genotypes were less pronounced in suspension culture, where suspensions from the same genotype often behaved differently. Some suspension cultures retained their capacity for green plant regeneration for almost two years, repeatedly producing 80–100% green regenerants during this period. Genotypes with high regeneration percentage and a large proportion of green plants from callus culture were also superior in suspension culture for both regeneration performance and longevity. Regeneration percentage and percentage of green plants were uncorrelated, and probably under different genetic control. While capacity for green plant formation from the different genotypes showed no correlation between anther culture and somatic in vitro culture, a positive correlation was observed between the regeneration percentages in somatic in vitro culture and anther culture (r=0.44^*–0.85^***), suggesting some common genetic control of the two systems.     

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

Summary A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l^-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l^-1 kinetin.Differences between leek genotypes in callus type, callus weight, shoot regeneration and primordia formation were observed. Histological observations showed that plant regeneration took place, both via the pathway of somatic embryogenesis and organogenesis.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog (1962) medium     

Selection of a highly-regenerative genotype of white clover (Trifolium repens L.) and plant regeneration from protoplasts derived from this genotype

Summary Callus cultures were induced from hypocotyl sections of 24 varieties of white clover (Trifolium repens L.). The calli did not show any significant difference of growth among the varieties. After the calli has been transferred to three regeneration media, green-spot formation was observed on calli derived from some seedlings. Remarkable intra- and intervarietal variations in the emergence of green spots and some trends between the origin of varieties and the frequency of green spots were observed. In most cases, the green spots turned brown without showing further differentiation, and only two genotypes formed shoots. A callus from a seedling of the Swedish variety Undrom has sustained high levels of plant regeneration throughout 24 months of culture. Protoplasts derived from this selected genotype were divided into cell colonies. 8P (Kao, 1977) medium containing 0.5 mg/1 2,4-D and 0.5 mg/1 kinetin was the most suitable medium for inducing divisions in protoplasts. When subcultured into solid B5 medium, the colonies produced calli, which when transferred to a regeneration medium, formed shoots. This genotype is expected to a useful subject for genetic engineering of white clover.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2ip 6-, -dimethylallylamino purine - NAA -naphthaleneacetic acid     

Regenerative trait and cold hardiness in highly productive cultivars of alfalfa and red clover

Summary In earlier work on improvement of persistance in forage legumes, we selected genotypes from highly productive cultivars of alfalfa, Algonquin and Apica (Euphytica 45: 105–112, 1990) and cv. Florex red clover (Plant Cell Reports 8: 395–398, 1989) capable of in vitro regeneration from callus and cell culture. The alfalfa germplasm and its F₁ progeny as well as an F₂ red clover population were tested for cold stress tolerance. Plantlets were hardened in culture tubes at 2 or 5°C, 8h photoperiod, for at least four weeks and then subjected to freezing temperatures, –16 or –10°C for alfalfa and red clover, respectively. Survival of regenerative genotypes was significantly higher than of the non-regenerative ones in both species. A strong oositive correlation (r=0.78) between the regenerative trait and plant survival was found in alfalfa. The experiments indicate that in vitro selection for regenerative trait may improve cold stress tolerance of alfalfa and red clover.     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

Trifolium ambiguum as a source of variation in the breeding of white clover

Summary Hybrids between T. ambiguum and T. repens were successfully established after ovule culture. The hybrids were male sterile but one of the hybrids produced a single seed when backcrossed to T. repens, while the other produced a significantly greater number of seeds. The majority of the backcross hybrids from the latter had 48 chromosomes, being produced from the fertilisation of unreduced gametes of the F1 hybrid by T. repens pollen. Regular meiosis in the BC1 hybrid resulted in the production of balanced gametes containing the full complement of T. repens chromosomes and the polyhaploid set of T. ambiguum chromosomes. There was no difficulty in establishing a substantial population of BC2 hybrids. The meiotic behaviour of the BC1 hybrid indicated that it should be possible to transfer genes from T. ambiguum into white clover T. repens. The significance of the results in terms of the use of alien variation in the improvement of white clover is discussed.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

High frequency plant-regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn)

Summary Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.Abbreviations CW Coconut water - 2,4-D 2,4-dichloro phenoxyacetic acid - Kn Kinetin - Z Zeatin     

Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of variety, seed position and seed source on screening for coleoptile length in bread wheat (Triticum aestivum L.)

A study was conducted to evaluate the usefulness of somaclonal variation as a means to obtain powdery mildew resistance in the background of an agronomically elite, high-yielding barley (Hordeum vulgare L.) cultivar. A total of 170 R_o-derived lines were regenerated from embryo-induced callus of the barley cultivar Léger. Forty-five lines were selected and evaluated in replicated field plots at two locations in Eastern Canada. In comparison with Léger, one of the 45 lines was higher yielding, one produced a greater test weight, two had a greater seed weight, and one was shorter in plant height. Three lines were found to segregate for resistance to powdery mildew (Erysiphe graminis DC ex Merat f. sp. hordei EM). Many of the single-plant selections from the three resistant lines showed resistance to powdery mildew under field conditions for two years. Three lines were eventually promoted to the official registration tests in Ontario. One of the three lines was subsequently registered as a new cultivar (AC Malone) in Canada. To our knowledge, AC Malone is the world's first barley cultivar selected from somaclonal variation. The results of this study demonstrate that it is possible to obtain high- yielding or disease-resistant lines from callus culture in barley. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Introduction of Transgenes to Control Blackheart in Pineapple (Ananas Comosus L.) cv. Smooth Cayenne by Microprojectile Bombardment

