Cultured Leaf Discs Research Articles

Pesticidal activity of extra cellular metabolites extracted from the green muscardian fungi Metarhizium anisopliae

AbstractThe present study was undertaken to screen the pesticidal activity of extra cellular metabolites derived from entomopathogenic fungus Metarhizium anisopliae against Spodoptera litura and Aphis crassivora an important aphid pest under laboratory and pot culture condition. Fungal strain used in the study was isolated from the soil by serial dilution technique. Isolated fungal strain was mass multiplied in a liquid medium sabouraud maltose yeast extract broth under optimum condition (28°C). Culture filtrate and the collected culture derived from the medium was used for the source of metabolites. The culture filtrate was extracted with ethyl acetate, acetone and the concentrated extract was screened for pesticidal activity against Spodoptera litura and Aphis crassivora adopting leaf disc and pot culture assay respectively. Extracted metabolites showed pesticidal activity against both insect pests. However, notable effect was recorded in ethyl acetate derived metabolites. The ethyl acetate extract treatment recorded 91.2% mortality at a concentration of 100 μg against Spodoptera litura and 100% mortality at a concentration of 100 μg against Aphis crassivora. Phytotoxicity was determined by measuring the germination index (GI) of groundnut seeds by exposing it to the crude metabolites extracted in ethyl acetate and acetone. The results showed that the germination index was high on ethyl acetate extract which was recorded to be 98.0% and germination index of acetone extract was found to be 90.0%. Production of insecticidal metabolites from M. anisopliae under laboratory condition proposed the possible utilization of metabolites as an effective, safe pesticidal agent against economic important insect pests. No impact on the seedling emergence revealed non phytotoxic effect of the metabolites.

An Efficient Agrobacterium-Mediated Genetic Transformation System for Persimmon (Diospyros kaki Thunb.)

Persimmon (Diospyros kaki Thunb.) is an important fruit crop in East Asia and has great commercial potential due to the high demand for fresh edible fruits or processed dried persimmon, as well as its being a source of raw materials for the chemical industry. Genetic engineering has shown great potential in persimmon fruit improvement and compensated for the limitations of conventional breeding. In this study, we developed an efficient transformation system and in vivo regeneration protocol by using the leaf disc culture of ‘Gongcheng Shuishi’ persimmon through the optimization of different basic media and hormone combinations. Based on the secondary induction system on 1/2DKW (DKW medium) + ZT (2.0 mg/L) + TDZ (0.5 mg/L) and MS (1/2 N) + ZT (2.0 mg/L) + IAA (0.1 mg/L), the callus induction rate and adventitious bud induction rate were 96.0% and 94.0%, respectively, and RNA interference of DkANR (ANR, anthocyanidin reductase) was achieved after transformation, thus resulting in a reduction in proanthocyanidin accumulation. The GFP (green fluorescent protein) fluorescence signal was visualized in the transgenic seedlings with GFP gene cotransformation. A total of 17.9% of the positive regenerated transgenic seedlings with empty vector were confirmed by both hyg gene and 35S promoter PCR analysis. Southern blotting assays showed that 20 positive transgenic persimmon seedlings were obtained from 135 regenerated transformants derived from DkANRi. Altogether, a reliable and highly efficient transformation system was achieved in persimmon, which may contribute to gene function identification and genetic improvement in the future.

Phytotoxic Responses of Soybean (Glycine max L.) to Botryodiplodin, a Toxin Produced by the Charcoal Rot Disease Fungus, Macrophomina phaseolina.

Toxins have been proposed to facilitate fungal root infection by creating regions of readily-penetrated necrotic tissue when applied externally to intact roots. Isolates of the charcoal rot disease fungus, Macrophomina phaseolina, from soybean plants in Mississippi produced a phytotoxic toxin, (−)-botryodiplodin, but no detectable phaseolinone, a toxin previously proposed to play a role in the root infection mechanism. This study was undertaken to determine if (−)-botryodiplodin induces toxic responses of the types that could facilitate root infection. (±)-Botryodiplodin prepared by chemical synthesis caused phytotoxic effects identical to those observed with (−)-botryodiplodin preparations from M. phaseolina culture filtrates, consistent with fungus-induced phytotoxicity being due to (−)-botryodiplodin, not phaseolinone or other unknown impurities. Soybean leaf disc cultures of Saline cultivar were more susceptible to (±)-botryodiplodin phytotoxicity than were cultures of two charcoal rot-resistant genotypes, DS97-84-1 and DT97-4290. (±)-Botryodiplodin caused similar phytotoxicity in actively growing duckweed (Lemna pausicostata) plantlet cultures, but at much lower concentrations. In soybean seedlings growing in hydroponic culture, (±)-botryodiplodin added to culture medium inhibited lateral and tap root growth, and caused loss of root caps and normal root tip cellular structure. Thus, botryodiplodin applied externally to undisturbed soybean roots induced phytotoxic responses of types expected to facilitate fungal root infection.

