Regeneration of androgenic embryos in apple (<Emphasis Type="Italic">Malus x domestica</Emphasis> Brokh.) <Emphasis Type="Italic">via</Emphasis> anther and microspore culture

Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

Induced androgenesis of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> L.

The aim of the research was to make a preliminary determination of the effectiveness of the induction of haploids in Capsicum frutescens L. In order to induce androgenesis red and yellow fruit forms of species were used, each bred by the researchers on their own. The experiment was performed in October. Anther cultures were conducted according to a modified method developed by Dumas et al. (1981) for C. annuum L. The anthers were laid on CP medium containing 0.01 mg dm⁻³ 2.4-D and 0.01 mg dm⁻³ kinetin, with the addition of 0.5 g dm⁻³ of activated carbon and 5 mg×dm⁻³ of silver nitrate, solidified with 8 g dm⁻³ of agar. The cultures were incubated in the dark at 35 deg C for 8 days. Next they were transferred to 25 deg C under a 12-hour photoperiod. After 14 days of induction, anthers were transferred to R₁ medium supplemented with 0.1 mg dm⁻³ kinetin. Obtained embryos were subsequently transplanted onto V₃ hormone-free medium and well growing plants were planted in greenhouses. The efficiency of androgenesis for both C. frutescens L. forms was relatively low and it did not exceed 5%. The ploidy level of the resulting plants was determined by flow-cytometric analysis. The regenerants consisted of about equal numbers of haploids and diploids. Additionally, among plants regenerated from anthers of yellow fruit forms, two mixoploids were observed.     

A single-base deletion mutation in <Emphasis Type="Italic">SlIAA9</Emphasis> gene causes tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) <Emphasis Type="Italic">entire</Emphasis> mutant

The entire (e) locus of tomato (Solanum lycopersicum L.) controls leaf morphology. Dominant E and recessive e allele of the locus produce pinnate compound and complex reduced leaves. Previous research had indicated that SlIAA9, an Aux/IAA gene, was involved in tomato leaf morphology. Down-regulation of SlIAA9 gene by antisense transgenic method decreased the leaf complex of tomato and converted tomato compound leaves to simple leaves. The leaf morphology of these transgenic lines was similar with leaf morphology of tomato entire mutant. In this paper, we report that a single-base deletion mutation in the coding region of SlIAA9 gene results in tomato entire mutant phenotypes.     

The effect of light quality on anther culture of <Emphasis Type="Italic">Citrus clementina</Emphasis> Hort. ex Tan.

This preliminary research reports results on the influence of light quality on anther culture of Citrus clementina Hort. ex Tan., cultivar Nules. After one month of cultivation in darkness, four light qualities were tested: White, Red, Far-Red and Blue. Continuous Darkness and White light under photoperiod of 16 hrs were used as a control conditions. Gametic embryoids and embryogenic callus were obtained only under photoperiodic conditions of White light, suggesting that the alternation of light and dark can be used for the process of gametic embryogenesis in Citrus.     

Doubled haploids in production of male sterility maintaining triticale (<Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) lines

The androgenetic response of several selected male sterility-maintainer genotypes of triticale was investigated. Androgenesis induction was obtained in all cultivars, but a large genotypic variation in green plant regeneration was observed. The number of regenerated triticale plants varied from 0.1 to 4.7 green plants per spike, depending on genotype. Spontaneous doubling of chromosomes varied from 14 to 60% for particular genotypes and, on average, reached the value of 34% for all genotypes. Fertile DH lines obtained in this study will find practical application in the development of triticale male sterile lines that are desirable in hybrid breeding.     

Plant regeneration from leaf protoplasts of <Emphasis Type="Italic">Solanum virginianum</Emphasis> L. (<Emphasis Type="Italic">Solanaceae</Emphasis>)

Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 10⁶/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.     

Improvements in the production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C₁₇ induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F₁ crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.     

