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The synergistic combination of hydrodynamic-based gene delivery and ultrasound was investigated to achieve improved gene transfer to the kidney. Plasmids encoding firefly luciferase and Erythropoietin (EPO) gene were delivered into the left kidney of rats by single or combinative application of renal vein hydrodynamic injection and ultrasound treatment with or without the addition of ultrasound contrast agents (UCA). Ultrasound exposure was found to enhance the efficiency of hydrodynamic-based gene delivery for both luciferase and EPO expression. An ultrasound exposure intensity of 2 W/cm2 at 10% duty cycle for 15 min, produced a maximal gene expression 4.5 times higher than hydrodynamic delivery alone. Duration, location, and tissue-specificity of gene expression were not changed by ultrasound exposure. Application of UCA reduced the intensity and exposure duration of ultrasound treatment needed for optimal expression. Appropriate application of ultrasound and UCA did not alter histological structure or impair physiological function of the treated kidney.

To correlate thermal dose from focused ultrasound (FUS) with gene expression and tissue injury, a temperature plateau strategy was employed. Plasmids encoding luciferase gene under the control of hsp70B promoter were transfected into the right gastrocnemius muscle in a rat via electroporation. One day after transfection, hind limbs were treated with 3.3-MHz focused ultrasound, using one of four different temperature plateaus with spatial-peak time-average focal temperatures (T(SPTA)) of 46 degrees C, 48 degrees C, 51 degrees C and 62 degrees C. The treatment duration at the plateau temperature was varied from 0 to 30s. Gene expression was analyzed in vivo one day following FUS treatment, and H&E staining was employed to assess tissue injury. Gene activation and tissue damage correlated closely with thermal dose. The highest level of gene activation was induced by FUS at T(SPTA)=51 degrees C for 20s, which was found to be statistically equivalent to that produced by water-bath hyperthermia.

This study aims to assess the risk of high intensity focused ultrasound (HIFU) therapy on the incidence of distant metastases and to investigate its association with HIFU-elicited anti-tumor immunity in a murine melanoma (B16-F10) model. Tumor-bearing legs were amputated immediately after or 2 days following HIFU treatment to differentiate the contribution of the elicited anti-tumor immunity. In mice undergoing amputation immediately after mechanical, thermal, or no HIFU treatment, metastasis rates were comparable (18.8%, 13.3%, and 12.5%). In contrast, with a 2-day delay in amputation, the corresponding metastasis rates were 6.7%, 11.8%, and 40%, respectively. Animal survival rate was higher and CTL activity was enhanced in the HIFU treatment groups. Altogether, our results suggest that HIFU treatment does not increase the risk of distant metastasis. Instead, HIFU treatment can elicit an anti-tumor immune response that may be harnessed to improve the overall effectiveness and quality of cancer therapy.

Background: High intensity focused ultrasound (HIFU) is an emerging non-invasive treatment modality for localized treatment of cancers. While current clinical strategies employ HIFU exclusively for thermal ablation of the target sites, biological responses associated with both thermal and mechanical damage from focused ultrasound have not been thoroughly investigated. In particular, endogenous danger signals from HIFU-damaged tumor cells may trigger the activation of dendritic cells. This response may play a critical role in a HIFU-elicited anti-tumor immune response which can be harnessed for more effective treatment., Methods: Mice bearing MC-38 colon adenocarcinoma tumors were treated with thermal and mechanical HIFU exposure settings in order to independently observe HIFU-induced effects on the host's immunological response. In vivo dendritic cell activity was assessed along with the host's response to challenge tumor growth., Results: Thermal and mechanical HIFU were found to increase CD11c+ cells 3.1-fold and 4-fold, respectively, as compared to 1.5-fold observed for DC injection alone. In addition, thermal and mechanical HIFU increased CFSE+ DC accumulation in draining lymph nodes 5-fold and 10-fold, respectively. Moreover, focused ultrasound treatments not only caused a reduction in the growth of primary tumors, with tumor volume decreasing by 85% for thermal HIFU and 43% for mechanical HIFU, but they also provided protection against subcutaneous tumor re-challenge. Further immunological assays confirmed an enhanced CTL activity and increased tumor-specific IFN-gamma-secreting cells in the mice treated by focused ultrasound, with cytotoxicity induced by mechanical HIFU reaching as high as 27% at a 10:1 effector:target ratio., Conclusion: These studies present initial encouraging results confirming that focused ultrasound treatment can elicit a systemic anti-tumor immune response, and they suggest that this immunity is closely related to dendritic cell activation. Because DC activation was more pronounced when tumor cells were mechanically lysed by focused ultrasound treatment, mechanical HIFU in particular may be employed as a potential strategy in combination with subsequent thermal ablations for increasing the efficacy of HIFU cancer treatment by enhancing the host's anti-tumor immunity.

