Analysis of growth in Sorghum callus cultures and association with a 27 kd peptide

A 27 Kd peptide was produced upon inducing callus formation from excised seedling tissues and immature embryos in vitro of Sorghum bicolor (L.) Moench. Callus formation was always preceded by production of this callus-associated peptide (CAP1), as detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). CAP1 and a second callus-associated peptide, CAP2, are more abundant in callus tissue which has lost the ability to regenerate organ primordia in comparison to organogenic callus. Anti-serum developed to CAP1 was found to be reactive with both CAP1 and CAP2. Concanavalin A (Con A) bound both CAP1 and CAP2, indicating the presence of glycosyl residues associated with these peptides. CAP1 has been detected in crown tissues of whole plants and was quantitatively enhanced in crown tissues following treatment of plants with 2,4-dichlorophenoxyacetic acid (2,4-D). CAP1 was not detected during a sampling of other whole plant tissues. The relationship of callus initiation frequency, callus dry weight and CAP1 accumulation was examined for eight genotypes of sorghum and is discussed.     

Increase of isoflavones in soybean callus by Agrobacterium-mediated transformation

Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition. We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection, transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through an alternative cell culture system to produce high concentrations of isoflavones.     

Transgenic sugarcane plants via microprojectile bombardment

Transgenic sugarcane plants were produced by bombardment of embryogenic callus with high-velocity DNA-coated microprojectiles, followed by a selection and regeneration procedure designed for this target tissue. Optimal bombardment conditions for embryogenic callus required microprojectile velocities higher than those previously found effective for sugarcane suspension culture cells. Bombardment of target tissues twice increased the number of transiently expressing cells in regenerable callus regions, to more than 300 per treated plate. Stable transformants were obtained following bombardment with the neomycin phosphotransferase (npt-II) gene under the control of the Emu strong monocot promoter. Stepped increases in antibiotic concentration during selection and regeneration allowed recovery of actively growing callus and plants on media containing geneticin concentrations completely inhibitory to untransformed controls. NPT-II levels in transformed plants were 20–50 times the background levels in control plants in ELISA assays, and Southern analysis revealed integration of one to three copies of the introduced gene in the sugarcane genome. The procedures described yield one to three transgenic plants per treated plate within 16 weeks of bombardment and provide a simple, efficient and broadly applicable system for genetic transformation of sugarcane. A similar approach should be applicable to other members of the Poaceae able to form embryogenic callus.     

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.     

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolyticaA1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

Alfalfa transformed with a portion of the leukotoxin gene from Mannheimia haemolytica was produced to test the feasibility of developing an edible vaccine capable of protecting cattle from pneumonic pasteurellosis. Leukotoxin (Lkt), has been identified as an important protective antigen of M. haemolytica, and a fragment, Lkt50, was shown to produce toxin-neutralizing antibodies in rabbits. The construct chosen for introduction into alfalfa carried lkt50 fused to a green fluorescent protein reporter gene, mgfp5-ER. The fusion gene was driven by either the cauliflower mosaic virus 35S promoter (35S) or the promoter from a rubisco small subunit (rbcS-3A) gene of pea. The constructs were introduced into alfalfa RSY27 germplasm using two Agrobacterium tumefaciens strains, LBA4404 and C58, producing a number of transformed lines with both A. strains. Although strain C58 had a slower initial response and produced less callus than strain LBA4404, it resulted in higher numbers of transformed embryos and plants. In total, 30 alfalfa lines (91% of those analyzed), each derived from a separate transformation event, produced detectable levels of Lkt50-GFP. Western analysis with anti-Lkt+66 antiserum revealed the presence of both full-length and truncated polypeptides in plants kept in magenta boxes, while plants transferred to the greenhouse produced only the full-length product. Immunoblotting with anti-GFP antiserum provided evidence that part of the GFP moiety was lost in the truncated protein. Southern blot analysis indicated a low number of insertion sites per event.     

Growth and chlorophyll production in plant callus tissues grown in vitro

Summary Growth, nutrition and chlorophyll development were studied in chlorophyllous callus tissues isolated from the following edible angiospermous plants: carrot root, crown gall of tomato, endive embryo, leaf petiole and stem of lettuce, leaf petiole of parsley, pea stem and rose stem. Growth patterns of these tissues in vitro were sigmoid. Synthetic media produced less growth, in terms of fresh weight increase, than media containing coconut milk, a highly complex and little understood natural substance. Murashige and Skoog's synthetic medium proved useful for satisfactory growth and chlorophyll production in a number of tissues. Its usefulness was further increased by additional amounts of copper sulphate, potassium nitrate and monobasic ammonium phosphate. Increased levels of iron and magnesium inhibited growth. Incorporation of yeast extract in the tobacco-high-salts-medium produced the highest amount of growth and chlorophyll formation in endive tissue. Presence of exogenous sucrose was essential for the continued good growth of the above callus tissues in vitro. Highest amount of growth took place either in white light or in the dark. Different tissues had different responses to high or low intensities of light. Endive and carrot tissues produced in vitro were palatable to human taste. Endive tissue was particularly good as it also differentiated many small rosettes of leaves, shoots and had a mild aromatic flavor typical of the endive plants grown in nature.     

