Green Plant Regeneration Research Articles

Development of doubled haploids from an elite indica rice hybrid (BS6444G) using anther culture

A total of 200 doubled haploids (DHs) were generated from an elite rice hybrid, ‘BS6444G’ for which an androgenic method was developed by manipulating the physical and chemical factors. The spike pretreated at 10 °C for 7–8 days was effective for callusing and green plant regeneration. The maximum callus frequency was achieved when the anthers cultured in N6 medium supplemented with 2.0 mg L−1 2,4-diclorophenoxyacetic acid, 0.5 mg L−1 6-benzylaminopurine and 3% maltose. Calli induced in N6 media also showed significant green shoot regeneration in MS medium supplemented with 0.5 mg L−1 1-napthalene acetic acid, 0.5 mg L−1 kinetin, 1.5 mg L−1 benzylaminopurine and 3% sucrose producing 210 green plants. Assessment of the ploidy status showed 95.71% fertile diploids and 4.2% polyploids; no haploids were observed. A total of 38 sequence-tagged microsatellite (STMS) markers proved able to discriminate a heterozygote from all the 200 DHs. The DHs grown in the field showed significant variation for their agronomic traits. Comparison of traits with control indicates homogeneity within each DH line and significant variance of traits between DH lines. Nine DH lines produce higher grain yield than the hybrid parent which suggests the possibility of exploiting hybrid vigor in indica rice through the development of DH lines of high yielding hybrids.

Solid-stemmed spring wheat cultivars give better androgenic response than hollow-stemmed cultivars in anther culture.

Solid-stemmed spring wheat cultivars (Triticum aestivum L.) are resistant to the stem sawfly (Cephus cinctus Nort.) and lodging. Anthers of 24 spring wheat cultivars with varying content of pith in the stem were used in the experiment. All were classified into three groups: solid, medium–solid and hollow stems. There was considerable influence of the cultivar on callus formation and green plant regeneration. The highest efficiency of green plant regeneration (24%) was observed for the solid-stemmed AC Abbey cultivar. There was no regeneration from the explants of four cultivars: CLTR 7027, Alentejano, Marquis and Bombona. Principal component analysis showed no differences between the cases under observation (callus induction and green plant regeneration) in their response to pre-treatment temperatures (4 and 8°C). The examination of the effects of various auxin types in the induction medium on callus formation and green plant regeneration revealed that the strongest stimulation of these processes was observed in the C17 medium with 2,4-D and dicamba. The efficiency of callus formation and green plant regeneration was greater in solid-stemmed cultivars than in hollow-stemmed cultivars.

Androgenic response of Brazilian wheat genotypes to different pretreatments of spikes and to a gelling agent

Abstract The objective of this work was to analyze the androgenic response of Brazilian wheat genotypes to different pretreatments of the spikes, prior to the culture of isolated microspores, and to the effect of a gelling agent in the induction culture medium. Five genotypes were evaluated for embryo formation, green plant regeneration, and spontaneous chromosome duplication. Wheat spikes were subjected to two pretreatments: cold, at 4ºC for 21 days; and 2-hydroxynicotinic acid, at 32ºC for two days. Culture media were evaluated with or without Ficoll as a gelling agent. Cold produced more embryos and green plants than the chemical pretreatment in four out of five genotypes. Only two genotypes treated with 2-hydroxynicotinic acid were able to produce plants, and one of them produced a single albino plant. Medium containing Ficoll produced more embryos than liquid medium and promoted a higher number of plants. Spontaneous chromosome duplication varies between genotypes and pretreatments, and shows high variability.