At present, there are limited methods of acquiring three-dimensional visualization of cardiac structure and function in real-time during interventional electrophysiology procedures. Images acquired for integration of computerized tomography and magnetic resonance imaging with electroanatomic mapping systems are static and are obtained earlier in time. The purpose of this study was to test the feasibility of real-time three-dimensional transesophageal echocardiography for the guidance of interventional electrophysiological studies. A matrix array transducer with 504 channels operating at 5 MHz in a 1 cm diameter steerable esophageal probe was used in conjunction with a scanner capable of real-time 3D scanning of pyramidal volumes from 65 degrees to 120 degrees at rates up to 30 volumes per second. This device has a spatial resolution of approximately 3 mm at 5 cm depth. The authors acquired real-time three-dimensional images of anatomic landmarks of value for electrophysiological procedures in five closed chest canines. Real-time, three-dimensional ultrasound imaging was also used for visualization and guidance of interventional catheter devices within the canine heart. Real-time three-dimensional images of the atria, pulmonary veins, and coronary sinus were acquired. Real-time 3-D color flow Doppler was employed to confirm patency. Multiple image planes of image volumes and rendered views were used to track catheter position and orientation. Images of left veno-atrial junctions have been confirmed by dissection. This study has demonstrated the feasiblity of using real-time three-dimensional transesophageal echocardiography for guiding interventional electrophysiology. The technology has the potential to fill a niche as an adjunct modality for cost-effective real-time interventional guidance and assessment, providing catheter and pacing lead visualization simultaneously with functional volumetric cardiac imaging.

The goal of this research is to determine the feasibility of using a single endoscopic probe for the combined purpose of real-time 3D (RT3D) ultrasound imaging of a target organ and the delivery of ultrasound therapy to facilitate the absorption of compounds for cancer treatment. Recent research in ultrasound therapy has shown that ultrasound-mediated drug delivery improves absorption of treatments for prostate, cervical and esophageal cancer. The ability to combine ultrasound hyperthermia and 3D imaging could improve visualization and targeting of cancerous tissues. In this study, numerical modeling and experimental measurements were developed to determine the feasibility of combined therapy and imaging with a 1 cm diameter endoscopic RT3D probe with 504 transmitters and 252 receive channels. This device operates at 5 MHz and has a 6.3 mm x 6.3 mm aperture to produce real time 3D pyramidal scans of 60-120 degrees incorporating 64 x 64 = 4096 image lines at 30 volumes/sec interleaved with a 3D steerable therapy beam. A finite-element mesh was constructed with over 128,000 elements in LS-DYNA to simulate the induced temperature rise from our transducer with a 3 cm deep focus in tissue. Quarter-symmetry of the transducer was used to reduce mesh size and computation time. Based on intensity values calculated in Field II using the transducer's array geometry, a minimum I(SPTA) of 3.6 W/cm2 is required from our endoscope probe in order to induce a temperature rise of 4 degrees C within five minutes. Experimental measurements of the array's power output capabilities were conducted using a PVDF hydrophone placed 3 cm away from the face of the transducer in a watertank. Using a PDA14 Signatec data acquisition board to capture full volumes of transmitted ultrasound data, it was determined that the probe can presently maintain intensity values up to 2.4 W/cm2 over indefinite times for therapeutic applications combined with intermittent 3D scanning to maintain targeting. These values were acquired using 8 cycle bursts at a prf of 6 kHz. Ex vivo heating experiments of excised pork tissue yielded a maximum temperature rises of 2.3 degrees C over 5 minutes of ultrasound exposure with an average rise of 1.8 +/- 0.2 degrees C over 5 trials. Modifications to the power supply and transducer array may enable us to reach the higher intensities required to facilitate drug delivery therapy.

