Variations in BARE-1 insertion patterns in barley callus cultures

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.     

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.     

Comparison of ISSR,IRAP and REMAP markers for assessing genetic diversity in different species of <Emphasis Type="Italic">Brassica</Emphasis> sp.

Molecular markers provide facilities in order to study genetic diversity and relationship among genotypes. In this study, genetic diversity among 35 genotype of Brassica sp. (belonging B. napus, B. juncea, B. rapa, B. nigra) were determined using 13 ISSR, 3 IRAP markers and 18 REMAP (primer combinations of ISSR and retrotransposon primer). The percentage of polymorphism for ISSR, IRAP and REMAP was 96.38, 94 and 96%, respectively. By comparison between markers, ISSRs indicated the highest expected heterozygosity (He) and Shannon’s information index (I) with value of 0.34 and 0.51, respectively, while REMAP marker had by far the highest number of polymorphic bands (340) and marker index (7.1) among all fragments scored over all markers. In pattern of clustering based on Bayesian methods, K = 8 was resulted for combined data clustering that was more organized clustering for genotypes compared to others. This research suggests the combined data of ISSR, IRAP and REMAP markers are most reliable than each solely marker whilst have been clustered genotypes in their taxonomic classification of Brassica without any mixture. Principle coordinate analysis (PCoA) separated 35 genotypes in four groups which all of genotypes were clustered correctly based on their taxonomic classification. The findings of this study provide the valuable insight into the Brassica species relationships in terms of similarity among genotypes which can be helpful in breeding programs, and also demonstrate that retrotransposon markers are legible for genetic diversity and next genetic analysis in Brassica genus.     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

The role of putrescine against the long terminal repeat (LTR) retrotransposon polymorphisms induced by salinity stress in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

This study aimed to research the impact of putrescine against the long terminal repeat (LTR) retrotransposon polymorphisms (Nikita-E2647, Sukkula, Stowaway, WLTR2105 and 5′LTR) induced by salinity stress in Triticum aestivum using inter-retrotransposon amplified polymorphism (IRAP) assay. The results showed that the LTR retrotransposon polymorphisms can be induced by all treated sodium chloride (NaCl) doses (0, 50, 100, 200 and 300 mM NaCl). On the other hand, the LTR retrotransposons polymorphisms were decreased effectively by treatment with putrescine (0, 0.01, 0.1 and 1 mM) together with NaCl. These results suggest that putrescine could effectively inhibit salt-induced LTR retrotransposon polymorphisms, and putrescine positively contributed to salt stress tolerance.     

Isolation and characterization of LEAFY COTYLEDON 1-LIKE gene related to embryogenic competence in Citrus sinensis

A member of the LEAFY COTYLEDON gene family encoding a HAP3 (heme activated protein 3) subunit of the CCAAT box-binding factor was isolated and termed as Citrus sinensis LEAFY COTYLEDON 1-LIKE (CsL1L). The deduced amino acid sequence shared a high similarity with LEAFY COTYLEDON 1-LIKE (L1L) in Arabidopsis thaliana, Phaseolus coccineus, Theobroma cacao, and Helianthus annuus. Quantitative RT-PCR results indicated that CsLIL was highly expressed in embryogenic callus, somatic embryos and immature seeds, but was rarely detected in non-embryogenic callus, vegetative and floral tissues. Ectopic expression of CsL1L in vegetative tissues could induce embryo-like structures, suggesting that CsL1L has the capability to transit cells from vegetative to embryogenic phase. Comparison of CsL1L expression in the newly formed and long-term subcultured embryogenic calli of W. Murcott tangor (C. sinensis × C. reticulata) and Hongkong kumquat (Fortunella hindsii Swingle) revealed that the potency of embryogenesis was related to the level of CsL1L expression. Sub-cellular localization analysis indicated that CsL1L was a nuclear protein in plant. A microsatellite in CsL1L was verified with polymorphism among the citrus species.     