Toxin Production in Soybean (Glycine max L.) Plants with Charcoal Rot Disease and by Macrophomina phaseolina, the Fungus that Causes the Disease.

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, results in major economic losses in soybean production in southern USA. M. phaseolina has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of M. phaseolina isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14–6.11 µg/mL), 37.8% produced traces below the limit of quantification (0.01 µg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 µg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in M. phaseolina culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, M. phaseolina isolates from soybeans with charcoal rot disease in southern USA.

Irradiance-based treatments of Quillaja brasiliensis leaves (A. St.-Hil. & Tul.) Mart. as means to improve immunoadjuvant saponin yield

Quillaja brasiliensis (soap tree) is native of southern Brazil. A purified fraction of saponins from leaves of Q. brasiliensis, QB-90, had significant immunoadjuvant activity in several experimental vaccines. The accumulation of QB-90 in cultured leaf disks and seedlings was previously shown to be responsive to different stresses, including UV-C and higher white light irradiance. An investigation on the regulation of QB-90 accumulation by irradiance treatments in young plants and in harvested leaves of trees of different ages was carried out. Young plants exposed to acute UV-C for 2–4d or to red light enrichment for 30d accumulated significantly more QB-90 in leaves than control plants. Red light treatment of plants did not increase carbohydrate content, indicating that its effects on saponin yield were not due to photosynthesis changes. The influences of irradiance treatments on QB-90 content of leaves from juvenile and adult trees, as well as the impact of temperature on saponin content of adult leaves, were investigated during post-harvest periods. QB-90 yield of adult leaves was not affected by various temperature treatments. A significant increase in the concentration of QB-90 both in juvenile and adult leaves was observed after exposure to UV-C radiation for 15min. Exposing leaves to visible irradiance enriched in red color was effective in increasing QB-90 yield after 48h of treatment in adult leaves and after 12h in juvenile leaves. QB-90 content in detached leaves was relatively stable over a 10d dark period, indicating that results of exposure to UV-C and red irradiance were a direct consequence of metabolic elicitation of bioactive saponins in leaves. Out of the best results of each light treatment on detached juvenile leaves, UV-C proved to be the most efficient to increase QB-90 content. Irradiance-based treatments can be a valuable tool to improve yields of immunoadjuvant saponins in soap tree.

A Novel System for Xylem Cell Differentiation in Arabidopsis thaliana

During vascular development, procambial and cambial cells give rise to xylem and phloem cells. Because the vascular tissue is deeply embedded, it has been difficult to analyze the processes of vascular development in detail. Here, we establish a novel in vitro experimental system in which vascular development is induced in Arabidopsis thaliana leaf-disk cultures using bikinin, an inhibitor of glycogen synthase kinase 3 proteins. Transcriptome analysis reveals that mesophyll cells in leaf disks synchronously turn into procambial cells and then differentiate into tracheary elements. Leaf-disk cultures from plants expressing the procambial cell markers TDRpro:GUS and TDRpro:YFP can be used for spatiotemporal visualization of procambial cell formation. Further analysis with the tdr mutant and TDIF (tracheary element differentiation inhibitory factor) indicates that the key signaling TDIF-TDR-GSK3s regulates xylem differentiation in leaf-disk cultures. This new culture system can be combined with analysis using the rich material resources for Arabidopsis including cell-marker lines and mutants, thus offering a powerful tool for analyzing xylem cell differentiation.

A novel system for xylem cell differentiation in Arabidopsis thaliana.