Laparoscopic ultrasound has seen increased use as a surgical aide in general, gynecological, and urological procedures. The application of real-time, three-dimensional (RT3D) ultrasound to these laparoscopic procedures may increase information available to the surgeon and serve as an additional intraoperative guidance tool. The integration of RT3D with recent advances in robotic surgery also can increase automation and ease of use. In this study, a 1-cm diameter probe for RT3D has been used laparoscopically for in vivo imaging of a canine. The probe, which operates at 5 MHz, was used to image the spleen, liver, and gall bladder as well as to guide surgical instruments. Furthermore, the three-dimensional (3-D) measurement system of the volumetric scanner used with this probe was tested as a guidance mechanism for a robotic linear motion system in order to simulate the feasibility of RT3D/robotic surgery integration. Using images acquired with the 3-D laparoscopic ultrasound device, coordinates were acquired by the scanner and used to direct a robotically controlled needle toward desired in vitro targets as well as targets in a post-mortem canine. The rms error for these measurements was 1.34 mm using optical alignment and 0.76 mm using ultrasound alignment.

Polyamines (PAs) are low molecular weight metabolites involved in various physiological and developmental processes in eukaryotic and prokaryotic cells. The cellular PA level is regulated in part by the action of amine oxidases (AOs) including copper diamine oxidases (DAOs) and flavoprotein polyamine oxidases (PAOs). In this study, the isolation and characterization of flavin amine oxidases (FAOs) from Brassica juncea (BJFAO) and Arabidopsis (ATFAO1) are reported that were clustered in the same group as polyamine oxidases from maize (MPAO) and barley (BPAO1) and monoamine oxidases from mammalian species. ATFAO1 was temporally and spatially regulated in Arabidopsis and showed distinct expression patterns in response to different stress treatments. To investigate the in vivo function of FAO, transgenic Arabidopsis plants expressing sense, antisense, and double-stranded BJFAO RNAs were generated and those with altered activity of FAOs were selected for further characterization. It was found that the shoot regeneration response in transgenic plants was significantly affected by the modulated PA levels corresponding to FAO activities. Tissues that originated from transgenic plants with down-regulated FAO activity were highly regenerative, while those from transgenic plants with upregulated FAO activity were poorly regenerative. The shoot regeneration capacity in these transgenic plants was related to the levels of individual PAs, suggesting that FAO affects shoot regeneration by regulating cellular PAs. Furthermore, it was found that the effect of FAO activity on shoot regeneration was exerted downstream of the Enhancer of Shoot Regeneration (ESR1) gene, which may function in a branch of the cytokinin signalling pathway.

Glutathione S-transferases (GSTs) are regulated by various stimuli at the transcriptional level. In this study, a 2,640-bp promoter sequence of a mustard GST gene, BjGSTF2, was cloned. Several truncated BjGSTF2 promoters were generated by 5'-deletion, fused to the beta-glucuronidase (GUS) coding sequence and the chimeric genes expressed in Arabidopsis thaliana. Transgene expression in GST2623::GUS plants carrying the longest promoter varied considerably. GUS activity was high in the roots, cotyledons, anthers and both ends of the silique, but it was low or barely detectable in the leaves, seeds, petals and stamens. Analysis of transgenic plants expressing the GUS gene under the control of different truncated BjGSTF2 promoters revealed several regions that possessed cis-acting elements required for the basal and induced expression by H(2)O(2), salicylic acid and 1-aminocyclopropane-1-carboxylate and down-regulation by spermidine. The results also showed that the GUS activity of GST2623::GUS coincided well with the H(2)O(2) accumulation pattern in cultured leaf-disc explants during the regeneration process.