Summary A transformation technique for the introduction of transgenes to control blackheart by particle bombardment has been developed for pineapple cv. Smooth Cayenne. Leaf callus cultures capable of high frequency organogenesis with a short regeneration time were used as explant material. Gus and gfp reporter genes were used to observe and determine transient and stable expression. The ppo gene, isolated from pineapple, was introduced to control blackheart. Co-transformation occurred with constructs containing the nptII gene conferring geneticin resistance. We have recovered 15 independent transgenic gus and gfp lines each from 8 separate experiments and 22 ppo lines from 11 experiments. Gus, gfp, ppo and nptII positive plants have been regenerated, which have been shown by Southern blot analysis to be stable transgenics containing multiple copies of the introduced genes. These results show that biolistic gene delivery in pineapple can be successfully achieved at an acceptable efficiency of 0.21–1.5% for genetic improvement of ’Smooth Cayenne’, the industry standard throughout the world.     

In vitro culture of species and varieties of four Amaranthus L. species

Summary Callus induction, callus growth and organogenetic processes were investigated in hypocotyl and stem cultures of four species of Amaranthus each of which comprised several varieties. Callus formation occurred in almost all the varieties in 100% of explants. The combinations of naphtaleneacetic acid plus benzylaminopurine or 2,4-dichlorophenoxyacetic acid plus Kinetin were very effective in causing callus formation; the results for callus growth in presence of the same growth regulator combinations revealed in some cases significant differences among the varieties.As far organogenesis-based on a few varieties-only A. caudatus and A. hypochondriacus responded well forming shoots from callus when cultured in presence of indoleacetic acid plus Kinetin and/or indoleacetic acid plus benzylaminopurine. Root regeneration was also observed in several varieties. Complete plantlets were obtained from rooted shoots.The results are discussed in relation to hormonal effect and the genotype importance.     

The induction in vitro of adventitious shoots in Rosa

Summary Adventitious shoots were formed on excised leaves, roots and callus of Rosa persica x xanthina and on excised leaves of R. laevigata and R. wichuraiana on culture media that included BAP and NAA as growth regulators. Shoots formed freely on freshly cultured callus of R. persica x xanthina but their production declined in successive cultures and ceased after twelve weeks. Transplantation to soil was improved by rooting plantlets in cellulose plugs in vitro and transferring plantlets to soil while still in the plugs.     

Genetic factors influencing the regeneration ability of rye (Secale cereale L.). II. Immature embryos

Summary Immature embryos of seven rye inbred lines were cultured on modified MS medium containing 3 mg/dm^–3 2,4-D. According to thein vitro response lines were divided into four groups: (1) those producing non-embryogenic callus (NEC) from above 30% of the embryos and having a high rate of non-responding (NR) embryos, (2) those producing non-embryogenic callus (NEC) from the majority of embryos, (3) those producing NEC by the majority of embryos with a high percentage of calli regenerating roots, (4) those producing embryogenic callus (EC) and regenerating plants by above 50% of the embryos. The inheritance of these response types was analysed in F₁, F₂, and F₃ generations of crosses of some lines. The results obtained indicate that EC production and both plant and root regeneration are determined by recessive genes whereas the reduced ability for NEC production most probably by dominant genes. The lack of response is controlled by at least two interacting genes.     

Inhibitory effect of streptomycin on rice tissue cultures and the selection of resistant callus clones

Summary Callus tissues were initiated from seed, radicle and anther cultures of rice (Oryza sativa L.) in order to study the effect of streptomycin on callus growth. Our results showed that the addition of 250 g/ml or more streptomycin to the culture medium caused a significant inhibition of callus proliferation. The degree of inhibition depended upon the genotype, the drug concentration and the tissue source of callus. Selection of resistant cell lines began with seed and immature embryo cultures grown on various levels of streptomycin. The fastest growing sectors of callus were subcultured for additional selection. In this way, a total of 11 comparatively vigorous callus clones were isolated after 7 or 12 subcultures. Some of these clones exhibited a significant increase of resistance index when compared with unselected starting material. After 5 or 6 selection cycles, 79 plantlets were regenerated from resistant callus, but none grew to maturity because all were white (albino).     

Barley lines showing prominent high green shoot regeneration in cultures derived from immature embryos

Eighty‐four cultivars of barley (Hordeum vulgare) and 95 wild strains (82 of H. spontaneum and 13 of H. agriocrithon) were surveyed for the production of callus, callus growth, and shoot regeneration in cultures derived from immature embryos. All cultivars except for ‘Turkey 381′, induced calli from more than 90% of embryos. On the other hand, the wild lines showed a large variation in the percentage of callus induction from 0 to 100%. Among the cultivars, those with the brittle rachis genotype, bt Bt₂, on chromosome 3H, regenerated shoots with a significantly higher percentage than the cultivars with the Bt bt₂ genotype. Green shoots were produced in a higher ratio (0.84) in the cultivars than in the wild lines (0.52). Among the lines examined,‘Lenins’ regenerated shoots efficiently (90.4%) and produced the highest number of calli with green shoots per embryo (4.77) followed by ‘Golden Promise’ (3.15). Examination of callus growth and shoot regeneration from embryos at different developmental stages revealed that scutellum development affected the quantity and quality of callus and shoot regeneration.     

Genetic basis of resistance against two Tospovirus species in tomato (Lycopersicon esculentum)

Summary A study was conducted to determine if the chlorsulfuron tolerance existing in barley could be improved with the use of in vitro tissue culture. Differential herbicide concentration led to variable culture response for callus culture.Plants regenerated from callus culture were evaluated for chlorsulfuron tolerance in the greenhouse. Regenerants showed improved tolerance when compared to the control or unselected tissue culture-derived lines.