ROLE OF POLYAMINES IN INHIBITION OF ETHYLENE BIOSYNTHESIS AND THEIR EFFECTS ON RICE ANTHER CULTURE DEVELOPMENT

The polyamines such as putrescine, spermidine, and spermine were reported to increase green plant regeneration in rice anther culture. Low response of anther culture of rice sub-species indica may be improved with the addition of putrescine in the culture media. Four experiments were conducted to study the role of polyamines in inhibition of ethylene biosynthesis and their effects on rice anther culture development. Anthers of two subspecies of rice, indica (IR64, Krowal, Jatiluhur) and japonica (Taipei 309) were cultured onto media supplemented with putrescine (N6P) and without putrescine (N6). Young<br />panicles containing the anthers at mid-to-late nucleate microspores were cold pretreated at 5 + 2°C and incubated in the dark for 8 days before the anthers were cultured. Results<br />showed that medium without putrescine produced an earlier senescence of indica rice anther than that of japonica. The addition of 10-3 M putrescine into the culture media inhibited ethylene biosynthesis as anther senescence delayed, increased the three polyamines contents, and decreased the ACC content as well as ACC oxydase activity in anther-derived calli. In the anther and anther-derived calli of subspecies indica, the total<br />polyamines content was lower (10.14 nM g-1 anther and 8.48 nM g-1 calli) than that of subspecies japonica (12.61 nM g-1 anther and 10.16 nM g-1 calli), whereas the ethylene production was higher (32.31 nM g-1 anther and 2.48 nM g-1 calli) than the japonica (31.68 nM g-1 anther and 1.76 nM g-1 calli). This study suggests that application of 10-3 M putrescine in anther culture of rice subspecies indica improves androgenesis by inhibiting<br />early senescence of cultured anthers and enhancing embryo or callus formation from microspores.

ROLE OF POLYAMINES IN INHIBITION OF ETHYLENE BIOSYNTHESIS AND THEIR EFFECTS ON RICE ANTHER CULTURE DEVELOPMENT

The polyamines such as putrescine, spermidine, and spermine were reported to increase green plant regeneration in rice anther culture. Low response of anther culture of rice sub-species indica may be improved with the addition of putrescine in the culture media. Four experiments were conducted to study the role of polyamines in inhibition of ethylene biosynthesis and their effects on rice anther culture development. Anthers of two subspecies of rice, indica (IR64, Krowal, Jatiluhur) and japonica (Taipei 309) were cultured onto media supplemented with putrescine (N6P) and without putrescine (N6). Young<br />panicles containing the anthers at mid-to-late nucleate microspores were cold pretreated at 5 + 2°C and incubated in the dark for 8 days before the anthers were cultured. Results<br />showed that medium without putrescine produced an earlier senescence of indica rice anther than that of japonica. The addition of 10-3 M putrescine into the culture media inhibited ethylene biosynthesis as anther senescence delayed, increased the three polyamines contents, and decreased the ACC content as well as ACC oxydase activity in anther-derived calli. In the anther and anther-derived calli of subspecies indica, the total<br />polyamines content was lower (10.14 nM g-1 anther and 8.48 nM g-1 calli) than that of subspecies japonica (12.61 nM g-1 anther and 10.16 nM g-1 calli), whereas the ethylene production was higher (32.31 nM g-1 anther and 2.48 nM g-1 calli) than the japonica (31.68 nM g-1 anther and 1.76 nM g-1 calli). This study suggests that application of 10-3 M putrescine in anther culture of rice subspecies indica improves androgenesis by inhibiting<br />early senescence of cultured anthers and enhancing embryo or callus formation from microspores.

Retrotransposon insertion variations in doubled haploid bread wheat mutants

The current study attempted to obtain candidate doubled haploid (DH) wheat lines by serially combining two approaches: conventional chemical mutagenesis and anther culture. Additionally, the salt tolerance levels were examined between stress-treated (100 mM NaCl) and non-treated DH groups. For the molecular analysis, IRAP markers were used to characterize retrotransposon insertion polymorphisms induced by haploidization, chromosome doubling, and/or mutagenesis in the DH lines. Various sodium azide (NaN3) concentrations (from 0 to 5 mM) were applied to seeds of the Pehlivan wheat cultivar to obtain an M1 generation mutant population. Anther culture was set up from the M1 mutant population. Green plant regeneration, the frequency of selected candidate mutants within the DH form and the levels of salt tolerance between samples were screened. A total of eight thousand anthers were cultured, and sixteen candidate salt-tolerant DH mutant lines, twenty-seven candidate DH mutant lines with different characteristics and one hundred and two candidate DH lines with morphologically normal appearances were obtained from the NaN3-mutagenized population. The IRAP patterns were quite similar between the control DH lines, and the genetic differences between the controls and DHs originating from possible mutants showed close relatedness. According to previous studies, chemical mutagenesis and anther culture were combined for the first time to detect candidate salt tolerant genotypes at the DH stage. This approach might also be useful for determining the threshold dose and efficiency of wheat mutagens.