Opine synthesis in wild-type plant tissue

Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid.     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

Transformation of tef (Eragrostis tef) by Agrobacterium through immature embryo regeneration system for inducing semi-dwarfism

Successful application of genetic transformation for integration of a transgene is much dependent upon availability of an efficient in vitro plant regeneration procedure and detection of transgene insertion and expression. Isolated immature embryos (IEs) of Eragrostis tef cultivar DZ-01-196 were used for embryogenic callus formation and the callus was transformed with GA inactivating gene PcGA2ox under the control of a triple CaMV 35S promoter using Agrobacterium transformation procedure. Embryogenic callus was induced from immature embryos in a medium containing KBP minerals in the presence of 2,4-dichlorophenoxiyacetic acid. The embryogenic calli were further inoculated with Agrobacterium and the calli were grown in co-cultivation medium (CCM) followed by selection in KBP and regeneration (K4NB) media. Putatively transformed E. tef embryogenic calli were tolerant to treatment with the selectable marker kanamycin, while 75 mg l^− 1 geneticin inhibited growth of non-transformed shoots derived from matured embryos completely after 12 days. A total of 55 plants were regenerated from all the embryogenic calli to fully viable plants setting seeds at maturity. Eight putatively transformed T₀ plants were produced carrying the transgene in their genome which was detected by PCR. Sequence analysis confirmed amplified PCR products to have 97.2 and 99.8% sequence identity to PcGA2ox and nptII, respectively. However, detection of the transgene, PcGA2ox or nptII, in T₁ plants was inconsistent although phenotypic analysis of T₁ plants showed changes in pheno-morphic and agronomic characters such as plant height, number of internodes, tillering, panicle length, biomass, yield as well as GA content. Culm reduction was due to absence of elongation of the upper-most internodes. Panicle length in semi-dwarfed plants showed no relation with culm length. GA analysis showed plants with semi-dwarf phenotype to be associated with a low level of bioactive GA₁ and its immediate precursors. Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.     

Cytokinin antagonist activity of substituted phenethylamines in plant cell culture

A series of structurally related substituted phenethylamines shows extreme toxicity toward wild-type callus tissue cultures of tobacco (Nicotiana tabacum), soybean (Glycine max), corn (Zea mays), and sunflower (Helianthus annuus L.), but tobacco crown gall cultures are resistant to the compounds. The essential components that result in toxicity of the phenethylamines include one aromatic hydroxyl and one primary aliphatic amino group. A series of attenuated crown gall cultures, generated by transformation of tobacco with various modified Agrobacterium strains, has been used to demonstrate that the resistance of crown galls to the phenethylamines is due to the expression in these tissues of isopentenyl transferase, a first step in cytokinin biosynthesis. The toxicity of the compounds to tissue cultures is very rapid, but can be overcome by prior exposure of the calli to exogenous cytokinin. Because of the relationships we have observed between cytokinins and these compounds, we propose that the substituted phenethylamines may represent a class of chemicals that can be used as specific probes to further an understanding of cytokinin metabolism in plant tissues.     

Production of transgenic maize plants and progeny by bombardment of hi-II immature embryos

Summary Production of transgenic maize (Zea mays L.) callus, plants, and progeny from microprojectile bombardment of 2–5-d cultured Hi-II immature embryos is described. Histological evidence indicates that these tissues are amenable to transformation due to surface layer cell division of the scutellum. Two out of every 100 bombarded embryos produced transgenic callus and R₀ transgenic plants were both male and female fertile. Expected segregation of transgenes was observed in progeny. The primary advantage of bombarding these tissues is increased male and female fertility of transgenic plants compared with those produced using long-term callus or suspension cultures.     

Type I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.)

To develop a less genotype-dependent maize-transformation procedure, we used 10-month-old Type I callus as target tissue for microprojectile bombardment. Twelve transgenic callus lines were obtained from two of the three anther-culture-derived callus cultures representing different gentic backgrounds. Multiple fertile transgenic plants (T₀) were regenerated from each transgenic callus line. Transgenic leaves treated with the herbicide Basta showed no symptoms, indicating that one of the two introduced genes, bar, was functionally expressing. Data from DNA hybridization analysis confirmed that the introduced genes (bar and uidA) were integrated into the plant genome and that all lines derived from independent transformation events. Transmission of the introduced genes and the functional expression of bar in T₁ progeny was also confirmed. Germination of T₁ immature embryos in the presence of bialaphos was used as a screen for functional expression of bar; however, leaf painting of T₁ plants proved a more accurate predictor of bar expression in plants. This study suggests that maize Type I callus can be transformed efficiently through microprojectile bombardment and that fertile transgenic plants can be recovered. This system should facilitate the direct introduction of agronomically important genes in to commercial genotypes.     

Expression of a humanS-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.     

Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection

This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.     

THE IN VITRO SECRETION OF GROWTH REGULATORS BY ISOLATED CALLUS TISSUES

Tissues from a wide variety of plants were surface sterilized, isolated, and grown on different media. These isolated tissues were bioassayed for growth regulatory activity. The secretions from four of the 20 callus tissues inhibited the growth of Bacillus subtilis. An aseptic method for measuring the growth of Lemna was developed and used to detect inhibitory materials in medium which had supported the growth of five isolated callus tissues. In the seed (Lycopersicon esculentum) germination test five callus tissues had an inhibitory influence while two callus tissues showed a stimulatory effect. The study also included expressed juices and extracts of callus tissues which secreted regulatory materials. The expressed juice of five callus tissues contained materials which inhibited the growth of Lemna. Two expressed juices inhibited the growth of Bacillus subtilis. The water extract of two callus tissues inhibited the growth of Lemna. Fifty percent of the plants which have been reported to produce growth regulatory materials in nature also produced callus tissue which was capable of regulating growth of assay organisms.     

Prolific plant regeneration from protoplast-derived tissues of Lotus corniculatus L. (birdsfoot trefoil)

Protoplasts isolated enzymatically from seedling roots, hypocotyls and cotyledons of Lotus corniculatus L. produced callus which underwent prolific shoot regeneration. The rapidity and ease of recovering plants from protoplast-derived tissues makes this forage legume an attractive experimental system for genetic manipulation.     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.