Green Plant Regeneration Research Articles

Efficient In Vitro Micropropagation and Regeneration of Humulus Lupulus on Low Sugar, Starch-Gelrite Media

Several Czech and foreign hop mericlones were tested in vitro for efficiency of green callus formation and plant regeneration from internodal or nodal explants. Modified MS media gelled either with agar, starch or a mixture of potato starch and Gelrite, supplemented with different concentrations of either glucose or maltose, were investigated. Two mericlones of Czech hop (Osvald 72 no. 5216 and SlĂĄdek no. 6908) were studied in more details because of their different regeneration capacities. The HPLC analysis of medium sugar concentrations after the explant cultivation has revealed slow uptake of sugar from the medium. Presence of glucose at concentration of 45 g dm-3 in agar medium resulted in a decreased number of nodes compared to the control with 30 g dm-3 of glucose. The use of a mixture of potato starch plus Gelrite instead of routinely used agar and decreasing the medium glucose concentration to 15 g dm-3 proved to be most efficient for multiplication rate. The use of this medium results in lower cost of micropropagation of healthy hop cultures without exhibition of vitrification.

Genetic Markers Associated with Green and Albino Plant Regeneration from Embryogenic Barley Callus

Genetic control of plant regeneration from cultured plant tissues has been documented for a number of species. The characterization and manipulation of loci that influence morphogenic responses may be useful for the development of highly regenerable germplasm or for physiological investigations. Quantitative trait loci (QTLs) for morphogenesis from barley cell cultures were identified on the basis of associations of mapped markers with the regeneration responses of 77 doubled haploid (DH) lines derived from the cross Steptoe/Morex. Two models were developed, one describing green plant regeneration, and one describing albino plant regeneration, measured as the numbers of green and albino plants regenerated per gram fresh weight of embryogenic callus. Approximately 62 and 12% of the observed variability for green and albino plant regeneration, respectively, was explained by the models. An independent data set was developed that consisted of the regeneration responses of 25 additional DH lines which were chosen randomly from the same segregating population. The models were tested for their ability to predict the responses of these independent DH lines. This study identified new QTLs for plant regeneration (one for green plants and at least one for albino plants), and confirmed previously reported associations of three QTLs with green plant regeneration.

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.

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Timothy ( Phleum pratense L.)

Somatic embryogenesis and green plant regeneration of timothy ( Phleum pratense L.) were successfully established using immature inflorescences and seedling stems. The capacity for somatic embryogenesis from immature inflorescences was higher than that from seedling stems. Immature inflorescences of 0.5-1.5 cm in size cultured in Murashige and Skoog (MS) basal medium containing 2 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D) gave the best performance in callus induction. Suspension cultures were initiated from friable nodule-forming callus in MS basal medium supplemented with 5.0 mg l -1 2,4-D and 0.5 mg l -1 abscisic acid. Fast-growing and finely dispersed suspension cultures were established. Kinetin at 1.0 mg l- 1 and 2,4-D at 0.1 mg l -1 was the best plant growth regulator combination in green plant regeneration from direct callus differentiation and suspension cultures. The highest green plant regeneration was achieved 5-7 weeks after initiation in suspension culture. More than 100 regenerated plants were obtained.

Chromosomal regions associated with green plant regeneration in wheat (Triticum aestivum L.) anther culture

Genetic capacity for green plant regeneration in anther culture were mapped in a population comprising 50 doubled haploid lines from a cross between two wheat varieties ‘Ciano’ and ‘Walter’ with widely different capacity for green plant regeneration. Bulked segregant analysis with AFLP markers and composite interval mapping detected four QTLs for green plant percentage on chromosomes 2AL (QGpp.kvl-2A), 2BL (QGpp.kvl-2B.1 and QGpp.kvl-2B.2) and 5BL (QGpp.kvl-5B).The three QTLs detected on chromosome 2AL and 2BL all derived their alleles favouring green plant formation from the responsive parent ‘Ciano’.The remaining QTL on chromosome 5BL had the allele favouring green plants from the low responding parent ‘Walter’. In a multiple regression analysis the four QTLs could explain a total of 80% of the genotypic variation for green plant percentage. None of the chromosomal regions with QTLs for green plant percentage showed significant influence on either embryo formation or regeneration frequencies from the anther culture. The three major QTLs located on group two chromosomes were fixed in a second DH population derived from two parents ‘Ciano’ and ‘Benoist’,both with high capacity to produce green plants. A QTL explaining31.5% of the genetic variation for green plant formation were detected on chromosome 5BL in this cross as well.

