Showing papers in "Plant Cell Tissue and Organ Culture in 1994"

An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue

Abstract: Cell viability or cell death is an important variable to monitor in many studies of host/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cell's temporary metabolic state rather than irreversible cell death. Evans blue has proven over the years to be a dependable stain for microscopic determination of cell death. We have used this stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells. This spectrophotometric procedure was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv. glycinea or tobacco/P. syringae pv. syringae. Relative increases in cell death during these interactions in suspension cell systems were measured by both the spectrophotometric and microscopic technique and found to be similar. The spectrophotometric procedure was also adapted for leaf disc assays.

Statistical methods suitable for the analysis of plant tissue culture data

Abstract: Statistical analyses are an essential part of biological research. Statistical methods are available to biological researchers that range from very simple to extremely complex. Therefore, caution should be used when selecting a statistical method. When possible it is best to avoid complicated statistical procedures that are difficult to interpret and may hinder the researcher's ability to make treatment comparisons. Instead a method should be chosen that compliments a logical and practical treatment design. Statistics should be used as a tool to compare treatments of interest and should not dictate the treatments. Experimental designs should take into account the eventual analysis, otherwise one could conceive of a design that could not be analyzed or, when analyzed, would not answer the desired questions. Therefore, time should be spent before conducting an experiment to plan an experimental design and analysis that best compliments the treatment scheme and questions to be answered. The purpose of this paper is to present examples of experimental designs, means separation procedures, data transformations and presentation methods suitable for plant cell and tissue culture data.

Factors that effect leaf regeneration efficiency in apple, and effect of antibiotics in morphogenesis

Abstract: Several factors that affect the frequency of organogenesis in apple leaf explants were examined for the scion cultivars ‘Empire’, ‘Freedom’, ‘Golden Delicious’, ‘Liberty’, ‘McIntosh’, and ‘Mutsu’ and for the rootstocks Malling 7A and Malling 26. The main factors affecting morphogenesis were BA concentration, basal medium, leaf explant origin and maturity, explant orientation, and photosynthetic photon flux. Depending on the genotype, optimal regeneration was obtained using either 22.2 or 31.1 μM BA and the N6 basal medium, with the exception of ‘Golden Delicious’ which regenerated better on MS medium. After 6 weeks, the average number of shoots per segment varied from 5 to 16, and the percentage of regeneration between 70 and 100%, depending on the genotype tested and the maturity of the explant. Regeneration capacity increased dramatically from the tip towards the base of the leaf, and was higher from the middle to the proximal end.

Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.)

Abstract: To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.

Micropropagation of safed musli (Chlorophytum borivilianum), a rare Indian medicinal herb

Abstract: In vitro clonal multiplication of safed musli (Chlorophytum borivilianum Sant. et. Fernand.), a rare Indian medicinal herb, has been achieved on Murashige and Skoog's (MS) medium supplemented with 22.2 μM benzyladenine using young shoot bases as explants. Shoots multiplied at a rate of four-fold every 3 weeks. All shoots rooted when transferred to MS medium with 3/4-strength inorganic and organic constituents and 9.8 μM indolebutyric acid and 67% of the micropropagated plants were successfully established in pots. Such plants produced normal fasciculated storage roots as in wild plants.

Effects of sucrose on photosynthesis and phosphoenolpyruvate carboxylase activity of in vitro cultured strawberry plantlets

Abstract: Photosynthesis and phosphoenolpyruvate carboxylase activity were investigated in 5, 10 and 28 day-old micropropagated strawberry plantlets (Fragaria x ananassa Duch. cv Kent) rooted in vitro with different levels of sucrose (0, 1, 3 and 5%) on cellulose plugs (Sorbarods). The photosynthetic capability was influenced by the level of sucrose in the culture medium with the largest rates of photosynthesis corresponding to the cultures with 0 and 1% sucrose. The apparent quantum yield and the ratio of variable fluorescence to maximum fluorescence were also reduced in plantlets cultured with 3 or 5% sucrose as compared to those with 0 or 1%. Phosphoenolpyruvate carboxylase activity was largest 5 and 10 days after the onset of culture and decreased in the absence of sucrose in the culture medium. At 28 days after the onset of culture, the activity of this carboxylating enzyme was lower than at the beginning of culture and independent of the concentration of sucrose in the culture medium. Phosphoenolpyruvate carboxylase appears to be an important carboxylating enzyme in micropropagated plantlets.

Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages

Abstract: The total duration of the plant regeneration process from cucumber (Cucumis sativus L.) cotyledonary explants was only six weeks, which included the induction of buds and their development into plants. Regeneration of shoots from cotyledons from three to five day-old seedlings ranged up to 100%. The regenerated plants were morphologically normal, flowered and set seed. The regeneration capacity of cotyledons from seven days-old and older seedlings was lowered dramatically. Most of those regenerated plants were polyploid/mixoploid and had an abnormal morphology. During seedling development (3 to 13 days), the DNA content of cotyledonary cells changed from 2C to 4C and more. The results show that the decrease in regeneration competence correspond with the change in DNA content of the cotyledonary cells.

Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae)

Abstract: Phalaenopsis or Doritaenopsis (Orchidaceae) flower stem sections cultivated in vitro on media containing 0.23–11.35 μM of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ) developed generally multiple shoots with the higher levels producing also protocorm-like bodies (PLB). Shoot and root development were reduced while proliferation increased with increasing concentration of TDZ. Similar effects were observed with Phalaenopsis protocorms at a lower range (0.23–1.14 μM) of TDZ.

An improved method for somatic plantlet production in hybrid larch (Larix × leptoeuropaea): Part 1. Somatic embryo maturation

Abstract: Somatic embryogenesis was induced from full-sib immature zygotic embryos of hybrid larch (Larix x leptoeuropaea) that were collected at three different dates. Analysis of variance showed interaction between the collection date and the induction medium. The highest response (55%) was observed from embryos that were at the precotyledonary stage. Twelve media containing various concentrations of abscisic acid and sucrose were used to promote the development of ‘high quality’ mature somatic embryos that would undergo a period of developmental arrest. Only media supplemented with abscisic acid (20, 40, and 60 μM), indolebutyric acid (1 μM), and 0.1 or 0.2 M sucrose supported such a development. The number of mature somatic embryos produced per gram fresh weight of embryonal mass was significantly affected by the three factors tested: embryogenic line, sucrose concentration, and abscisic acid concentration. Moreover, strong interaction effects among these factors existed, complicating the formulation of a universal maturation medium that would be optimal for all embryogenic lines.

Importance of the iron chelate formula for micropropagation of Rosa hybrida L. ‘Moneyway’

Abstract: In vitro propagation of the rose rootstock ‘Moneyway’ was investigated on the following media: Murashige and Skoog (MS), Quoirin and Lepoivre (QL) and Woody Plant (WP). Growth, which was measured as length of shoots after a 6-week period, was faster on MS and QL than on WP. In spite of the better growth, chlorosis of newly formed leaves occurred from the third week on and was correlated with a lower chlorophyll content of shoots. Replacement of FeEDTA by FeEDDHA in QL and MS resulted in the development of green shoots for more than 3 months. The occurrence of chlorosis was not pH directed since the pH of QL with FeEDTA or FeEDDHA had not changed after 6 weeks of growth. Addition of the light absorbing dye fast yellow 9 to QL with FeEDTA also resulted in green shoots with a higher chlorophyll content. It is suggested that FeEDDHA is a more photostable chelate than FeEDTA, resulting in a higher availability of iron for the rose shoots. The impact of the iron chelate formula on the micropropagation of plant species that are susceptible to iron deficiency is discussed.

Tissue culture studies on species of Passiflora

Abstract: The effect of different explant sources, growth regulators and coconut water concentrations and also light radiation on tissue cultures ofPassiflora edulis var.flavicarpa was evaluated.

The types of bioreactors used for shoots and embryos

Abstract: Plant regenerated organs such as shoots, bulbs, microtubers, corms, embryos, etc. have been successfully proliferated in the bioreactor. The use of a bioreactor leads to the development of technology suitable for large scale plant propagation. The basic construction and characteristics of various types of bioreactor systems are reviewed in relation to shoot and embryo cultures. A pilot scale 500 liter bioreactor system was applied to the production of large scale Stevia rebaudiana shoots.

