Commercial sugarcane, belonging to the genus Saccharum (Poaceae), is an important industrial crop accounting for nearly 70% of sugar produced worldwide. Compared to other major crops, efforts to improve sugarcane are limited and relatively recent, with the first introduction of interspecific hybrids about 80 yr ago. Progress in traditional breeding of sugarcane, a highly polyploid and frequently aneuploid plant, is impeded by its narrow gene pool, complex genome, poor fertility, and the long breeding/selection cycle. These constraints, however, make sugarcane a good candidate for molecular breeding. In the past decade considerable progress has been made in understanding and manipulating the sugarcane genome using various biotechnological and cell biological approaches. Notable among them are the creation of transgenic plants with improved agronomic or other important traits, advances in genomics and molecular markers, and progress in understanding the molecular aspects of sucrose transport and accumulation. More recently, substantial effort has been directed towards developing sugarcane as a biofactory for high-value products. While these achievements are commendable, a greater understanding of the sugarcane genome, and cell and whole plant physiology, will accelerate the implementation of commercially significant biotechnology outcomes. We anticipate that the rapid advancements in molecular biology and emerging biotechnology innovations would play a significant role in the future sugarcane crop improvement programs and offer many new opportunities to develop it as a new-generation industrial crop.

Applications of biotechnological tools, including genetic modification aimed at combating Dutch elm disease, are described. In vitro shoot cultures of Ulmus procera (English elm) SR4, U. glabra, U. americana, and U. parvifolia have been established and used as source material in genetic transfer experiments. Biolistic transformation of U. procera leaf material with cauliflower mosaic virus 35S-promoted constructs resulted in transient gusA (β-glucuronidase) expression. Subsequently, regenerant U. procera have been obtained following transformation of stem pieces with wild-type tumor-inducing or root-inducing plasmid-harboring Agrobacterium strains. Genetically modified elms expressing gusA, gfp (green fluorescent protein), and nptII (neomycin phosphotransferase II) genes have been produced using disarmed vectors. Regenerant English elms have been produced following transformation with anti-fungal genes, transferred to soil, and are currently being tested for their ability to resist the Dutch elm disease fungus Ophiostoma novo-ulmi. Future prospects for fighting this fungal wilt using biotechnological tools are discussed.

The problem of environmental nitrogen enrichment is most likely to be solved by reducing the inputs of synthetic nitrogen fertilizers through the creation of cereals that, like legumes, are able to fix nitrogen. In legumes, rhizobia present intracellularly in vesicles in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Rhizobia within these membrane-bounded compartments are supplied with energy from plant photosynthates and, in return, the bacteria provide the plant with biologically fixed nitrogen. Recently, we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of the root meristems of maize, rice, wheat, and other major non-legume crops can be colonized intracellularly by the non-rhizobial, non-nodulating, nitrogen-fixing bacterium, Gluconacetobacter diazotrophicus, that occurs naturally in sugarcane. G. diazotrophicus expressing nitrogen-fixing genes is present in membrane-bounded compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume species, similar to the intracellular colonization of legume root nodule cells by rhizobia. In order to obtain an indication of the likelihood of adequate growth and yield of maize, for example, with reduced inputs of synthetic nitrogen fertilizers, we are determining the extent to which nitrogen fixation is correlated with systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs of synthetic nitrogen fertilizer.

The MuDR/Mu transposons of maize are the most aggressive DNA transposons known in terms of their ability to increase the forward mutation frequency by ∼100-fold. Their activities are held in check by multiple host mechanisms that restrict movement to late in development and that minimize the impact on the maize genome. Aspects of MuDR gene expression that are modulated include transcriptional start site choice, alternative intron splicing, and translational activity. Although not all features of MuDR transposase biology are understood, this element has been used to mobilize endogenous Mu elements and genetically engineered Mu transposons to conduct efficient transposon mutagenesis throughout the maize genome. RescueMu, which contains a copy of the pBluescript bacterial plasmid, has been used to sequence maize genes and to generate a large population of tagged alleles for subsequent genetic analysis. Derivatives of this element are in the testing phase for enhancer trap and activation gene tagging in maize.

Embryo development is a very key phase in the life cycle of seed plants. At maturity, the embryo contains the complete machinery to elaborate the entire plant body. While the embryogenic process is an innate feature of the zygote, gametic and somatic cells can undergo embryogenesis under the appropriate culture conditions. Embryogenesis is a highly regulated process and the use of mutants, especially in Arabidopsis, has allowed the identification of genes regulating pattern formation during this process. The use of such mutants has revealed the critical roles of auxin levels and transport in the establishment of embryo axis. Root and shoot apical meristem function and integrity have been defined by examination of genes involved in their identity and function. Further knowledge of the molecular and biochemical aspects of zygotic embryogenesis should contribute to our understanding of the underlying regulatory pathways and networks and also provide critical insights into unique totipotent features of the plant cell.