Anther culture-derived regenerants of durum wheat and their cytological characterization.

Anther culture is being increasingly used in cereal crop improvement both as a source of haploids and for inducing new genetic variation. We studied the androgenetic ability and regenerability of 10 cultivars of durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB), using three different growth conditions and four media. From a total of 86,400 anthers cultured, 324 plants were obtained: 248 green and 76 albino. Genotype, growth condition, and media significantly affected anther response and callus production; interactions were also significant. Green plant regeneration was influenced significantly by genotype and growth condition, as well as by genotype and growth condition interactions. Albino plant regeneration was significantly affected only by growth condition. Regenerants showed gametoclonal/somaclonal variation. Differences in morphology, growth habit, adult plant height, spike size, and development of spikes at nodes were observed. Mitotic and meiotic chromosomes were studied by conventional staining and fluorescent genomic in situ hybridization techniques. Chromosome numbers of the regenerants ranged from 14 to 70. All 76 haploid plantlets (2n = 2x = 14; AB) were albino. Some of the 28-chromosome regenerants were also albino. Chromosome number in the green plantlets ranged from 28 to 70. Chromosome number also varied in regenerants originating from the same callus. Both intergenomic and intragenomic multivalents were observed. An interesting feature was the preferential multiplication of B-genome chromosomes, which formed multivalents (trivalents, quadrivalents, and hexavalents). We observed several chromosomal abnormalities, which seemed to increase with the level of polyploidy. Translocations, dicentric chromosomes, chromatid exchanges, and Robertsonian translocations involving the A- and B-genome chromosomes were observed. Chromosome breakages resulting in centric and acentric fragments, and telocentrics were observed. Chromosome multiplication and structural aberrations induced during culture may constitute the bases of gametoclonal and somaclonal variations.

A Two-Step Approach to Scale Up Green Plant Regeneration Through Somatic Embryogenesis from in vitro Cultured Immature Inflorescences of a Male Sterile Line and a Maintainer Line of Rice

ABSTRACT A novel, two-step protocol for rapid propagation of cyto-plasmic male sterile (CMS: A line) and maintainer (B line) line of rice through somatic embryogenesis was developed and more than 100-170 green plantlets from A and B lines were regenerated per single immature inflorescence after 8 weeks of culture. Immature inflorescence explants of IR58025A and IR58025B were cultured on an induction medium supplemented with 2 mg l−1 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l−1 6-benzyl amino purine (BAP) for a period of 2 weeks. The medium under appropriate conditions of explant age and hormones allowed predetermination of partially differentiated cells of the individual spikelet into an embryogenically competent proembryogenic group. Differentiation of these proembryogenic groups was achieved by transferring them to embryo proliferation medium containing a high concentration of BAP, proline and maltose for a period of 2 weeks. About 800-1200 green plants could be regenerated after 8 weeks of cu...

Isolated microspore culture and plant regeneration in rye (Secale cereale L.).

An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration.

Étude de la réponse à l’androgenèse in vitro des lignées de substitution disomique chez le blé dur

The variety ‘Langdon’ and its substitution series were used to evaluate the effect of each substituted chromosome of the A and B genomes on the in vitro androgenetic potential. This study showed the implication of chromosomes 1B and 5B in repressing embryogenesis. Genes located on these chromosomes seem to have an inhibitor effect. The substitution of these chromosomes by their homeologous ones from the D genome increased the number of embryos while with the presence of the original genes the number of embryos was less than in the control. Chromosome 5B is also especially involved in the regeneration of green plants. The genetic control is inhibitory; this explains the difficulty of obtaining good levels of in vitro androgenesis in durum wheat. In this study no effect of the D genome on the androgenetic response of the substitution lines was observed.