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Abstract: Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g-1 DW, or 7.0 and 3.0 mg l-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro- and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.

Comparison of selective agents for use with the selectable marker gene bar in maize transformation

Abstract: The effectiveness of four phosphinothricin (PPT)-based selective agents were evaluated for use in maize transformation: glufosinate, bialaphos, Basta® and Herbiace®. Glufosinate and its commercial formulation, Basta®, were less effective in controlling growth of non-transgenic corn callus than the tripeptide, bialaphos, or its commercial formulation, Herbiace®. Addition of 25 mM l-proline had no significant effect on selection when using bialaphos. However, when l-proline was included with the selective agent glufosinate, selection was inhibited and callus growth was enhanced. At four weeks, callus growth on 0.3, 1.0 and 3.0 mg l-1 glufosinate in the presence of proline was 76, 43, and 21% of control growth, respectively, and in the absence of proline was only 32, 9, and 6% of control growth. Optimized selection protocols for Basta® and bialaphos yielded comparable numbers of transformants. Using these protocols, fertile transgenic plants were regenerated from transformed callus cultures.

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures - Medium optimization and elicitation

Abstract: Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g-1 DW, or 7.0 and 3.0 mg l-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.

Genetic transformation of the apple scion cultivar ‘Delicious’ viaAgrobacterium tumefaciens

Abstract: Transformed shoots of the major apple scion cultivar ‘Delicious’ (Malus × domestica Borkh.) were obtained by cocultivation withAgrobacterium tumefaciens carrying disarmed plasmids. The transformation efficiency was influenced by the type of plasmid and by the inoculation temperature. Initial selection involved a callus stage followed by shoot regeneration. Shoot regeneration occurred only in the dark. Shoots grew in the light and were rooted in the presence of 100 mg l−1 kanamycin. Of the range of plasmids tested, the cointegrates pGV 3850::1103neo and pGV 3850::1103gus gave a higher frequency of transformation than the binary vector pGV 3111 × pKIWI. Elongation of transformed shoots was enhanced by culture in a mixture of the cytokinins 6(γ-γ-dimethylallylamino)purine and 6-benzyladenine. Up to 60% of the elongated shoots rooted in 100 mg l−1 kanamycin. Transformation was indicated by kanamycin resistance, β-glucuronidase assay, nopaline synthesis, and by integration of the T-DNA as judged by Southern analysis.

Synthetic seeds as an application of mass production of somatic embryos

Abstract: Production of encapsulatable unit, scale-up of the production system, and encapsulation technologies were developed as an integrated technology for transplant production using somatic embryos of celery and carrot. Encapsulatable units, somatic embryos improved in their quality for encapsulation, showed 80% conversion without encapsulation and any nutrient supply on a nursery medium in a greenhouse. The high conversion ability was also shown by carrot encapsulatable unit produced with a newly developed 8-liter culture jar, where 5.09×105 torpedo stage embryos were produced and 4.10×104 encapsulatable units were obtained from them.

Influence of auxin type and concentration on peanut somatic embryogenesis

Abstract: Somatic embryogenesis in peanut (Arachis hypogaea L) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l−1) and naphthaleneacetic acid (NAA) (20 to 50 mg l−1) levels Percent embryogenesis ranged from 31 to 94% As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants However, with higher 2,4-D induction levels (40 mg l−1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation

In vitro clonal propagation of Capsicum spp.

Abstract: The effect of various concentrations of benzyladenine (BA 4.4–177.5 μM) or kinetin (4.7–185.9 μM) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 μM BA or 92.9 μM kinetin in C. praetermissum, and 88.8 μM BA or 116.2 μM kinetin in C. annuum after 4 weeks of culture. Combining 1 μM 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 μM indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.

Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures

Abstract: Transformed organ cultures formed following transformation of plant tissues with Agrobacterium species owe their phenotypes to alterations in hormone metabolism. Exogenously supplied hormones have been used to probe the relationship between the growth and morphology of transformed root cultures of a number of species and their ability to accumulate secondary products. Auxins in the presence of low levels of kinetin induce the rapid disorganisation of transformed roots of Nicotiana rustica ultimately toform suspension cultures of transformed cells and this process is associated with a decrease in nicotine content of the cells. This is related to cells in the culture losing competence in alkaloid biosynthesis. In contrast, exogenously supplied GA3 enhanced branching in two transformed root clones of the tropane-alkaloid producing species, Brugmansia candida and so enhanced their typical “hairy root” phenotype. This growth substance had the effect of reducing the overall alkaloid accumulation but in one case significantly altered the relative concentrations of different tropine esters.