During vascular development, procambial and cambial cells give rise to xylem and phloem cells. Because the vascular tissue is deeply embedded, it has been difficult to analyze the processes of vascular developmen in detail. Here, we establish a novel in vitro experimental system, in which vascular development is induced in Arabidopsis thaliana leaf-disk cultures using bikinin, an inhibitor of glycogen synthase kinase 3 proteins. Transcriptome analysis reveals that mesophyll cells in leaf disks synchronously turn into procambial cells and then differentiate into tracheary elements. Leaf-disk cultures from plants expressing the procambial cell markers TDRpro:GUS and TDRpro:YFP can be used for spatiotemporal visualization of procambial cell formation. Further analysis with the tdr mutant and TDIF indicates that the key signaling TDIF- TDR-GSK3s regulates xylem differentiation in leaf-disk cultures. This new culture system can be combined with the analysis using the rich Arabidopsis material resources including cell-marker lines and mutants, thus offering a powerful tool for analyzing xylem cell differentiation.

In vitro culture and fructan production by Vernonia herbacea (Asteraceae)

Vernonia herbacea is a native species from the Brazilian Cerrado that accumulates about 80 % of inulin-type fructans in the underground reserve organs, the rhizophores. This work aimed at establishing a protocol for in vitro culture of V. herbacea, using seeds (achenes) and leaf discs as explants. Following germination and seedling growth, stem nodes from 6-month-old in vitro germinated plants were isolated and incubated on culture medium free of growth regulators for plant propagation and rhizophore formation. Fructan content and composition were evaluated in leaves, stems, roots and rhizophores from plants grown in vitro and compared with those of greenhouse-grown plants, in order to evaluate inulin production in vitro. Fructan contents of aerial organs and roots from in vitro plants were higher, compared with greenhouse plants, while in rhizophores, the opposite was observed. High performance anion exchange chromatography/pulsed amperometric detection profiles revealed the presence of the inulin homologous series in the aerial organs exclusively for in vitro plants, while in roots and rhizophores, this series was detected in plants grown in both conditions. These results indicate a modification in the source/sink ratio, leading to changes in the distribution of carbohydrates in in vitro plants. The leaf disc cultures on medium supplemented with indole-3-butyric acid induced the formation of roots (0.24, 0.49 µM) and friable callus (2.46 µM), while 6-benzylaminopurine (from 1.1 through 4.43 µM) induced compact callus. However, no shoot formation was observed. The use of seeds allowed the establishment of a protocol for in vitro culture and provides a model system for a better understanding of fructan metabolism in V. herbacea.

Efficient In vitro Plant Regeneration Protocol from Leaf Explant of Jatropha curcas L — A Promising Biofuel Plant

An efficient in vitro plant regeneration from leaf-disc culture of Jatropha curcas L has been established. Adventitious shoot buds along with callus were induced from leaves of 2-year-old J. curcas plants cultured on Murashige and Skoog’s (MS) medium supplemented with TDZ (2 μM) BAP (2 μM) and IBA (1 μM), wherein 63.3% leaf explants responded. The multiplication of shoots was achieved from the adventitious shoot buds after transferring them to shoot induction medium. The highest number of shoots (9.7/explant) was achieved after 6 weeks of culture on MS medium containing 3 μM of BAR The welldeveloped shoots were rooted on MS medium supplemented with IBA (1.5 μM) with the rooting frequency of 53.3%. Addition of phloroglucinol (200 μM) to the medium enhanced the frequency of rooting to 76.7%. Regenerated plantlets were successfully transferred to field after initial acclimatization.

Brassinolide influences the regeneration of adventitious shoots from cultured leaf discs of tobacco

When several concentrations of brassinolide (BL) were added to a shoot induction medium (SIM) that contained only BA, redifferentiation of adventitious shoots from tobacco leaf discs was unaffected at low BL levels (10-10~10-8 M), but was inhibited at higher concentrations. In comparison, when BL was applied without BA, only cell expansion occurred and no shoots formed. The determination time for shoot formation was shortened at low BL concentrations, but their formation was postponed (i.e., time was lengthened) at higher concentrations. Elongation of shoots incubated for 30 d was unaffected at low BL concentrations, but was inhibited as that amount increased.NTH1, a tobacco homeobox gene that is expressed in the central zone of the tobacco shoot apex, showed greater expression levels in the SIM over time, and its expression was stronger in media treated with low concentrations of BL compared with the SIM control at the same time point. Expression ofNTH1 was postponed at higher BL concentrations. In conclusion, at low concentrations, brassinolide has no effect on shoot formation. However, it inhibits their formation at high concentrations when cytokinin is included in the media.

