Showing papers in "Plant Cell Reports in 1992"
TL;DR: High levels of transient expression of the ß-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of corn and soybean, and leaf tissue of cowpea, using the Particle Inflow Gun.
Abstract: A simple and inexpensive particle bombardment device was constructed for delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles using pressurized helium in combination with a partial vacuum. The particles are accelerated directly in a helium stream rather than being supported by a macrocarrier. Bombardment parameters were partially optimized using transient expression assays of a s-glucuronidase gene in maize embryogenic suspension culture and cowpea leaf tissues. High levels of transient expression of the s-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of corn and soybean, and leaf tissue of cowpea. Stable transformation of embryogenic tissue of soybean has also been obtained using this bombardment apparatus.
511 citations
TL;DR: This protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121 and used to test three other vectors, finding that CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.
Abstract: Leaf or stem explants of a hybrid poplar clone (Populus tremula X Populus alba), sensitive to Agrobacterium tumefaciens, were co-cultivated either by an octopine or a nopaline disarmed A. tumefaciens modified strain. Transformed poplar shoots were readily regenerated from explants. The protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121. This protocol was then used to test three other vectors. The first one, possessing a nptII gene fused to the CaMV 19S promoter, permitted regeneration of transformed shoots in presence of 50 to 100 mg/l kanamycin. The two other vectors carried an additional nptII gene under the control of the CaMV 35S or CaMV 35S promoter with a double enhancer sequence (CaMV 70). CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.
315 citations
TL;DR: It is shown that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.
Abstract: Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5′ region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTATM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.
253 citations
TL;DR: A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported and two plants established in soil were shown to be transgenic by Southern analysis.
Abstract: A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker s-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage (≤ 5%) of the shoots regenerated in the presence of 100 μg/ml kanamycin were GUS+. Polymerase chain reaction (PCR) analysis confirmed that GUS+ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis.
200 citations
TL;DR: Histochemical analysis showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection.
Abstract: The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the s-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80-90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.
189 citations
TL;DR: Cell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium faldparum in vitro both in the n-hexane Extract of the plant cell culture medium and in the chloroform extract of the cells.
Abstract: Cell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium faldparum in vitro both in the n-hexane extract of the plant cell culture medium and in the chloroform extract of the cells. Trace amounts of the antimalarial sesquiterpene lactone artemisinin may account for the activity of the n-hexane fraction but only the methoxylated flavonoids artemetin, chrysoplenetin, chrysosplenol-D and cirsilineol can account for the activity of the chloroform extract. These purified flavonoids were found to have IC50 values at 2.4 – 6.5 × 10−5M against P. falciparum in vitro compared with an IC50 value of about 3 × 10−8M for purified artimisinin. At concentrations of 5 × 10−6M these flavonoids were not active against P. falciparum but did have a marked and selective potentiating effect on the antiplasmodial activity of artemisinin.
179 citations
TL;DR: Fourteen soybean genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63, and a total of thirty five plants have been transplanted into pots in the greenhouse.
Abstract: Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1–2 mm diameter colonies were formed in 5–6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l−1 each of 2,4-D, BA and KN and 500 mg l−1 CH for further growth. Once the colonies had become green, compact and nodular, and were 8–10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8–12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.
131 citations
TL;DR: This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.
Abstract: A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene s-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.
125 citations
TL;DR: Investigation of kinetics showed that a change in the medium caused shifts in peaks of growth and production, and modifications of the cell metabolism, that led to the discovery ofRosmarinic acid, a natural antioxidant produced by cell suspension cultures of sage.
Abstract: Rosmarinic acid (RA) is a natural antioxidant produced by cell suspension cultures of sage (Salvia officinalis L.). The growth and production of RA by these cells can be modified by the type of culture medium. Production can be increased 10-fold to attain 6.4 g.1(-1) under optimal conditions. Investigation of kinetics showed that a change in the medium caused shifts in peaks of growth and production, and modifications of the cell metabolism. RA production can be correlated with growth or begins only when growth has stopped.
117 citations
TL;DR: Plants were regenerated from encapsulated shoot tips of banana regenerated in vitro on different substrates using White's medium, which resulted in 100% conversion of encapsulatedshoot tips into plantlets.
Abstract: Plants were regenerated from encapsulated shoot tips of banana. Shoot tips (ca 4 mm) isolated from multiple shoot cultures of banana cv. Basrai were encapsulated in 3% sodium alginate containing different gel matrices. The encapsulated shoot tips regenerated in vitro on different substrates. Use of White's medium resulted in 100% conversion of encapsulated shoot tips into plantlets. The plantlets were successfully established in soil.
109 citations
TL;DR: Results support use of particle bombardment for transformation of this important ornamental monocot.