Media Composition and Anther Plating for Production of Androgenetic Green Plants from Cultivated Rye ( Secale cereale L.)

Media composition and anther plating environment were studied in rye anther culture. In the studies on media the effects of basal media composition, plant growth regulators, glutamine, sucrose and maltose, and solidification of media were investigated. Increase in the size of the induction dish improved regeneration of green plants. The basal media W14 and 190-2 were superior in promoting callus induction and green plant regeneration. Genotype by medium interactions for induction were not recorded. Dicamba auxin in the induction medium seemed beneficial for the green to albino plant ratio. Glutamine improved induction generally and green plant regeneration with a responsive genotype. Adding 10% ficoll type 400 was favourable for induction with one rye line, but this effect was not confirmed in subsequent testing with two different ryes. Solid medium promoted the highest production of green plants. Maltose in induction medium was superior to glucose and the maltose concentration of 6% seemed optimal for induction in comparison with higher concentrations and most cost-effective for production of green plants.

Assessment and Improvement of Wheat Microspore derived Embryo Induction and Regeneration

Summary In this study we present an efficient protocol for the production of embryos in isolated wheat microspore culture and discuss parameters determining the efficiency of embryo induction and green plant regeneration. Microspores were isolated from the spring wheat genotypes DH83Z118.32 and DHBW3 showing high and low androgenetic response, respectively. The washing (WM) and induction/culture medium (AMC) were optimised and led to an average embryo yield of 1350 embryos per 10 5 microspores with DH83Z118.32. One single culture dish yielding 7250 embryos per 10 5 microspores demonstrated that the androgenetic potential of this genotype is even higher. With DHBW3 an average of 82 embryos per 10 5 microspores was achieved. We developed a non-destructive UV fluorescence image processing system that revealed a correlation between size parameters of microspore populations and their embryo yield. For DH83Z118.32 we found the ability of embryos to regenerate green plants (embryo quality) to be strongly determined by embryo age and size. The highest yield of regenerated green plants was obtained when large embryos (\s>4 mm) were transferred to regeneration medium 25 days after microspore isolation. The formation of albinos was negatively correlated with the embryo size. The early assessment of microspore size and embryo quality combined with optimised culture methods demonstrated that the potential of wheat haploid induction by isolated microspore culture is not yet fully exploited.

Increasing anther culture efficiency in rice ( Oryza sativa L.) using anthers from ratooned plants

Anther culture response for the variety Taipei-309 was compared using anthers of ratooned and non-ratooned plants. Anthers were plated onto N6 liquid media with 0, 2, 5, and 10 mg l −1 ABA, respectively. After 5 days in culture, the media were removed and N6 medium without ABA was added. A higher number of pre-mitotic P-pollen grains were observed in anthers from ratooned plants. A significant synergistic effect was found between anthers from ratooned plants/ABA concentrations and the increase in embryogenic-like structures (ELS) induction and green plant regeneration efficiency. The highest frequency of anthers producing ELS was found in N6 medium (6% Sucrose and 2 mg l −1 2,4-D) with 5 and 10 mg l −1 ABA for anthers derived from ratooned plants. Mean green plant regeneration efficiencies were raised from 24.2 to 42% (non-ratooned), and from 30 to 70%, (ratooned donor plants) using a combination of 10 mg l −1 ABA in induction media, and MS with 2 mg l −1 BAP, 1 mg l −1 NAA, 2 mg l −1 kinetin and 0.45% agarose. The highest number of green shoots per ELS were obtained from ratooned-derived anthers (up to 82). The use of ratooned plants in anther culture, its importance and relative advantages are also discussed.

Comparison of anther and isolated microspore cultures in barley. Effects of culture density and regeneration medium.

