BAGY2 Retrotransposon Analyses in Barley Calli Cultures and Regenerated Plantlets

The stability of aging barley calli and regenerated plantlets from those calli was investigated by the BAGY2 retrotransposon-specific IRAP technique. Mature embryos of barley (Hordeum vulgare cv. Golden Promise) were cultured in Murashige and Skoog medium supplemented with 4 mg/L dicamba and maintained on the same medium for 45 and 90 days. Two IRAP-based primers were used, and the levels of variation of DNA isolated from 45- and 90-day-old calli and regenerated plantlets were found to be increased 0–21%, depending on the mature embryo material and the age of the callus. It has been observed that culture conditions cause genetic variations and evident BAGY2 retrotransposon alterations. Internal domains of BAGY2 were also analyzed by qPCR, and copy numbers were found to be increased. These findings are expected to contribute to understanding of how retrotransposons affect features like tissue culture (especially callus tissue) formation and genetic engineering studies.     

Sukkula retrotransposon insertion polymorphisms in barley

Tissue culture could be considered as one of the stress factors that affect the activation of retrotransposons. In this study, mature barley embryos (Hordeum vulgare L., cv. Tokak 157/37) were cultured for callus formation. Sukkula (a non-autonomous retrotransposon) polymorphism was investigated in calli with different culturing time (40- and 80-day-old), which was derived from the same embryo in MS medium supplemented with 3 mg/L of Dicamba. Polymorphism was detected using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Inter-Primer Binding Site Amplification (iPBS) techniques with two different Sukkula-specific primers. Noncultured five mature embryos were used as a control. While up to 14% polymorphism rate was observed in the case of noncultured embryos, 61 and 0% polymorphism rates were observed with IRAP analyses for 40- and 80-day-old calli, respectively. iPBS analysis revealed the polymorphism ratio up to 29% in noncultured material, and up to 58 and 70% in 40- and 80-day-old callus materials, respectively. In addition, there were 50 and 74% polymorphism rates between 40- and 80-day-old calli by IRAP and iPBS analyses, respectively. The results obtained showed that tissue culture conditions and callus age affected Sukkula retrotransposon movements, and all individuals did not present the same effect.     

Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques

The Sequence-Specific Amplification Polymorphism (S-SAP) method, and the related molecular marker techniques IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism), are based on retrotransposon activity, and are increasingly widely used. However, there have been no systematic analyses of the parameters of these methods or of the utility of different retrotransposon families in producing polymorphic, scorable fingerprints. We have generated S-SAP, IRAP, and REMAP data for three barley (Hordeum vulgare L.) varieties using primers based on sequences from six retrotransposon families (BARE-1, BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula). The effect of the number of selective bases on the S-SAP profiles has been examined and the profiles obtained with eight MseI+3 selective primers compared for all the elements. Polymorphisms detected in the insertion pattern of all the families show that each can be used for S-SAP. The uniqueness of each transposition event and differences in the historic activity of each family suggest that the use of multiple retrotransposon families for genetic analysis will find applications in mapping, fingerprinting, and marker-assisted selection and evolutionary studies, not only in barley and other Hordeum species and related taxa, but also more generally.     

The effect of cefotaxime on the growth and regeneration of callus from four varieties of barley (Hordeum vulgare L.)

Recently it has been reported that the cephalosporin antibiotic cefotaxime increases growth, regeneration and embryogenesis in wheat calli. We investigated the effect of cefotaxime on callus initiated from immature embryos of four barley (Hordeum vulgare L.) varieties. In calli cultured in the presence of antibiotic callus growth was up to 45% greater than in controls and the frequency of regenerating calli was increased by up to 80%. There was an apparent interaction of the antibiotic with genotype and the 2,4-D in the medium.     

The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution

Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.      

Relative DNA content of cells in Festuca Arundinacea and Dactylis Glomerata calli of different ages

Experiments were conducted to determine changes in nuclear DNA content in cells of tall fescue (Festuca arundinacea) and orchardgrass (Dactylis glomerata) embryo-derived calli ranging in age from 3 to 24 weeks. Calli were induced and maintained on a modified Murashige-Skoog medium containing 22.6 μM of 2,4-dichlorophenoxyacetic acid. Calli were divided with one pieces being fixed in 3:1 ethanol: acetic acid and the other transfered too fresh maintenance medium at 3, 6, 9, 12, 18 and 24 weeks after initial plating of mature embryos. Fixed calli were processed through a cold hydrolysis technique and Feulgen stained. Stained callus pieces were squashed in 45% acetic acid and relative DNA contents were measured with a microscope cytophotometer. Results showed predominately 2C nuclei in calli of both species regardless of callus age. More cells with high DNA contents (4C) were found in orchardgrass than in tall rescue calli. The proportion of 2C cells increased with increasing callus age, especially in tall fescue. Cells of various sizes and shapes were observed in calli of both species and very large cells with small (2C) nuclei were common in callus tissue of all ages. The mitotic index was low and decreased with increasing callus age, especially in tall fescue. Nuclei with 2C---4C, 4C---8C, or less than 2C, amounts of DNA may be due to anuploidy.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