Abstract: Transformed dendrobium orchids (Dendrobium x Jaquelyn Thomas hybrids) were recovered from protocorms bombarded by particles coated with the plasmid pGA482GG/cpPRV4, which contains the plant expressible Nos-NPT II and papaya ringspot virus (PRV) coat protein (CP) genes. Approximately 280 protocorms from four crosses were bombarded and potentially transformed tissues were identified by growth and green color on half-strength Murashige and Skoog medium supplemented with 2% sucrose and 50–100 mg 1−1 kanamycin sulfate. Kanamycin concentrations that prevented growth of nontransformed tissues could not be used for long-term selection because such levels suppressed the regeneration of potentially transformed tissues. PCR and restriction analysis 21 months after treatment found 13 of 13 plants from two crosses, which appeared kanamycin-tolerant, to contain the Nos-NPT II gene, while only one of these plants carried the vector-linked PRV CP-gene. These results support use of particle bombardment for transformation of this important ornamental monocot.
TL;DR: The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivarsof B. oleifera plants and showed segregation of the kanamycin-resistance trait in the progeny.
Abstract: Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1–9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.
TL;DR: HPLC, GC and GC-MS analyses have shown that concentrations of carbenicillin and penicillin G, commonly used in plant tissue culture, break down to give physiologically active levels of the auxin phenylacetic acid, which offers a mechanism for the stimulation of growth caused by these two antibiotics.
Abstract: The effects of various antibiotics on the development of hypocotyls of Antirrhinum majus in tissue culture have been studied. The penicillins, carbenicillin and penicillin G, have been shown to stimulate callus growth, have little impact on shoot production and may stimulate root formation. The cephalosporins, cephotaxime and cephalosporin, have no effect on callus production and reduce shoot and root formation. HPLC, GC and GC-MS analyses have shown that concentrations of carbenicillin and penicillin G, commonly used in plant tissue culture, break down to give physiologically active levels of the auxin phenylacetic acid. This offers a mechanism for the stimulation of growth caused by these two antibiotics.
TL;DR: A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris, however colchicine could be effectively used to achieve chromosome doubling.
Abstract: A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris. Bud size, genotype, media constituents, and incubation time and temperature were examined. Donor plants were grown in a growth cabinet at a day/night temperature of 10/5°C. Microspores were isolated from buds 2.0 – 2.9 mm in length and cultured in modified Lichter (1982) medium containing 17% sucrose, pH 6.2. After 48 h at 32°C, the incubation medium was replaced with NLN (Lichter 1982) medium containing 10% sucrose. Microspores were cultured at 24°C in darkness and embryos developed after three weeks. More than 1000 plants have thus far been regenerated. Genotypic differences were observed for microspore embryogenesis. The majority of the regenerants were haploid, however colchicine could be effectively used to achieve chromosome doubling.
TL;DR: Cytokinins were found to stimulate the alkaloid synthesis induced by removing auxin from the medium of a cell line of Catharanthus roseus, and addition of high levels of Ca2+, Mg2+ or Sr2+ to B5 media in which the mineral salts were diluted to 5–40%, increased the alkAloid production.
Abstract: Cytokinins were found to stimulate the alkaloid synthesis induced by removing auxin from the medium of a cell line of Catharanthus roseus. Diluting the mineral salts of the culture medium decreased the alkaloid production but increased the “sensitivity” of the cells. Addition of high levels of Ca2+, Mg2+ or Sr2+ to B5 media in which the mineral salts were diluted to 5–40%, increased the alkaloid production. The latter effect is related only partially to enhanced osmotic potential.
TL;DR: The ability to deliver and detect reporter gene activity in maize microspores was tested and the CaMV 35S + Adh1-I6 and Ubiq 1-I1 promoters produced high levels of CAT activity in corn microspore.
Abstract: The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adh1-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adh1-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adh1 promoters did not promote gene expression in maize microspore. The CaMV 35S + Adh1-I6 and Ubiq1-I1 promoters produced high levels of CAT activity in maize microspores.
TL;DR: Cotyledonary node explants of Acacia nilotica subspecies indica Brenan differentiated multiple shoots on Gamborg et al.
Abstract: Cotyledonary node explants of Acacia nilotica subspecies indica Brenan, differentiated multiple shoots on Gamborg et al.' s medium (B5, Gamborg et al. 1968) supplemented with cytokinins like N6-benzyladenine, 6-(γ, γ-Dimethylallylamino)-purine, kinetin or zeatin. Of the four, BA supported maximum multiple shoot differentiation; the highest average number of shoots (6.3) per expiant was in 1.5 mg/l. The number of shoots was further enhanced by (i) using nodal explants of in vitro regenerated shoots as microcuttings, and (ii) repeated subculture of the original expiants (stumps) on the same medium after excising the shoots. Thus, over seven hundred shoots could be obtained from a single cotyledonary node explant. Individual shoots, when transferred to 2 mg/l indole-3-acetic acid augmented medium organised healthy roots in 100% cultures. Such test tube grown plantlets have been successfully transferred to soil, where they grow well up to eight weeks.
TL;DR: A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract to the suspension cultures, reaching a maximum at 24 hr.
Abstract: A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both 14C-phenylalanine and 14C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.
TL;DR: It is demonstrated that expression of TDC and SS enzyme activities is preceeded by the transient appearance of mRNAs for both enzymes, suggesting transcriptional control of these events.