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key rate-limiting enzyme located in the polyamine biosynthesis pathway. When compared with other organisms, the plant SAMDC genes possess some distinct features because they are devoid of introns in the main open reading frame (ORF) but have an intron(s) in their 5' untranslated leader sequences, in which two overlapping tiny and small upstream ORFs (uORFs) are present. Our results show that the presence of the 5' leader sequence plays important roles in transcriptional and posttranscriptional regulation of SAMDC expression. This sequence may help to keep the transcript of its downstream cistron at a relatively low level and function together with its own promoter in response to external stimuli or internal changes of spermidine and spermine to initiate and regulate SAMDC expression. Under stress and high spermidine or spermine conditions, the tiny uORF shows the same function as its overlapping small uORF, which is involved in translational repression and feedback controlled by polyamines. The presence of introns is necessary for the SAMDC up-regulation process when the internal spermidine level is low. Our results suggest that plants have evolved one network to adjust SAMDC activity through their 5' leader sequences, through which transcriptional regulation is combined with an extensive posttranscriptional control circuit.

The enzymes glutathione-S-transferases (GSTs, E.C.2.5.1.18) have been associated with detoxification of xenobiotics, limiting oxidative damage and other stress responses in plants. In this study, we report the isolation of a mustard gene, BjGSTF2, homologous to the phi class GSTs and changes in plant growth in vivo and shoot regeneration in vitro were related to GST expression. GST transcripts accumulated differentially in mustard organs, where transcript was most abundant in root. Tissues incubated at high temperature or in the presence of exogenous H2O2, HgCl2, 1-aminocyclopropane-1-carboxylate, salicylic acid and paraquat upregulated GST expression, whereas spermidine was inhibitory. To investigate the in vivo function of GST, transgenic Arabidopsis thalianaplants expressing sense (GST-S6), antisense (GST-A4) and double-stranded BjGSTF2 (GST-DS1) RNAs were generated. GST-S6 was shown to flower two days earlier and was relatively more tolerant to HgCl2 and paraquat, whereas GST-DS1 with least stress tolerance flowered one week later compared to WT and GST-A4. In shoot regeneration response, tissues originated from GST-S6 were highly regenerative, whereas no shoot regeneration was observed in GST-DS1 tissues after 30 days of culture. Results of this study provide the evidence showing that GST plays a role in plant growth and development in vivo and shoot regeneration in vitro.

Transesophageal echocardiography (TEE) is an essential diagnostic tool in patients with poor transthoracic echocardiographic windows or when detailed imaging of structures distant from the chest wall is necessary. A real-time 3D TEE probe has been fabricated in our laboratory in order to increase the amount of information available during a transesophageal procedure. The 1 cm diameter esophageal probe utilizes a 2-dimensional, 5 MHz array at its tip with a 6.3 mm diameter aperture, including 504 active channels. The array has a periodic vernier geometry with an element pitch of 0.18 mm, built onto a multilayer flexible (MLF) interconnect circuit. In order to accommodate 504 channels within the device, a 1 m long Gore MicroFlat cable was utilized for wiring the MLF to the corresponding system connectors. Pulse-echo tests in a water tank have yielded a -6 dB bandwidth of 25.3%. Fully connected to the system through 3 m of cable, the probe shows an average 50 omega insertion loss of-85 dB with a standard deviation of 4 dB, as determined through pitch-catch measurements for a sampling of 10 elements. Using the completed 3D TEE probe with the Volumetrics Medical Imaging 3D scanner, real-time volumetric images of in vivo canine cardiac anatomy have been acquired, displaying atrial views, mitral valve function and interventional catheter guidance.

