Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation

The development of vegetative and generative buds on thin-layer expiants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.     

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.     

Wound healing and regenerative response of fragments of the Drosophila wing imaginal disc cultured in vitro

Fragments of imaginal discs of the fruitfly Drosophila undergo growth and pattern regulation when cultured in vivo in adult female hosts for several days prior to metamorphosis in host larvae. Pattern regulation results in either regeneration of excised pattern elements or duplication of elements whose fate map positions lay within the fragment. Initial wound healing along the cut edge of a fragment is thought to be a crucial first step in the process of pattern regulation. We have examined the capacity for wound healing and pattern regulation of fragments (distal halves) of the wing disc cultured in vitro, using the culture system recently reported to support extensive growth and transdetermination of slightly wounded whole imaginal discs in vitro. Our results suggest that disc fragments and whole discs apparently respond differently in the culture system. With disc fragments, wound healing did not occur in vitro. When fragments were first cultured overnight in adult female hosts to allow initial wound healing prior to explantation in vitro, then some volume increase and regeneration of excised portions occurred during 2–3 weeks of culture in vitro. The extent of apparent growth was much less than that reported for whole discs, and the frequency of regeneration in vitro (19%), while highly significant relative to controls not cultured in vitro (0%), was much less than that observed for fragments cultured in vivo (84%). Furthermore the extent of regeneration which occurred in vitro was considerably smaller than that which occurs during regeneration in vivo.     

Genetic analysis of leaf explant regenerability inSolanum chacoense

An F₁ population consisting of 51 genotypes, derived from two unresponsive parental lines ofSolanum chacoense Bitt., was examined for shoot regeneration from leaf explants. Fourteen genotypes failed to respond whereas, among the responsive genotypes, four produced multiple shoots on over 90% of the explants. Estimates of broad-sense heritability were high for both frequency of responsive explants (83%) and the number of shoots per responsive explant (82%). The segregation of the F₁ hybrid progeny was in agreement with the theoretical ratios of a genetic model for tissue culture responsiveness involving three unlinked genes. This study confirms earlier findings concerning the genetic control ofin vitro shoot regeneration from leaf explants inS. chacoense.     

Differences in in vitro plant regeneration ability among four Arabidopsis thaliana ecotypes

Summary The Arabidopsis ecotypes Columbia (Col), Landsberg erecta (Ler), Cape Verde Island (Cvi) and Wassilewskija (WS) have been tested for their regeneration response in vitro. A characteristic morphology of leaf-derived calluses has been found for each ecotype. Differences in regeneration ability have been detected depending on the plant strain. the explant source and on the culture medium composition. In CIR/SIR media, which contain 0.5 mg l⁻¹ (2.26 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and glucose, root explants from the four ecotypes are able to reach a considerable regeneration level, while leaf explants do not regenerate beyond a basal level (5% approximately). In CIH/SIH media, which contain 2.2 mg l⁻¹ (9.95 μM) of 2,4-D and suerose, leaf explants from all the ecotypes, with the exception of Col, are able to regenerate, but they do it at variable levels (Ler 5.75%, WS 75.09%, and Cvi 27.53% as regeneration rates). With these media all root explants are able to regenerate, but again the four ecotypes show different rates (Col 27.7%, Ler 57.25%, WS 98.54%, and Cvi 42.25%). The variation of the different medium components affects differentially the regeneration ability of the four ecotypes depending also on the kind of explant. Thus, when the 2,4-D concentration is raised WS duplicates its regeneration rate in both leaf and root explants. Changing glucose for sucrose in CIR/SIR media diminishes to the basal level the regeneration of Cvi root explants, while the CIH/SIH salts and vitamin concentration permit the regeneration of leaf explants from all the ecotypes except Col. The genes responsible for those observed differences in regeneration ability could be identified and mapped by analyzing the in vitro regeneration behavior of the recombinant inbred lines (RILs) obtained by crossing these ecotypes.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation

Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 g/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.     

Assessment of gene expression profiles in primary and secondary zoospores of Plasmodiophora brassicae by dot blot and real-time PCR

Information on the molecular basis of pathogenicity of the clubroot pathogen Plasmodiophora brassicae is very limited. Although the sequences of more than 100 P. brassicae genes are available in GenBank, their expression and regulation are largely unknown. In this study, specific primers were designed and used to amplify genomic fragments of 118 P. brassicae genes that represent all database-available proteins and ESTs. The PCR products were blotted on membranes and hybridized with digoxigenin-labeled double-stranded cDNA, derived from either primary or secondary zoospores of P. brassicae. The same primers were also used in real-time PCR against the single-stranded cDNA synthesized from the two types of zoospores. Both dot blot and real-time PCR identified up- and down-regulated genes and the correlation between these two techniques was confirmed. Real-time PCR indicated that 58 genes were up-regulated in the secondary zoospores relative to the primary zoospores, whereas 55 were down-regulated. These data suggest that different mechanisms are utilized by the pathogen in causing primary and secondary infections. The expression patterns of genes with known or putative functions suggest the relative importance of these genes during pathogenesis. In contrast, highly expressed or regulated genes with unknown function can be further studied in identifying pathogenicity factors.     

Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants

Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

Biochemical status of in vitro regenerated Lilium bosniacum and Lilium cattaniae plantlets

Lilium cattaniae (Vis.) Vis. and Lilium bosniacum (G. Beck) Beck ex Fritsch, endemic species of Balkan Dinaric Alps, were micropropagated from seeds collected from their natural habitats. The relationship between peroxidase activity, photosynthetic status and differentiation of Lilium cattaniae and L. bosniacum in vitro was investigated. Peroxidase activity recorded for somatic embryos of Lilium cattaniae obtained on Murashige and Skoog (MS) medium containing 9.05 mM 2.4-dichlorophenoxyacetic acid (2.4-D) and 4.44 mM N6-bezyladenin (BA), was about two times higher than for any other treatment. Photosynthetic status of plantlets obtained through regeneration was explant-specific and generally higher for plantlets regenerated from basal leaf explants than from bulb explants. The accumulation of anthocyanin was detected in some regenerated shoots and more often in plantlets obtained through regeneration from bulb explants. High frequency of somatic embryo formation was recorded for L. cattaniae on MS medium containing 9.05 mM 2.4-D and 4.44 mM BA. The peroxidase activity for L. bosniacum plantlets obtained through regeneration showed treatment-specific elevations. We consider that basal leaf parts are suitable for successful regeneration of these two lilies and that elevation in peroxidase activity is a good indicator of somatic embryogenesis in both lilies.