IRAP,a retrotransposon-based marker system for the detection of somaclonal variation in barley

The retrotransposon-based marker system, inter-retrotransposon amplified polymorphism (IRAP), and inter-simple sequence repeats (ISSRs) were used to detect somaclonal variation induced by tissue culture. IRAPs use a single primer designed to amplify out from the 5′ LTR sequence of the BARE-1 retrotransposon combined with a degenerate 3′ anchor, similar to that of ISSR primers. We analysed DNA polymorphisms in 147 primary regenerants and parental controls from three cultivars of barley (Hordeum vulgare). The ISSR marker system generated an average of 218 bands per primer, with 29 polymorphisms of which 12 were novel non-parental bands. In comparison, the IRAP system generated an average of 121 bands per primer, with 15 polymorphisms of which nine were novel non-parental bands. Polymorphism detected for IRAP and ISSR markers was more than twofold higher in Golden Promise than Mackay and Tallon cultivars. However, there was no significant difference in the frequency of novel non-parental bands. Cluster analysis revealed that the level of polymorphism and genetic variability detected was comparable between IRAP and ISSR markers. This suggests that retrotransposon-based marker systems, such as IRAP, based on retrotransposons such as BARE-1, are valuable tools for the detailed characterisation of mutation profiles that arise during tissue culture. Their use should improve our understanding of processes influencing mutation and somaclonal variation and allow for the design of methods that yield fewer genome changes in applications where maintaining clonal integrity is important.     

In vitro selection of barley and wheat for resistance against Helminthosporium sativum

Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.     

Improved Regeneration Efficiency from Mature Embryos of Barley Cultivars

A reliable protocol for plant regeneration from mature embryo derived calli of nine barley (Hordeum vulgare) cultivars has been developed. The auxins 2,4-dichlorophenoxyacetic acid, picloram and dicamba proved effective in inducing callus from mature embryos of most of the barley cultivars. The induced primary callus was loose, friable and translucent. It ultimately yielded creamy white and compact callus after 2 - 3 transfers on fresh medium of the same composition. Callus induction and regeneration capacity were highly cultivar dependent. Addition of a high concentration of picloram (4 mg dm^-3) promoted regeneration in 3 cultivars (Tallon, Grimmett and Sloop). In cv. Arapiles, abscisic acid and betaine were crucial in generating morphogenic callus from the mature embryos. Plants regenerated from these calli were hardy and developed roots readily when transferred to hormone free medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley

Net blotch, which is caused by the fungus Pyrenophoral teres Drechs. f. teres Smedeg., presents a serious problem for barley production worldwide, and the identification and deployment of sources of resistance to it are key objectives for many breeders. Here, we report the identification of a major resistance gene, accounting for 65% of the response variation, in a cross between the resistant line C19819 and the susceptible cv. Rolfi. The resistance gene was mapped to chromosome 6H with the aid of two recently developed systems of retrotransposon-based molecular markers, REMAP and IRAP. A total of 239 BARE-1 and Sukkula retrotransposon markers were mapped in the cross, and the 30-cM segment containing the locus with significant resistance effect contained 26 of the markers. The type and local density of the markers should facilitate future map-based cloning of the resistance gene as well as manipulation of the resistance through backcross breeding.     

Leaf callus induction and suspension culture establishment in lychee ( Litchi chinensis Sonn.) cv. Huaizhi

This study reports a protocol for leaf callus induction and suspension culture establishment in lychee cv. Huaizhi. The results showed that 12-day-old leaf explants cultured under a photoperiod of 16/8 h with their adaxial side touching the medium were the optimum conditions for leaf callus induction. Globular embryos were formed when the induced calli were kept on the callus induction medium without 2,4-D for 24 weeks. Friable calli were induced after 2–3 subcultures at 4 weeks intervals on the Murashige and Skoog medium supplemented with 3 mg/L IAA and 2 mg/L BAP. Suspension culture was established when these friable calli were subcultured six times in liquid callus induction medium.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

银杏愈伤组织培养及其黄酮类化合物的测定(简报)

采用银杏胚、胚乳及3个月苗龄的苗叶为外植体,在附加不同激素的培养基上诱导出愈伤组织,从愈伤组织中提取黄酮类化合物并测定其含量。结果表明,由胚诱导的愈伤组织中黄酮类化合物含量最高。