Artemisia annua L.: dedifferentiated and differentiated cultures

Abstract: Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l-1), myoinositol (100 mg l-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 μM : μ0.02 d-1) and NAA (5.4 μM : μ 0.06 d-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 μM) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 μM)+NAA (0.54 μM); Zeatin (45.62 μM)+NAA (5.37 μM) or BA (8.9 μM) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 μM)+NAA (1.08 μM) or BA (13.32 μM) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.

Foreed flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

Abstract: The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70–300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 μM of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.

Constitutive and elicitation induced metabolism of isoflavones and pterocarpans in chickpea (Cicer arietinum) cell suspension cultures

Abstract: Constitutive phenolics of chickpea cell suspension cultures are the isoflavones formononetin and biochanin A, the isoflavanones homoferreirin and cicerin and the pterocarpans medicarpin and maackiain. They accumulate as vacuolar malonylglucosides. The biosynthetic pathways to isoflavones, pterocarpans and malonylglucoside conjugates together with their enzymes are explained. Elicitation of cell cultures leads to pronounced increases in the activities of biosynthetic enzymes with differential effects on the enzymes involved in conjugate metabolism. Low elicitor doses favour pterocarpan conjugate formation whereas high doses lead to pterocarpan aglycone accumulation accompanied by vacuolar efflux of formononetin and pterocarpan malonylglucosides. Elicitor-induced changes in enzyme activities and vacuolar efflux of conjugates are prevented by application of 10-3M concentrations of cinnamic acid. Cinnamate is alternatively metabolized to a glucose ester, a S-glutathionyl conjugate and to cell wall bounds forms; these reactions are intensified by elicitation. Isoflavone and pterocarpan biosynthesis and conjugate metabolism as regulated by elicitation and cinnamate is depicted in a metabolic grid to explain the complex regulatory pattern of phenolic accumulation in chickpea cell cultures.

The methylation status of DNA derived from potato plants recovered from slow growth

Abstract: The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.

Expression of human α-interferon cDNA in transgenic rice plants

Abstract: The plasmid pIG3031 containing human α-interferon cDNA and the neomycin phosphotransferase II coding sequence was successfully transferred into rice protoplasts (Indica type rice) by Lipofectinmediated transformation. Assays for NPT II enzyme activity indicated that the transformation frequency was 10%. Transgenic plants were regenerated from transformed calli. Southern blots showed that the human α-interferon cDNA sequence was present in rice DNA. RNA slot blots indicated apparent transcription of human α-interferon cDNA under the control of the plant-active promoter PI'. Extracts of transgenic cell cultures and plants contained apparent interferon activity as measured by resistance of a human amniotic cell line to viral infection in the presence of plant extracts. These studies demonstrate that human α-interferon cDNA may be correctly expressed in rice cells.

An evaluation of antibiotics for the elimination ofXanthomonas campestris pv.pelargonii (Brown) fromPelargonium x domesticum cv. ‘Grand Slam’ explants in vitro

Abstract: A range of antibiotics was evaluated for activity againstXanthomonas campestris pv.pelargonii (Xep) on diagnostic sensitivity testing and plant tissue culture media. Many of the antibiotics showed reduced or no activity on the latter. Tetracycline and cefotaxime, chosen for further investigation, were screened for light stability under plant culture regimes. Tetracycline was inactivated in photosynthetic photon fluxes of 22 μmol m-2 s-1 and above. The minimum bacteriocidal concentration of cefotaxime was determined in bacteriological and plant tissue culture media. Cefotaxime was further tested for phytoxicity and ability to eliminate Xcp from deliberately infected explants. Cefotaxime was shown to eliminate contamination and stimulate the growth of the plant tissue cultures up 500 mg l-1.