Abstract: When cell suspension cultures of Catharanthus roseus are treated with autoclaved elicitor from the fungus Pythium aphanidermatum, they respond with the rapid transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) enzyme activities, followed by the accumulation of indole alkaloids (Eilert et al., 1987). In this report, we demonstrate that expression of TDC and SS enzyme activities is preceeded by the transient appearance of mRNAs for both enzymes, suggesting transcriptional control of these events. The strong transient accumulation of both TDC and SS enzyme transcripts observed in elicitor-treated cell suspension cultures contrasts with the barely detectable level of TDC transcripts and the undetectable level of SS transcripts observed in developing seedlings.
TL;DR: A novel type of somatic embryogenesis characterized by an efficient and highly synchronized embryo formation was observed in embryogenic callus of Coffea arabica initiated on Murashige and Skoog medium containing kinetin and 2,4-D, and the term self-Controlled Somatic Embryogenesis (SCSE) is proposed.
Abstract: A novel type of somatic embryogenesis characterized by an efficient and highly synchronized embryo formation was observed in embryogenic callus of Coffea arabica initiated on Murashige and Skoog medium containing kinetin (4 mg/l) and 2,4-D (1 mg/l). It occurs in suspension and goes along with the suppression of “High Frequency Somatic Embryo Induction” (HFSE). This is achieved by favoring during cultivation senescence-or necrosis-like processes which apparently do not impair the competence for embryogenesis. Since the resulting embryos germinate at a rate of 94.5 % without the need of a maturation step, we propose the term “Self-Controlled Somatic Embryogenesis” (SCSE). In addition, HFSE was optimized using half-strength liquid medium with 0.1 mg/l kinetin and 0.25 mg/l 2,4-D for proliferation of embryonic tissue, and 2.6 mg/l ABA for maturation of embryos. Yields as well as germination rates of HFSE embryos were markedly lower as compared to SCSE.
TL;DR: The influence of PAA appears at the embryogenic phase of the culture system and the possible mechanisms by which PAA may improve regeneration are discussed.
Abstract: The effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar Veery‘S’, while the average response from twelve F1 hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.
TL;DR: Relatively higher levels of BAP, 50–80 μM, were found to be optimal for inducing regeneration while lower concentrations were ineffective and Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1 μM naphthaleneacetic acid.
Abstract: A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6–8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N6-benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50–80 μM, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40–70 shoots and 70–250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1 μM naphthaleneacetic acid.
TL;DR: Results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism, and this response was dependent on the level of BAP.
Abstract: The morphogenetic response of Brassica campestris genotype R500 to inhibitors of ethylene biosynthesis and action was investigated. A medium containing 1.0 mg.l(-1) NAA, 2.0 mg.l(-1) BAP, and 30 or 60 μM AgNO3 significantly enhanced both the percentage shoot regeneration and the number of shoots per cotyledon expiant. Although callus proliferation occurred on hypocotyl segments, no shoots were formed in response to AgNO3 with expiants older than five days. Cotyledons older than six days formed shoots only with AgNO3. Cobalt chloride at 20 and 30 μM increased cotyledon shoot regeneration but was inferior to AgNO3. Hypocotyl segments were unresponsive. Salicylic acid at 25 and 50 μM prevented both shoot regeneration and callusing without any obvious toxic effects. Removal of expiants from AgNO3 after 12 days did not alter the percentage of shoot regeneration but increased the number of shoots per expiant. This response was dependent on the level of BAP. Percentage shoot regeneration and number of shoots per cotyledon explant were not affected by removal of CoCl2. These results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism.
TL;DR: Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv.
Abstract: Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. 'Pusa Kalyani'. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and β-glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7-13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated with Agrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450-625 μ from the cut surface. Such shoots were negative for GUS activity.
TL;DR: To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium with a 24 h dehydration treatment.
Abstract: To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.
TL;DR: Some somatic embryos converted into normal plants capable of greenhouse survival when induced on peanut leaflets from aseptically germinated embryo axes, and other treatments had lower percent embryos, no secondary embryogenesis, and embryos with single axes with two cotyledons.
Abstract: Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets Maximum embryogenesis of 146% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +02 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+02 mg/l kinetin Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons Some somatic embryos converted into normal plants capable of greenhouse survival
TL;DR: Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis and phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.
Abstract: Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the s-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.
TL;DR: The inclusion of a desiccation period during embryo development enhanced conversion four-fold and the cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential.
Abstract: A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.
TL;DR: A method of clonal propagation via somatic embryogenesis of date palm, cultivar Barhee, which has potential for large scale commercial application as well as for developmental studies on embryos is described.
Abstract: A method of clonal propagation via somatic embryogenesis of date palm, cultivar Barhee, which has potential for large scale commercial application as well as for developmental studies on embryos is described. Cultures were initiated from shoot tip and immature inflorescence explants, both of which were capable of development into embryogenic callus. When the embryogenic callus was cultured in liquid suspension on a rotary shaker, hundreds of embryos developed from milligram quantities of callus in a fairly synchronous manner. Scanning electron microscopy showed globular, heart-shaped and torpedo-shaped embryos. Green leaves emerged from a white cotyledonary sheath.
TL;DR: The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.
Abstract: Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.