Effect of D Genome on Wheat Anther Culture Response After Cold and Mannitol Pretreatment

AbstractThe present study was conducted to determine the effect of the D genome on embryoid induction and green plant regeneration in wheat anther culture and how it is influenced by low temperature and mannitol treatment. For this reason, the anther culture response of two Canadian bread wheat cultivars and their extracted tetraploids (AABB) was studied. As controls two cultivars well responding to anther-culture (i.e. cvs. Kavkaz/Cgn and Acheron) and a no-responding cultivar (cv. Vergina) were used. Approximately 3000 anthers of these cultivars were cultured and three pre-treatments were applied: cold pre-treatment for 7 and 18 days at 4°C, and 0.3M mannitol for seven days at 4°C. W14 and 190-2 were used as induction and regeneration media, respectively, and the basic MS medium as the rooting medium. No green plants were produced from the tetraploids, which supports the view that the D-genome chromosomes are necessary for androgenic response in wheat. Furthermore, the Canadian cultivars performed better after 18-day pre-treatment at 4°C. The extracted tetraploids produced fewer embryoids and performed better after seven days of cold pre-treatment. The controls well responding to anther culture performed better than the Canadian cultivars, although their best response was recorded after seven-day cold pre-treatment. Cultivar Vergina produced no green plants. The presence of mannitol influenced negatively both embryoid and green plant production. It was concluded that the D genome plays a crucial role in anther culture response of wheat and that this response is influenced by both the genotype and the duration of cold pre-treatment.

Doubled Haploids generated through anther culture from an elite long duration rice hybrid, CRHR32: Method optimization and molecular characterization

An improved procedure has been developed for high frequency androgenesis in an elite long duration indica rice hybrid. The effects of cold temperature pretreatment, duration of treatment and media with different plant growth regulators on callus induction and shoot regeneration were examined for generation of doubled haploids. N6 medium supplemented with 2.0 mg l−1 2,4-D, 0.5 mg l−1 BAP and 30 g l−1 maltose was found to be most effective for callusing when compared with MS and SK1. The N6 media grown calli showed maximum green shoot regeneration frequency in MS medium supplemented with 0.5 mg l−1 NAA, 0.5 mg l−1 Kinetin, 1.5 mg l−1 BAP and 30 g l−1 sucrose after 2 week of culture. The cold temperature treatment of spike at 10°C for 2 days alongside by 8 days was found to be most suitable conditions for callusing and green shoot regeneration producing 186 green plants from indica rice hybrid, CRHR32. The ploidy status assessed on the basis of morpho-agronomic characters revealed fertile diploids at a frequency of about 81.10%; 16.10%, 2.68% and 1.08% were polyploids, haploids and mixploids respectively. Microsatellite marker analysis showed 1 : 1 ratio of the alleles of CMS and restorer lines used for development of CRHR32. Homozygosity was detected for all the marker loci in 150 DHs and only one plant was identified as heterozygote. This investigation identified the favorable media composition and condition for callus induction and green plant regeneration which would further increase the knowledge and better understanding in rice hybrids for development of DHs.

Anther culture as an effective tool in winter wheat (Triticum aestivum L.) breeding.

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.

Androgenic capability among genotypes of winter and spring barley

AbstractVariable efficiency of androgenesis remains a serious problem in many species of cereals. It is still unclear what makes certain genotypes more amenable to androgenesis than others. This study was undertaken to quantify the previously suspected advantage of winter barley genotypes over spring ones with regard to regeneration efficiency in anther culture. The material consisted of 40 barley hybrids originating from Polish breeding companies. The number of androgenic structures per 100 anthers did not differ significantly between analysed groups (119 vs. 152 non‐significant), but the average regeneration of green plants per 100 anthers was five times higher in winter genotypes (6.4 vs. 1.3). The incidence of albinism was lower for the winter than for the spring materials (70% vs. 90%), while the rate of spontaneous chromosome doubling was similar in both groups (58% vs. 56%). The results strongly support the notion that winter genotypes are more amenable to androgenesis and this may be a consequence of their better adaptation to stress conditions.

Dimethyl tyrosine conjugated peptide prevents oxidative damage and death of triticale and wheat microspores

Functional damage of mitochondria and chloroplasts under stress contributes to reactive oxygen species (ROS) induced cell death. A high percentage of cell mortality during microspore culture is a limiting factor in haploid and doubled haploid plant production. In the present study, we studied the contribution of dimethyl tyrosine (DMT) conjugated short peptides to microspore embryogenesis. These DMT-peptides, which are known to translocate to subcellular targets and scavenge ROS, mitigate the effect oxidative stress plays on microspore viability and embryogenesis. The number of viable microspores was significantly higher in the presence of SS-31 and caspase-3-inhibitor (Ac-DEVD-CHO). In particular, the total number of green plant regeneration was increased by 42 % in the presence of SS-02, and by 55 % in the presence of SS-31, in triticale. Conversely, lower caspase-3-like activities were observed in the presence of SS-31 and Ac-DEVD-CHO, and intracellular ROS was reduced in the presence of SS-31, supporting the involvement of SS-31 in reducing microspore cell death by mitigating ROS and caspase-3-like activity. This study further supports the concept that antioxidant conjugated peptides offer a useful strategy for reducing ROS in plant cells.