A comparison of anther and microspore culture efficiency for green doubled haploid plant production was undertaken using 17 F1 crosses with potential agronomic performance. Green doubled haploid plants were produced from all F1 crosses by anther and microspore cultures, although there was a great variation among crosses. On average, anther culture resulted in a production of green plant twice that of isolated microspore culture (30 and 14 greenplants/100 anthers, respectively).The effect of microspore culture density on green plant regeneration was studied with the cultivars Igri, Reinette and Hop which have a high, medium and low androgenic response. The highest number of dividing microspores was obtained at a density of 2.4 ×105 viable microspores/ml for the three cultivars. However, the optimal density for the percentage of embryos/dividing microspores and greenplants/103 microspores depended on the cultivar. The highest number of green plants/103microspores was produced at 1.2 × 105 viable microspores/ml for cv. Igri and 2.4 × 105 for cultivars Reinette and Hop. Percentage of green plants/total plants was raised when the culture density was increased up to 6.0 × 105 viable microspores/ml, especially for cv. Reinette. Six regeneration media differing in maltose concentration, organic nitrogen and type of auxin were assayed with embryos from cultivar Reinette. Media without organic nitrogen containing 31 g l-1maltose and the auxins IAA or NAA produced more vigorous green plants.

Efficient Anther Culture Method of the Japonica Rice Cultivar Koshihikari.

The japonica rice cultivar Koshihikari has been used as the crossing parent for breeding of high-eating quality cultivars. An anther culture method has been developed to breed a new rice cultivar with in a short period, and several cultivars have been released in recent years. However, the efficiency of anther culture of Koshihikari was low. We developed a culture medium suitable for Koshihikari callus culture to increase the anther culture efficiency. The medium was a modification of the R2 medium, i.e., the concentrations of potassium nitrate (KNO3) and ammonium sulfate ((NH4)2SO4) in the R2 medium were diluted to 1/5, 5mM aspartic acid and 5mM glutamine were added, and the vitamins were replaced by those of B5 medium. We designated the medium as DKN (dilute KNO3) medium. The anthers of Koshihikari were cultured by using the three-step anther culture method in which DKN medium was used as the basal medium. The green plant regeneration rate peranther on DKN medium was 4.3 times (donor plants cultivated in green house) and 21.4 times (donor plants grown in field) higher than those on 1/2R2 medium which had been used for rice breeding by anther culture. It is concluded that the combination of the three-step anther culture method and DKN medium is efficient for anther culture of Koshihikari.

Optimization of media nitrogen and copper concentrations for regeneration of green plants from polyembryogenic cultures of barley ( Hordeum vulgare L.)

In recent years particle bombardment has become the most used method for gene transfer to barley ( Hordeum vulgare L.). Transformation efficiency depends greatly on the ability of the target material to regenerate into green plants. In this work we improved the regeneration efficiency of polyembryogenic cultures of barley (cv. Kymppi) by optimizing the nitrogen and copper concentrations in the media.

Stress treatments and ficoll for improving green plant regeneration in triticale anther culture

Anther culture of 10 winter triticale cultivars of diverse origin was studied. Two-week cold pretreatment was applied to all cultivars. In addition, cold pretreatment in combination with heat shock or mannitol starvation and prolonged cold stress of three weeks were tested on some of the genotypes. Field and greenhouse conditions were compared for growing the mother plants. Anthers for stress treatment testing were cultured on solid W14. Ficoll-supplemented induction medium was tested with three cultivars. A total of 378 green regenerants were analysed for DNA-ploidy with flow-cytometry. Morphological observations and seed-set were recorded for 317 plants grown to maturity. Large genotypic differences were noted between the cultivars. Seven out of 10 cultivars exhibited a differential response to the treatments and it was possible to improve green plant production considerably by applying increased stress or by culture on a medium containing ficoll. Cold pretreatment complemented with heat shock improved androgenetic response of the three cultivars tested. Prolonged cold pretreatment increased generally both the proportion of green plants and the proportion of spontaneous doubling. Morphological abnormalities, possibly caused by aneuploidy and associated with loss of fertility, were observed in 23% of the plants.