In patients who are obese or exhibit signs of pulmonary disease, standard transthoracic scanning may yield poor quality cardiac images. For these conditions, two-dimensional transesophageal echocardiography (TEE) is established as an essential diagnostic tool. Current techniques in transesophageal scanning, though, are limited by incomplete visualization of cardiac structures in close proximity to the transducer. Thus, we propose a 2D curvilinear array for 3D transesophageal echocardiography in order to widen the field of view and increase visualization close to the transducer face. In this project, a 440 channel 5 MHz two-dimensional array with a 12.6 mm aperture diameter on a flexible interconnect circuit has been molded to a 4 mm radius of curvature. A 75% element yield was achieved during fabrication and an average -6dB bandwidth of 30% was observed in pulse-echo tests. Using this transducer in conjunction with modifications to the beam former delay software and scan converter display software of the our 3D scanner, we obtained cylindrical real-time curvilinear volumetric scans of tissue phantoms, including a field of view of greater than 120 degrees in the curved, azimuth direction and 65 degrees phased array sector scans in the elevation direction. These images were achieved using a stepped subaperture across the cylindrical curvilinear direction of the transducer face and phased array sector scanning in the noncurved plane. In addition, real-time volume rendered images of a tissue mimicking phantom with holes ranging from 1 cm to less than 4 mm have been obtained. 3D color flow Doppler results have also been acquired. This configuration can theoretically achieve volumes displaying 180 degrees by 120 degrees. The transducer is also capable of obtaining images through a curvilinear stepped subaperture in azimuth in conjunction with a rectilinear stepped subaperture in elevation, further increasing the field of view close to the transducer face. Future work includes development of an array for adapting these modifications to a 6 mm diameter endoscope probe.

As part of a study to understand the molecular basis of fruit ripening, this study reports the isolation and characterization of a banana cytochrome P450 (P450) cDNA, designated as MAP450-1, which was associated with fruit ripening of banana. MAP450-1 encoded a single polypeptide of 507 amino acid residues that shared an overall identity of 27-45% with that of several plant P450s, among which MAP450-1 was most related phylogenetically to the avocado P450 CYP71A1. The polypeptide that possessed residue domains conserved in all P450s was classified as CYP71N1. Expression of CYP71N1 varied greatly between banana organs. Transcripts were detected only in peel and pulp of the ripening fruit and not in unripe fruit tissues at all developmental stages or other organs (root, leaf, ovary and flower). During ripening, transcripts were barely detectable in pre-climacteric and climacteric fruits but, as ripening progressed, they began to accumulate and reached a maximum in post-climacteric fruits. CYP71N1 expression in pre-climacteric fruit could be upregulated by exogenous application of ethylene (1-5 ppm) and treatment of overripe fruit with exogenous sucrose (50-300 mM) but not glucose downregulated the expression. These results indicate that P450s may not play a role in fruit development and its expression is associated with ripening, which may be regulated, in part, by ethylene and/or sucrose, at the transcript level.

Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.

Arginine decarboxylase (ADC) is a key enzyme involved in the synthesis of polyamines, which have been implicated in a wide range of plant responses, including stress. However, regulation of polyamine levels in relation to ADC in response to stress at the molecular level is not well understood. In an attempt to address this question, we first cloned two cDNAs in mustard (Brassica juncea[L.] Czern & Coss var. Indian Mustard), designated MADC2 and MADC3, encoding predicted ADC. MADC2 and MADC3 encode polypeptides of 692 and 680 amino acid residues, respectively. A comparison of deduced amino acid sequence revealed that both were highly homologous to MADC1 (77%), a mustard ADC, and other plant ADCs (63-84%). Northern analysis revealed that ADC transcripts in mustard were generally more abundant in stem and root but were barely detectable in leaf. However, ADC expression in the leaf was up-regulated differentially in response to stress such as chilling, salt and mannitol and to treatments with exogenous polyamines. While chilling induced expression of all three ADC genes, salt predominantly resulted in increased accumulation of MADC3 transcript. Leaves exhibited a similar response to exogenous putrescine, spermidine and spermine, all of which stimulated accumulation of MADC2 and MADC3 transcripts but not MADC1. Furthermore, exogenous putrescine also increased the endogenous levels of spermidine and spermine, while a higher endogenous putrescine and spermidine content was detected in leaf incubated with exogenous spermine. Leaves also responded to chilling, salt and mannitol by increasing the levels of the cellular polyamine content, in which the level of spermine in free and conjugated forms increased most profoundly.