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA3) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.

Paraquat Resistance in Leaf Discs of P SAG12- IPT Modified Gerbera Is Related to the Activities of Superoxide Dismutase, Catalase, and Dehydroascorbate Reductase

In this paper, using in vitro leaf disc culture system, the changes of contents of chlorophylls, carotenoids, soluble protein, thiobarbituric acid reactive substance (TBARS), and activities of antioxidant enzymes were investigated during the incubation of leaf discs of P SAG12- IPT modified gerbera in 0, 25, 50 μmol L −1 paraquat (PQ) under continuous light intensity of 130 μmol m −2 S −1, compared with the control plant (wild type). The results showed that PQ treatment significantly decreased the contents of chlorophylls, carotenoids, and soluble protein, therefore, promoted leaf senescence. However, the decreases in the leaf discs of modified gerbera were considerably smaller. The activities of superoxide dismutase (SOD), catalase (CAT), and dehydroascorbate reductase (DHAR) were significantly increased by PQ treatment and with the increasing of PQ concentration, particularly in the modified plants. The activities of ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) could not be detected in the leaf discs of PQ treatments, which suggested that they were labile to the oxidative stress induced by PQ. As a product of lipid peroxidation, TBARS significantly increased in content with the increase of PQ concentration, while its concentration in the modified plants was significantly lower than that of control plants. Therefore, it could be concluded that the chimeric gene P SAG12- IPT transformed gerbera leaves had higher antioxidative potential, thus causing the delay of senescence under oxidative stress induced by PQ.

Involvement of a tobacco leucine-rich repeat-extensin in cell morphogenesis

The unique mutant nolac-K4 (non-organogenic callus with loosely attached cells), which was generated by T- DNA transformation using leaf-disk cultures of haploid Nicotiana plumbaginifolia, has lost the ability to form adventitious shoots and also shows decreased intercellular attachment. The gene tagged with the T-DNA in this line, named NpLRX1 (LRR-EXTENSIN 1), is a novel tobacco gene that encodes a cell-wall protein containing chimeric leucine-rich repeat and extensin domains. The gene is highly similar to the Arabidopsis LRX genes, and phylogenetic analysis places it in the vegetative LRX clade. NpLRX1 is expressed ubiquitously in vegetative tissues, strongly in the leaf and root vascular bundles, and in emerging lateral roots and root tips. Tobacco leaf disks transformed with an NpLRX1-RNAi construct displayed aberrant adventitious buds and disorganized cell morphogenesis with large intercellular spaces. The shapes of NpLRX1- RNAi-transformed BY-2 cells were irregular, and the cells showed disorganized cortical microtubules. These results suggest that NpLRX1, the lack of which may be responsible for the nolac-K4 mutation, is a new tobacco LRX gene that has regulatory roles in cell morphogenesis that are essential for plant tissue development.

(242) Callus Induction and Regeneration in Hydrangea macrophylla (Thunb.) Ser.

Development of a reliable method for Hydrangea macrophylla (Thunb.) Ser. organogenesis is critical for developing an in-vitro mutagenesis protocol. Container-grown (11.8 L) H. macrophylla `Nikko Blue' plants were maintained in a controlled environment greenhouse, with supplemental lighting (1600 hr to 2400 hr mercury vapor lamp), fertilized with 65 g Nutricote total (18N–2.6P–6.6K, Agrivert, Inc., New York, N.Y.) and hand-watered. To reduce fungal contamination, stock plants were sprayed to run-off biweekly with Alliette WDG (375 mg·L-1, aluminum tris), Bayleton (250 mg·L-1, triadimefon), and Heritage (25 mg·L-1, azoxystrobin). Leaf explants were sterilized with 0%, 10%, 15%, or 20% bleach (5.25% sodium hypochlorite) (by volume) for 10 or 15 min, and stem explants were sterilized with 0%, 10%, 25%, or 50% bleach (5.25% sodium hypochlorite) for 10 or 15 min. About 97% of fungal contaminates were eliminated from leaf and stem explants when treated with 10% bleach for either 10 or 15 min. Leaves were plated on Gamborg B5 media at pH 5.7 containing 0, 2.5, 5, or 10 μM 2,4-D and 0, 0.25, 0.5, or 1.0 μM BAP and placed in a controlled environment growth room under a 14-h photoperiod or in a dark growth chamber. Callogenesis followed by root organogenesis was observed on explants treated with a variety of concentrations and combinations of 2,4-D and BAP. Strongest callogenesis was observed on media supplemented with 10 μM 2,4-D. A greater callus concentration was observed along the edges of dark cultured leaf discs. Indirect root induction was greatest on 10 μM 2,4-D.