Androgenetic green plants from winter rye, Secale cereale L., of diverse origin

AbstractTwenty varieties and breeders’ lines of winter rye, Secale cereale L., including Finnish, Polish, Russian, Swedish and Danish germplasm, were tested for their anther culture ability. To establish a reference point, the initial culture method was based on published results from rye anther culture. Following results from methodological studies, a modified protocol was tested on the same 19 winter rye lines. The modifications to the protocol included growth of mother plants under controlled greenhouse conditions, prolonged cold pretreatment of spikes for 3 weeks at 4°C, excision of anthers with microspores in the late uninucleate/binucleate developmental stage and induction on liquid medium supplemented with a buoyancy increasing component, Ficoll. With the modified protocol, the total production of green androgenetic plants increased nearly fivefold; it reached an average of 2.75% with the best line yielding 12.5%, while the proportion of green to albino plants increased by 50% to 25.1%. Eighteen rye lines showed green plant regeneration ability. Several genetically different winter rye lines with relatively good anther culture ability, yielding ≤ 1/% green plants per 100 anthers, were identified for the use in further methodical studies and for researching doubled haploidy in rye breeding.

Promotion of Androgenesis in Rice Anther Cultures by Substitution of Sucrose with Maltose and Mannitol

Maltose was four times more effective than sucrose for androgenesis in anther cultures of an indica rice cultivar IR 43. Partial substitution of maltose by mannitol considerably enhanced the regeneration of green plants.

Donor‐Plant Environment Effects on Regeneration from Barley Embryo‐Derived Callus

High levels of green plant regeneration are necessary for efficient barley (Hordeum vulgare L.) transformation. Experiments to optimize regeneration of calli derived from immature embryos have examined the effects of genotype and media components, but research on the effects of donor-plant growth environment is lacking. Defined growth conditions in growth chambers are known to give consistent regeneration rates, but space often is limited. The objective of this project was to evaluate regeneration from barley tissue cultures of ‘Golden Promise’ and ‘Morex’ grown under greenhouse conditions, planted on four different dates, to determine optimal greenhouse planting times. Results were compared with cultures from plants grown in the growth chamber planted on the same four dates. Genotype, growth environment, and planting date had significant effects on green plant regeneration. Cultures of embryos from plants sown in the greenhouse in December resulted in significantly fewer green regenerants per plate. Regeneration was more dependent on the average solar radiation than on temperature. The best greenhouse regeneration rates for Morex were not significantly different than regeneration from growth chamber-grown donor plants. Green plant regeneration was higher and less variable from growth chamber-grown donor plants. Sufficient regeneration from greenhouse-grown donor plants can be reached when natural light levels are high and temperatures are moderate.

Use of anthocyanin biosynthesis stimulatory genes as markers for the genetic transformation of haploid embryos and isolated microspores in wheat

Experiments were undertaken to study the use of anthocyanin biosynthesis stimulatory genes under the control of CaMV35S promoter and first intron of maize alcohol dehydrogenase (Adhl), as marker genes for genetic transformation of wheat haploid embryos and microspores by microprojectile bombardment. Transient expression was monitored for isolated microspore cultures of 1 and 8 days, after bombardment at 650 and 1100 psi. Anthocyanin expression in 1 day cultured microspores was only recovered at 650 psi, while a higher expression efficiency was obtained with 8 days old microspore culture at 1100 psi. Experiments conducted on embryos show that total regeneration as well as albino and green plant regeneration of embryos were not affected by bombardment. 64.5 % embryos presented spots and 6.2 % of the regenerated plants were chimeric, with sectorial expression of anthocyanin on leaves. The comparison of GUS and anthocyanin expression was realized and we have shown that anthocyanins are more reliable markers when compared with GUS. Besides being more practical, they give a better estimation of transformation procedure efficiency. Results show that anthocyanin biosynthesis stimulatory genes should be used as markers in haploid embryos and isolated microspores transformation in wheat.