Metallothioneins (MTs) are cysteine-rich polypeptides that are involved in metal detoxification and homeostasis in both prokaryotes and eukaryotes. In this study, we report the isolation and characterization of three members (MT2A, MT2B and MT3) of the MT-like gene family from ripening banana fruit and their differential expression in various banana organs and during fruit development and ripening. All members of the MT-like gene encode small cysteine-rich polypeptides of 65-79 amino acid residues. MT2A shared a high sequence similarity (54-77%) with several type-2 MTs in plants, while MT3 was highly homologous (51-61%) with type-3 MTs. The three members expressed differentially in various organs but transcripts were generally more abundant in reproductive than vegetative organs. During fruit development, the MT2A transcript was barely detectable in ovary but increased to a high level in young fruit at 20 days after shooting (DAS) and declined gradually thereafter as fruit developed. In contrast, both MT2B and MT3 expressed poorly in young fruits (20-60 DAS) and transcripts were detected only in fruits at later stages of development. As ripening progressed, expression of MT2A decreased but that of MT3 increased. Expression of MT members during ripening appeared to be differentially regulated by ethylene, whose levels were low in FG and TY fruit but surged climacteristically in MG and declined sharply as ripening advanced further. Exogenous application of ethylene at 5 ppm or higher concentrations down-regulated MT2A expression and the inhibitory effect of ethylene could be partially suppressed by the presence of norbornadiene, an inhibitor of ethylene action. Ethylene had no effect on transcript accumulation of MT2B and MT3. However, MT3 expression was greatly enhanced in response to metals such as CdSO4, CuSO4 and ZnSO4. These results suggest that increased MT3 expression may be associated with excess metal ions present in ripening fruit tissues. This study also provided evidence, for the first time, that ethylene and metals play a regulatory role in expression of MT-like genes in banana.

The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20-30 μM aminoethoxyvinylglycine (AVG) or AgNO3] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N(6)-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO3 in combination with AVG. Culture medium solidified with agarose in the presence of AgNO3 but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10-25 mM putrescine in combination with 30 μM AgNO3 or AVG. Explants with high regenerability grown in the presence of AgNO3 or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1-5 mg/l were more effective than NAA.

The promotive effect of ethylene inhibitors (Els), i.e. AgNO3 and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1-3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO3 at 30 μM did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 μM AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1-20 mM putrescine, 0.1-2.5 mM spermidine, or 0.1-1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90-100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5-2 mg/L BA in combination with 0.01-0.05 mg/L 2,4-D or 0.1-1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.

The promotive effect of AgNO(3) and aminoethoxyvinylglycine (AVG) on in vitro shoot regeneration from cotyledons of Brassica campestris ssp. pekinensis in relation to endogenous 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, ACC, and ethylene production was investigated. AgNO(3) enhanced ACC synthase activity and ACC accumulation, which reached a maximum after 3 to 7 days of culture. ACC accumulation was concomitant with increased emanation of ethylene which peaked after 14 days. In contrast, AVG was inhibitory to endogenous ACC synthase activity and reduced ACC and ethylene production. The promotive effect of AVG on shoot regeneration was reversed by 2-chloroethylphosphonic acid at 50 micromolar or higher concentrations, whereas explants grown on AgNO(3) medium were less affected by 2-chloroethylphosphonic acid. The distinctive effect of AgNO(3) and AVG on endogenous ACC synthase, ACC, and ethylene production and its possible mechanisms are discussed.

The presence of 1-10 μM aminoethoxyvinylglycine (AVG) or 5-30 μM AgNO3 markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 μM), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO3 and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO3 was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 μM.

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown in the light on shoot-forming medium in the presence of Na2SO4 for over a year. An increase in Na2SO4 concentration resulted in decreasing callus growth, decreasing percentage of calli producing shoots and number of shoots per callus, and increasing callus percent dry weight. Regeneration of shoots from callus grown in the absence of Na2SO4 began to decline after 14 months in culture, and shoot regeneration capacity was completely lost after 18 months. In contrast, 18-month old callus continuously grown in the presence of Na2SO4 retained the ability to form shoots. The highest percent of callus pieces that formed shoots and the maximum number of shoots per callus occurred at 70 mM (1%) Na2SO4. All plants arising from the 18-month old callus were polyploid. Both 9- and 18-month old callus exhibited more negative water and osmotic potentials in the presence of increasing Na2SO4 concentration.