<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Efficient and stable regeneration from protoplasts of <Emphasis Type="Italic">Cyclamen coum</Emphasis> Miller via somatic embryogenesis

Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic embryos per gram fresh mass. Up to 4.24 × 10⁵ protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis. Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated from the embryogenic cultures.     

Osmotic shock improves <Emphasis Type="Italic">Tnt1</Emphasis> transposition frequency in <Emphasis Type="Italic">Medicago truncatula</Emphasis> cv Jemalong during in vitro regeneration

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.     

Enhanced shoot organogenesis and regeneration in the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Four cultivars of common bean (Phaseolus vulgaris L.) were tested for regeneration efficiency. Embryo axes from mature seeds were incubated on Murashige and Skoog or Gamborg media containing 6-benzyladenine (10 mg/l), without and with adenine hemisulphate (20 mg/l). Efficient regeneration was achieved when explants were incubated on Gamborg media amended with 6-benzyladenine, without adenine hemisulphate. This medium provided high regeneration efficiency in the four cultivars tested: Apetito G13637 (98–100%), Flor de Mayo Anita (96–98%), ICA Palmar G4523 (88–97%) and Pinto Saltillo (83–84%). The division and transfer of organogenic shoot of all cultivars to induction and multiplication medium every 15 days resulted in the formation of three to five new organogenic embryo axes per transfer. A single 5-mm cluster formed up to 20 shoots, from which two to three whole plants were regenerated. Regeneration efficiency differed significantly between the two basic media; Gamborg induced high organogenic shoot formation (98–100%) and whole plant regeneration (93%), whereas Murashige and Skoog media showed lower and inconsistent organogenic shoot formation (15–73%) and whole plant regeneration (29%). The protocol that included Gamborg media show high regeneration efficiency across different bean genotypes, resulting in whole plants comparable to seed-produced plants.     

Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants. Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO₃). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from parthenogenetic melons.     

Efficient plant regeneration from protoplasts of <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis> via organogenesis

A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 × 10⁵ protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial density of 1 × 10⁵ protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 μM 2,4-dichlorophenoxy acetic acid (2,4-D), 5.4 μM 1-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzyladenine (BA). Cell wall regeneration was observed within 4 days of culture and cell division began after 5–7 days. When cultured in a liquid medium with 5.4 μM NAA and 8.9 μM BA, protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 μM indole-3-acetic acid (IAA) and successfully established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions found gave a plant regeneration efficiency of 6.4 plants per 1 × 10⁵ protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.     

Development of an efficient plant regeneration system for Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>)

Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants.     

Plant regeneration from carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) anther culture derived embryos

The research concerned of the regeneration of plants from embryos obtained from anther cultures of seven carrot (Daucus carota L.) cultivars. The aim was to determine the influence of the regeneration medium on the efficiency of the regeneration process. The optimization of the adaptation of the obtained plants was also carried out. Embryogenesis occurred on four of the tested media: B5 and MS without hormones, MS with charcoal, and MS with 1 mg dm⁻³ BA and 0.001 mg dm⁻³ NAA. Embryos obtained from the anther cultures produced secondary embryos, from which the regenerations of plants was observed. Secondary embryos were formed most extensively on the B₅ medium without hormones. The efficiency of the regeneration process depended on the cultivar. Most of the secondary embryos were formed by androgenetic embryos of the cultivar ‘Feria F₁’. The highest number of plants (102) regenerated from one embryo during 12 weeks of culture was also obtained in case of the cultivar ‘Feria F₁’. Secondary embryogenesis and plant regeneration from embryos allow to omit the difficult stage of root induction applied when plants are regenerated form shoots' explants. This makes the plant regeneration process quicker and easier. The plants regenerated by the conversion of embryos are better adapted to the ex vitro conditions than those obtained in the two-stage organogenesis involving the regeneration of shoots and in second stage roots induction.     

Haploid plant regeneration from unpollinated ovule cultures of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.)

Haploid plants were regenerated in vitro from unpollinated ovules of niger (Guizotia abyssinica (L. f.) (Cass.) on Murashige and Skoog nutrient medium (MS) supplemented with 10 μM naphthaleneacetic acid or 10 μM NAA + 1.5 μM kinetin and 30 g/l sucrose. Gamborg (B5) medium was the best for plant regeneration (in comparison with MS, Nitsch and Nitsch (NN), and Chu (N6) media) from cultured ovules, and 6.66 and 7.33 ovules of JNC-6 and Ootacamund cultivars were involved in direct plant regeneration on this medium. Matured ovules (ovules collected one day before anthesis or on the day of anthesis) only responded to cultural regimes and involved in direct plantlet development. Cytological preparation of root tips and chloroplast counts in the guard cells of leaf stomata of regenerated plants confirmed their haploid nature. This text was submitted by the authors in English.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

Selection of ethionine-resistant variants with increased accumulation of methionine from embryogenic protoplasts of the forage legume <Emphasis Type="Italic">Astragalus adsurgens</Emphasis>

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7–8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5–9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.     

Improved <Emphasis Type="Italic"> Agrobacterium </Emphasis>-mediated transformation of sunflower (<Emphasis Type="Italic"> Helianthus annuus </Emphasis>L.): assessment of macerating enzymes and sonication

Agrobacterium -mediated transformation of shoot apices of sunflower (Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (beta-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold. The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment. Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation. Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene. Regenerated plants were fertile and seeds could be harvested.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

Protoplast isolation and plant regeneration of different genotypes of <Emphasis Type="Italic">Petunia</Emphasis> and <Emphasis Type="Italic">Calibrachoa</Emphasis>

Protoplasts were isolated from leaves of in vitro-grown shoot cultures of different Petunia and Calibrachoa genotypes by enzyme digestion with 0.6% macerozyme R-10 and 2.0% cellulase R-10. Shoot regeneration was achieved in five out of nine Calibrachoa and three out of four Petunia genotypes. Protoplast yield and frequency of shoot regeneration varied among experiments and genotypes. Among all regenerants, few morphological changes, such as chlorophyll deficiency, variegated leaves, and polyploidization, were observed.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Identification of highly regenerative plants within sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines for molecular breeding

Summary Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration response, line FC607 also had the highest mean number (8.3±1.1) of shoots per explant. Individual plants within each line exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs.     

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.