Effects of brassinosteroids on microspore embryogenesis in <Emphasis Type="Italic">Brassica</Emphasis> species

Summary Experiments were conducted to determine the effects of brassinosteroids on microspore embryogenesis in Brassica species. Two compounds, 24-epibrassinolide (EBR) and brassinolide (BL), were evaluated. An increase in embryogenesis was observed in all Brassica napus lines evaluated, including Topas 4079 and several recalcitrant cultivars: Garrison, Westar, and Allons. Microspore embryogenesis, calculated as the number of embryos at 21 d of culture, was increased in the recalcitrant cultivars up to 12 times that of the control. An increase in microspore embryogenesis was also observed for B. juncea when EBR or BL was added to the culture medium. In constrast, no significant increases in embryogenesis was observed for several other Brassica species evaluated (i.e. B. carinata, B. nigra, and B. rapa). The addition of brassinosteroids to the induction media did not affect the subsequent conversion of the embryos to plantlets, but did appear to influence chromosome doubling.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

Interspecific somatic hybrids <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.     

The recessive potyvirus resistance gene <Emphasis Type="Italic">pot-1</Emphasis> is the tomato orthologue of the pepper <Emphasis Type="Italic">pvr2-eIF4E</Emphasis> gene

The translation initiation factor 4E (eIF4E) has been implicated in naturally occurring resistance to Potato virus Y (PVY) determined by the pvr2 locus in pepper (Capsicum annuum). Here, the molecular basis of the recessive resistance to PVY and Tobacco etch virus (TEV) controlled by the pot-1 locus in tomato (Lycopersicon esculentum; now Solanum lycopersicum) was investigated. On the basis of genetic mapping data that indicated that pot-1 and pvr2 are located in syntenic regions of the tomato and pepper genomes, the possible involvement of eIF4E in pot-1-mediated resistance was assessed. Genetic mapping of members of the eIF4E multigenic family in tomato introgression lines revealed that an eIF4E locus indeed maps in the same genomic region as pot-1. By comparing eIF4E coding sequences between resistant and susceptible Lycopersicon genotypes, a small number of polymorphisms that co-segregate with the pot-1 locus were identified, suggesting that this gene could be involved in resistance to potyviruses. Functional complementation experiments using Potato virus X-mediated transient expression of eIF4E from a susceptible genotype in a resistant pepper genotype confirmed that a small number of amino acid substitutions in the eIF4E protein indeed account for resistance/susceptibility to both the PVY and TEV, and consequently that pot-1 and pvr2 are orthologues. Taken together, these results support the role of this eIF4E gene as a key component of recessive resistance to potyviruses, and validate the comparative genomic approach for the molecular characterization of recessive resistance genes.     

Granulosain I,a Cysteine Protease Isolated from Ripe Fruits of <Emphasis Type="Italic">Solanum granuloso</Emphasis><Emphasis Type="Italic">-leprosum</Emphasis> (Solanaceae)

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Morphogenesis in Isolated Microspore Cultures of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility.     

Survey of resistance gene analogs in <Emphasis Type="Italic">Solanum caripense</Emphasis>, a relative of potato and tomato,and update on <Emphasis Type="Italic">R</Emphasis> gene genealogy

The resistance (R) proteins of the TIR- and non-TIR (or CC-) superfamilies possess a nucleotide binding site (NBS) domain. Within an R gene, the NBS is the region of highest conservation, suggesting an essential role in triggering R protein activity. We compared the NBS domain of functional R genes and resistance gene analogs (RGA) amplified from S. caripense genomic DNA via PCR using specific and degenerate primers with its counterpart from other plants. An overall high degree of sequence conservation was apparent throughout the P-loop, kinase-2 and kinase-3a motifs of NBS fragments from all plants. Within the non-TIR class of R genes a prominent sub-class similar to the potato R1 gene conferring resistance to late blight, was detected. All non-TIR-R1-like R gene fragments that were sequenced possessed an intact open reading frame, whereas 22% of all non-TIR-non-R1-like fragments and 59% of all TIR-NBS RGA fragments had an interrupted reading frame or contained transposon-specific sequence. The non-TIR-R1-like fragments had high similarity to Solanaceae R genes and low similarity to RGAs of other plant species including A. thaliana and the cereals. It is concluded that appearance of the non-TIR-R1-like NBS domain represents a relatively recent evolutionary development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Influence of bacterial density during preculture on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato

An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD_600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and resuspended in a liquid cocultivation medium-I, adjusted to OD_600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency is stable and highly efficient.