AFLP Analysis and Improved Phytoextraction Capacity of Transgenic gshI-Poplar Clones (Populus x canescens L.) for Copper in vitro

Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10(-1) to 10(-5) M) incubated for 21 days on aseptic tissue culture media WPM containing 1 microM Cu. Zn2+ caused phytotoxicity only at high concentrations (10(-1) to 10(-2) M). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks.

To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differential stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differential phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen.

Production of Non-organogenic and Loosely Attached Callus in Leaf Disk Cultures of Haploid Nicotiana plumbaginifolia by 14N Ion Beam Irradiation.

The leaf disks of Nicotiana plumbaginifolia were irradiated by an 14N heavy-ion beam and then were cultured in shoot-inducing medium in order to produce mutants for elucidating the functions of the cell wall in plant morphogenesis. Non-organogenic and loosely attached callus, which had the same morphological features as non-embryogenic callus of carrot was formed on 11.8% of the haploid leaf disks irradiated by 14N heavy-ion beam (5 Gy) at high frequency. The cells stimulated by the heavy-ion beam loosely attached to each other resulting in a random morphology of cell clusters, whereas the non-irradiated cells formed a tight callus with multiple shoots.

A tobacco homologue of the Ri-plasmid orf13 gene causes cell proliferation in carrot root discs.

The tobacco genome contains genes, called cellular rol (c-rol) genes, that are very similar in sequence to genes present in the T-DNA of the Agrobacterium rhizogenes Ri-plasmid. We have cloned two homologues (torf13-1 and torf13-2) of the Ri-plasmid orf13 gene from Nicotiana tabacum L. cv. Havana 425. The clone torf13-1 has a 594-bp open reading frame (ORF) which is similar in sequence (77-82% for DNA and 67-77% for the deduced amino acid sequence) to orf13 genes of the agropine, mikimopine, and mannopine Ri-plasmids and the N. glauca homologue Ngorf13. Southern analyses showed that there are at least two torf13 genes derived from the N. tomentosiformis ancestor of tobacco, strongly suggesting that torf13 resulted from an ancient transfer between ancestors of modern A. rhizogenes and tobacco. Steady-state expression of torf13 mRNA is high in sepals, petals, shoot tips and in younger leaves, but considerably lower in stem tissues, lower leaves and roots. Treatment of cultured leaf discs for 5-20 days on medium containing auxin (10.7 microM alpha-naphthaleneacetic acid) and cytokinin (1.4 microM kinetin) resulted in a marked down-regulation of torf13 mRNA accumulation. Therefore, torf13 is transcriptionally active in normal tobacco tissues and the steady-state mRNA level is regulated. Inoculation of carrot-root discs with A. tumefaciens strains carrying the mannopine Ri-plasmid orf13 and torf13-1 regulated by the strong cauliflower mosaic virus 35S RNA promoter induced the formation of dense green callus on the disc surface. These findings indicate that at least one function of the orf13 ORF is conserved in the tobacco homologue, and provide direct evidence that a c-rol gene can influence cell proliferation.

Factors affecting shoot organogenesis in leaf disc culture of African violet

Shoot organogenesis in African Violet was affected by factors including leaf age, wounding and leaf disc orientation. A gradient of regenerative potential was found among individual leaves on a plant. This regenerative potential was not directly proportional to the juvenility of leaf tissues. Wound treatment increased shoot organogenesis only when the wound was in direct contact with the medium, possibly by increasing nutrient and hormone uptake. Leaf disc orientation did not affect shoot number per disc but did determine the position of shoot organogenesis. Reduced gas exchange significantly reduced shoot production. This study also showed that adventitious roots originated from spongy mesophyll cells.