Дослідження андрогенезу in vitro в культурі пиляків сортів та реципрокних гібридів пшениці м’якої озимої

Testing of haploprodution ability in vitro anther culture of bread winter wheat was studied. The different nature of indicator inheritance of forming of tumors and regeneration of green plants for F1 hybrids were detected: effects of heterosis and depression, intermediate nature, and the inheritance of signs at the level of the parents. The 283 green plant-regenerants were obtained by anther culture of winter wheat.

Hormonal requirements for effective induction of microspore embryogenesis in triticale (× Triticosecale Wittm.) anther cultures.

Key message Effective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11–39 nmol g−1 DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121–424 pmol g−1 DW) and zeatin ryboside (cZR, 146–432 pmol g−1 DW). Surprisingly, a relatively high level (10–64 pmol g−1 DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of ‘responsive’ DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with ‘recalcitrant’ genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-014-1686-4) contains supplementary material, which is available to authorized users.

The influence of combinations of alien translocations on in vitro androgenesis in near-isogenic lines of spring bread wheat

The features of in vitro androgenesis were studied in Cultured anthers of spring bread wheats L503 and Dobrynya, having 7DS-7DL-7Ae#1 L translocation with genes Lrl9/Sr25 (Lrl9 translocation) from Agropyron elongatum (Host.) P.B. and their near-isogenic lines carrying combinations of Lrl9 translocation with translocations: 1BL-IR#1S with genes Pm8/Sr31/Lr26/Yr9 (Lr26translocation) from Secale cereal L., 4BS-4BL-2R#1L with genes Lr25/Pm7 (Lr25 translocation) from Secale cereal, 3DS-3DL-3Ae#1L with genes Lr24/Sr24 (Lr24 translocation) from Agropyron elongatum and 6BS-6BL-6U#1L with gene Lr9 (Lr9 translocation) from Aegilops umbellulata Zhuk. In comparison with those varieties having received the Lrl9 translocation, the following was established: (1) the combination of translocations Lr19+26 increased embryo frequency and green plant regeneration; (2) the combination of translocations Lr19+9 decreased embryo frequency but increased green plant regeneration; (3) the combination of translocations Lr19+24 decreased embryo frequency but increased green and albino plant regeneration; (4) the combination of translocations Lr19+25 increased embryo frequency and green plant regeneration but decreased albino plant regeneration. Thus, on near-isogenic lines of spring bread wheat, the influences of genotypes of four alien translocation combinations on in vitro androgenesis were determined.

Improving the efficiency of isolated microspore culture in six-row spring barley: I-optimization of key physical factors

An improved isolated microspore culture protocol alleviating the recalcitrance typically observed in six-row spring barley was developed by optimizing four key physical factors to increase embryogenesis and reduce albinism. Doubled haploid (DH) plants are completely homozygous individuals that can be generated in just a few months via androgenesis in vitro. DHs are useful tools in genetic research and in plant breeding. Isolated microspore culture (IMC) is the most efficient way to produce DHs, but a strong genotype dependency imposes limitations to its wide application. Six-row, spring barley genotypes are considered as particularly recalcitrant due to a low frequency of embryogenesis and a high rate of albinism. Seeking to develop an efficient IMC protocol for this type of barley, we explored four important factors: (1) the harvest stage of immature spikes, (2) the type of pretreatment applied, (3) the osmotic potential in the induction medium, and (4) the plating density of microspores. This work was first performed using four barley genotypes: two typical six-row spring cultivars (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) cultivar. First, by optimizing the harvest stage for each genotype we obtained a twofold to fourfold increase in the yield of embryogenic microspores. Second, two pretreatments (0.3 M mannitol for 2 days, or a combination of cold and heat over 15 days) both performed significantly better than the commonly used cold pretreatment (28 days at 4 °C). Third, an induction medium-containing mannitol (32 g/l) doubled green plant regeneration. Fourth, a plating density of 10(6) microspores/ml yielded the highest number of green regenerated plants. Our most important findings were then confirmed using sets of F1s from a six-row, spring-type breeding program.

Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism

Two alternative cytokinins, thidiazuron and meta-topoline, were tested in isolated microspore culture on recalcitrant barley genotypes (six-row, spring), and green plant regeneration was improved substantially. Doubled-haploid (DH) plants are coveted in plant breeding and in genetic studies, since they are rapidly obtained and perfectly homozygous. In barley, DHs are produced mainly via androgenesis, and isolated microspore culture (IMC) constitutes the method offering the greatest potential efficiency. However, IMC can often be challenging in some genotypes because of low yield of microspores, low regeneration and high incidence of albinism. Six-row spring-type barleys, the predominant type grown in Eastern Canada, are considered recalcitrant in this regard. Our general objective was to optimize an IMC protocol for DH production in six-row spring barley. In particular, we explored the use of alternative hormones in the induction medium (thidiazuron and dicamba), and in the regeneration medium (meta-topoline). This optimization was performed on two typical six-row spring (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) barley cultivar. When 6-benzyl-aminopurine (BAP) was replaced by a combination of thidiazuron and dicamba in the induction medium, a 5.1-fold increase (P < 0.01) in the production of green plants resulted. This increase was mainly achieved by a reduction of albinism. Moreover, a 2.9-fold increase (P < 0.01) in embryo differentiation into green plants was obtained using meta-topoline instead of BAP in the regeneration medium. Together, these innovations allowed us to achieve a substantial improvement in the efficiency of IMC in this recalcitrant type of barley. These results were later successfully validated using sets of F1s from a six-row spring barley breeding program.

Stress treatments influence efficiency of microspore embryogenesis and green plant regeneration in hexaploid triticale (× Triticosecale Wittmack L.)

Doubled haploid (DH) production is a key technology in plant breeding and research. One emerging method of choice for DH production is microspore culture, which requires reprogramming of the microspores from their normal gametophytic development to a sporophytic development resulting in embryo formation. This commonly requires the application of stress such as cold, heat, or starvation. Here, we report the effect of different stress treatments on embryo formation and the proportion of green plants in triticale microspore culture. We observed different responses to the applied stress treatments among three studied genotypes. In general, a 3-wk cold stress treatment performed best with regard to the two criteria. For one genotype, the application of a 24- or 48-h heat stress gave similar or slightly better results and consequently may be an alternative for genotypes that are recalcitrant to the cold stress treatment.

The Evaluation of Response to Isolated Microspores Culture in Some Iranian Hexaploid Wheat (Triticum aestivum L.) Cultivars

The most important method of haploid plant production is isolated microspores culture. To be successful in this method, genetic potential of cultivars is very important. In this research, ten Iranian hexaploid wheat cultivars (exclusive of MV17) on the basis of their characters such as heat, drought and salinity resistant have been selected and their responses to isolated microspore culture were evaluated. Meanwhile the effects of two pretreatments (cold and heat + chemical) and embryo induction media (NPB99 and CHB-2) on androgenic traits including embryogenesis, total plant regeneration and green plant regeneration were studied in selected cultivars. There were significant interactions between cultivars and pretreatments and also between cultivars and media in most cases. The interaction between cultivars Chamran and Navid with 21 days cold pretreatment produced the highest number of embryos and the interaction between cultivar Hamoon and 21 days cold and heat + chemical pretreatments produced the most green plant regeneration percentage. Also, the results showed that the interaction between cultivar Chamran and CHB-2 medium produced the highest number of embryos while the interaction between cultivar Hamoon and NPB99 medium had the highest green plant regeneration.

Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.

Developing tools for investigating the multiple roles of ethylene: identification and mapping genes for ethylene biosynthesis and reception in barley

The plant hormone ethylene is important to many plant processes from germination through senescence, including responses to in vitro growth and plant regeneration. Knowledge of the number and function of genes that are involved in ethylene biosynthesis and reception is necessary to determine the role of specific genes within gene families known to influence ethylene biosynthesis and other aspects of ethylene function in plants. Our objective was built on previous studies that have established the critical role of ethylene in the in vitro response of barley (Hordeum vulgare L.), and that have identified ethylene-related QTL in the barley genome. In this study, we have identified the locations of genes in the barley 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and ethylene receptor (ETR) gene families. Specific primers for PCR amplification of each gene were developed and used to map these genes in the Oregon Wolf Barley mapping population. Five ACS, 8 ACO, and 7 ETR genes were identified and mapped to six of the barley chromosomes. Gene locations were syntenous to the orthologs in rice except for two that mapped to chromosome 6H. Gene duplication was evident for ACO genes on chromosomes 5H and 6H. Gene-specific primers will be useful for determining expression of each gene under various environmental conditions, including in vitro environments, to better understand the role of ethylene. Of the six known QTL for green plant regeneration in barley, three were located near the genes mapped in this study.