Abscisic acid promotes shoot regeneration in Arabidopsis zygotic embryo explants

An efficient shoot organogenesis protocol for Arabidopsis zygotic embryo explants of Landsberg erecta ecotype was established. This de novo shoot organogenesis protocol has three different steps, i.e., induction of callus in an auxin-rich callus induction medium, the formation of green-organogenic callus in the shoot induction medium (SIM), and the final morphological differentiation of shoot in the hormone-free shoot development medium (SDM). Abscisic acid (ABA), auxin, and cytokinin (CK) were used in the SIM. Individual plant growth regulators as well as their combination were studied to understand their importance in the shoot induction treatment. We found that a combination of ABA + CK and ABA + CK + auxin induced higher shoot organogenic ability in the callus than ABA, CK, and auxin alone. Optimum ABA concentration on shoot organogenesis was determined to be 10^?5 M. Morphological characterization of callus induction and shoot organogenesis events indicated that calli were derived from the cotyledons of zygotic embryo explants and the formation of green organogenic calli was specific to the exogenous inclusion of ABA + CK in the SIM. During the time of shoot development, the green organogenic callus became darker green due to the formation of anthocyanins. Shoot organogenic calli in the SIM and the SDM were easily identified by the green-colored calli and anthocyanin pigments, respectively. Furthermore, we demonstrated the significance of exogenous and endogenous ABA in shoot organogenesis by fluridone treatments. The inclusion of ABA in SIM has a significant effect on shoot formation.     

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.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

AgNO3 increases type II callus production from immature embryos of maize inbred B73 and its derivatives

Incorporating 10 to 100 M AgNO₃ into Phytagel (0.2%) solidified N6 medium containing 1 mg/L 2,4-D, 100 mg/L casamino acids and 25 mM praline (N6 1-100-25) promoted type II callus production from cultured Zea mays L. immature embryos of FRB73, B73 X A188 and a proprietary B73 BC6 genotype. Under these conditions, approximately 15, 80 and 80% of the respective FRB73, B73 X A188 and B73 BC6 explants produced type II calli after 2 to 3 weeks incubation in the dark at 28 C. In the absence of AgNO₃, the type II culture response from B73BC6 immature embryos was 25% on N6 1 100-25 solidified with Phytagel (0.2%) as compared to 0% for that solidified with 0.8% agar. Duncan's medium was tested using 10 to 100 m AgNO₃ and generally promoted type I callus initiation, although up to 6% of the explants produced type II cultures in the presence of 0.2% Phytagel. Ethylene emanation rates of up to 370 and 115 nL g-1 h-1 were detected from B73 X A188 immature embryos and calli, respectively, cultured on N6 1-100-25.     

Retrotransposons in <Emphasis Type="Italic">Betula nana</Emphasis>, and interspecific relationships in the Betuloideae,based on inter-retrotransposon amplified polymorphism (IRAP) markers

The Betulaceae family comprises two subfamilies, Betuloideae and Corylaceae. The subfamily Betuloideae contains two genera, Alnus Mill. and Betula L. Twenty putative long terminal repeat (LTR) retrotransposons were mined from 171 scaffolds containing 5,208,995 bp of dwarf birch (Betula nana) genome sequences. Five retrotransposons were finally selected after filtering the retrotransposon canonical features and nucleotide similarities between left and right LTR sequences. Of the five retroelements, three elements were found to be Ty1/Copia retrotransposons; identity of the other two elements could not be ascertained due to sequence undetermined ‘N’ bases in the sequence database. Inter-retrotranposon amplified polymorphism (IRAP) analysis, based on the LTR sequences of the mined LTR-retrotransposons, produced 179 discernible IRAP bands among the Alnus and Betula genera. Sequence analysis revealed no size homoplasy among the homologous IRAP bands. Phylogenetic and principle coordinate analysis, based on the band sharing among the taxa, showed the species in two different genera were clearly separated. The subgenera in each genus of Alnus and Betula were also distinguishable from the IRAP profiles. In the genus Betula, the species in subgenus Betula showed mixed clustering between species. This is incongruent with the phylogeographical distribution of the species.     