Effect of induction medium pH and maltose concentration on in vitro androgenesis of hexaploid winter triticale and wheat

Abstract: Low levels of in vitro androgenesis limit the utility of anther culture as a routine tool for the improvement of triticale. The objectives of this research were to determine the effect of induction medium modifications on the embryoid induction (EI) and green plant regeneration (GPR) of three hexaploid winter triticales and a hexaploid winter wheat. Medium modifications were factorial combinations of gelling agents, pH and maltose concentration. There was a significant difference in the response of wheat and triticales. An induction medium pH of 4.8 vs. 5.8 led to higher EI percentages in all cases and in Ficoll containing medium to a significantly higher GPR in the wheat, but it had no effect on the response of the triticales. Maltose at 0.26 M vs. 0.21 M increased the EI of both the triticales and the wheat but led to higher GPR only in the case of wheat. Averaged over genotypes, induction media with agar and Ficoll 400 were superior to the liquid form. In terms of GPR the wheat gave the highest response: 9.1%. The GPR percentages for the triticales ranged from 1.4 to 7.1. Genotype specificity must be overcome if anther culture is to be routinely used in diverse arrays of triticale germplasm.

Microtuberization of layered shoots and nodal cuttings of potato : The influence of growth regulators and incubation periods

Abstract: A protocol is presented for the rapid induction of microtubers on micropropagated, layered potato shoots of ‘Kennebec’, ‘Russet Burbank’ and ‘Superior’ in medium devoid of growth regulators. Layered shoots microtuberized more rapidly and produced significantly larger microtubers compared with nodal cuttings. The addition of coumarin or (2-chloroethyl)-trimethylammonium chloride and benzyladenine to microtuberization medium, either had no effect or significantly reduced microtuber weight per shoots compared with medium containing only 80 g × 1-1 sucrose and minimally affected the number of microtubers per shoot. Increasing the incubation period from 28 to 56 days did not affect the number but significantly increased the weight of microtubers per shoot and substantially increased the proportion, up to 20%, of microtubers heavier than 1 gram.

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

Abstract: A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata cv. ‘Crandon’) suspension cultures and regeneration of transformed plants is described. Transformants were recovered when suspension cultures were inoculated with Agrobacterium tumefaciens at a density of 107 colony-forming units ml-1, cocultivated for 48 h, and plated to cellulose acetate filters on Woody Plant Medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 250 mg l-1 cefotaxime. Levels of cefotaxime greater than 250 mg l-1 were unnecessary for control of residual bacteria and inhibited callus growth. Transgenic plants were regenerated by culturing the transformed callus on media containing 0.11 to 27 μM thidiazuron. In contrast to thidiazuron, N6-benzyladenine had a negative effect on shoot regeneration; the callus became necrotic when we attempted to induce shoots with concentrations of 1.1 to 8.9 μM, and growth was inhibited when concentrations of 0.11 or 0.22 μM were used to regenerate callus from suspension cultures. Following cocultivation of poplar suspension cultures, we recovered transgenic plants containing the maize transposon Ac, and callus containing an insect toxin gene from Bacillus thuringiensis.

The effects of silver nitrate, colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat ( Triticum turgidum )

Abstract: Anther culture of four tetraploid wheat (Triticum turgidum) genotypes was studied using ten different culture medium treatments in a randomized block design with four replicates. Each replicate consisted of 2 pots with 3 plants. Anther donor plants were grown in a greenhouse with a 16 h day/8 h night at 25°C and 15°C, respectively. The first treatment which was considered as the control, was potato 2 medium modified by adding 0.5 g l−1 glutamine and solidified by gelrite (4 g l−1). The nine test treatments differed from the control by addition of 3 different concentrations of silver nitrate (1, 2.5 and 5 mg l−1), colchicine (10, 100 and 200 mg l−1) or cupric sulfate (2,5 and 10 mg l−1). The study of about 2000 anthers per genotype and treatment showed that both genotype and treatment affected embryoid formation. The presence of cupric sulfate (10 mg l−1) and silver nitrate (2.5 and 5 mg l−1) usually increased the frequency of embryoid formation in 3 genotypes out of the 4 studied. On the contrary, colchicine had a significant and enegative effect on anther culture responses for three out of the four genotypes studied. Because of the large genotype x medium interaction, it is very difficult to identify the best medium for embryo production by all genotypes studied.