Molecular characterization of Fagaceae species using inter-primer binding site (iPBS) markers

Retrotransposons (RTNs) contribute for genome evolution, influencing its size and structure. We investigated the utility of the RTN-based markers inter-primer binding site (iPBS) for the molecular characterization of 25 Fagaceae species from genera Castanea, Fagus and Quercus. The assessment of genetic diversity, relationships and structure, as well as taxonomic classification of Fagaceae based on molecular data is important for definition of conservation, forestry management strategies and discrimination among natural hybrids and their parents since natural hybridization may increase with the climate changes. Here, iPBS primers designed by other authors were tested alone and combined. Some of them were discriminative, revealed polymorphism within and among taxa allowing the production of a total of 150 iPBS markers. In addition, several monomorphic iPBS markers were also amplified in each taxon. The UPGMA dendrogram based on the pooled iPBS data revealed 27% of genetic similarity among species. The individuals were clustered per genus and most of the oaks per infrageneric group corroborating the adopted taxonomy. Globally, the iPBS markers demonstrated suitability for DNA fingerprinting, determination of phylogenies and taxonomic discrimination in Fagaceae, and could constitute a useful and alternative tool for germplasm characterization, and for definition of conservation strategies and forestry management. Moreover, these markers would be useful for fingerprinting natural hybrids that share morphological similarities with their parents. Since iPBS markers could also enable insights about RTNs evolution, an eventual correlation among iPBS polymorphism, variability of RTN insertions and/or genome size in Fagaceae is discussed.     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

Regeneration of fertile plants from excised immature zygotic embryos of Arabidopsis thaliana

Summary We present protocols for the regeneration of fertile plants from immature zygotic embryos of Arabidopsis thaliana ecotype Zürich either directly by multiple shoot formation from the apical region or indirectly by shoot induction from embryo derived calli. The regeneration efficiency by multiple shooting depended on the developmental stage of the cultured embryos and ranged from 15% for early heart shaped to 90% for early torpedo shaped and further developed embryos. 85% callus induction was achieved from embryos in the early torpedo shaped or a later stage of development. The efficiency of shoot induction from embryo derived calli varied between 25% and 75% in different experiments.Abbreviations BAP 6-benzylaminopurine - CIM callus inducing medium - 2,4-D 2,4-dichlorophenoxyacetic acid - Gln glutamine - IAA indole-3-acetic acid - Kin kinetin - NAA l-naphthaleneacetic acid - 2ip 2-isopentenylaminopurine - Pro proline - RIM root inducing medium - SIM shoot inducing medium     

Plant regeneration via somatic embryogenesis in sugarcane

Plants, regenerated from callus cultures of sugarcane (Saccharum officinarum L.) clone IJ76-316, originated through somatic embryogenesis. Callus cultures were established from primordial leaves and apical meristems on Murashige and Skoog medium (MS) supplemented with 3 mg 1^?1 2,4-dichlorophenoxy acetic acid and 100 ml 1^?1 coconut water (MSC₃). Nodular calli formed within 2 weeks of culture. Calli were maintained on MSC₃ medium by transfer every 3 to 4 weeks. Somatic embryogenesis occurred after 10 weeks culture of callus on MSC₃ medium. Somatic embryogenesis was also observed in cell suspension cultures initiated from calli maintained on MSC₃ and then cultured in half strength MS liquid medium supplemented with 0.5 mg 1^?1 2,4-D. Somatic embryos produced coleoptiles and shoots 2 to 4 weeks after transfer to MS medium supplemented with 100 ml 1^?1 coconut water (MSC), and produced complete plantlets within 4 weeks of further culture on half-strengh MS medium (half-MS) with 30 g 1^?1 sucrose. Calli grown on MSC₃ medium, when transferred to half-MS medium containing 15 g 1^?1 sucrose, produced tiny plantlets, circa 4–10 mm, without forming coleoptiles, suggesting precocious germination of somatic embryos. The regenerates included morphological variants.     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.