The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.

The study of plant viruses and their interaction with the plant host has contributed greatly to our understanding of plant biology. The recent development of plant viruses as transient expression vectors has not only enhanced our understanding of virus biology and antiviral defense mechanisms in plants, but has also led to the use of plant viral-based vectors as tools for gene discovery and production of recombinant proteins in plants for control of human and animal diseases. An overview of the state-of-the-art of viral expression systems is presented, as well as examples from our laboratory on their use in identifying nuclear targeting motifs on viroid molecules and development of therapeutic proteins for control of animal diseases.

The development of robust plant regeneration technology in cereals, dicots and ornamentals that is in turn coupled to a high-frequency DNA transfer technology is reported. Transgenic cereals that include maize, Tripsacum, sorghum, Festuca and Lolium, in addition to dicots that include soybean, cotton and various ornamentals such as petunia, begonia, and geranium have been produced following either somatic embryogenesis or direct organogenesis independent of genotype. Coupled with these regeneration protocols, we have also identified several interesting genes and promoters for incorporation into various crops and ornamentals. In addition, the phenomenon of direct in vitro flowering from cotyledonary nodes in soybean is described. In in vitro flowering, the formation of a plant body is suppressed and the cells of the cotyledonary node produce complete flowers from which fertile seed is recovered. This in vitro flowering technology serves as a complementary tool to chloroplast transformation for developing a new transgenic pollen containment strategy for crop species. Recently, the center has undertaken to screen the expression response of the 24 000 Arabidopsis genes to nitric oxide. This signaling molecule upregulated 342 genes and downregulated 80 genes. The object here was to identify a population of promoters that can be manipulated by using a signaling molecule. In addition, in keeping with the mission of enhancing greenhouse profitability for North West Ohio growers, we cloned a number of genes responsive for disease resistance from ornamentals that play an important role in disease management and abiotic stress. We have constructed a plant transformation vector with CBF3 gene under the rd29A promoter for engineering cold and freezing tolerance in petunia. Leaf discs of Petunia×hybrida v26 were used for Agrobacterium-mediated transformation, and 44 hygromycin-resistant T0 plants were obtained. The presence of CBF3 gene was confirmed in all the transgenic plants by PCR and Southern analyses.

Bioengineering approaches provide unprecedented opportunities for reducing poverty, food insecurity, child malnutrition, and natural resource degradation. Genetic engineering offers outstanding potential to increase the efficiency of crop improvement. Thus agricultural biotechnology could enhance global food production and availability in a sustainable way. Small farmers in developing countries are faced with many problems and constraints which biotechnology may assist. Yet, there are varying levels of opposition to the use of this technology in most countries and it is especially intense in Europe. While there is certain public apprehension with the use of bioengineering in food improvement, the primary hurdles facing this technology are the stringent and burdensome regulatory requirements for commercialization, opposition from the special interest groups, apprehension by the food industry especially with the whole foods, and trade barriers including rigid policies on traceability and labeling. Bioengineered crops such as soybean, maize, cotton, and canola with a few traits have already made a remarkable impact on food production and environmental quality. But, in the developing world, bioengineering of crops such as bananas, cassava, yams, sweet potatoes, sorghum, rice, maize, wheat, millet, and legumes, along with livestock, can clearly contribute to global food security. However, the integration of biotechnology into agricultural research in developing countries faces many challenges which must be addressed: financial, technical, political, environmental, activism, intellectual-property, biosafety, and trade-related issues. To ensure that developing countries can harness the benefit of this technology with minimal problems, concerted efforts must be pursued to create an awareness of its potential benefits and to address the concerns related to its use through dialog among the various stakeholders: policy makers, scientists, trade groups, food industry, consumer organizations, farmer groups, media, and non-governmental organizations. Biotechnology holds great promise as a new tool in the scientific toolkit for generating applied agricultural technologies; however, per se it is not a panacea for the world's problems of hunger and poverty.

A modified encapsulation–dehydration cryopreservation protocol based on the replacement of cold acclimation with high-sucrose pretreatment was assessed for the long-term storage of Ribes germplasm. Four steps in the procedure were examined for eight genotypes: (1) pregrowth of shoot tips in sucrose-supplemented solid growth medium for 1 wk; (2) pretreatment of alginate-encapsulated shoot tips in sucrose-supplemented liquid culture medium for 21 h; (3) evaporative desiccation of encapsulated–dehydrated shoot tips; and (4) exposure to liquid nitrogen (LN). Differential responses were observed for black currant and gooseberry genotypes. Recovery of growing shoots was high (72–100%) at all four steps for the five black currants tested. Evaporative desiccation slightly decreased viability for some black currants and in some cases LN exposure reduced regrowth. In contrast, three gooseberry species had poor recovery from the initial sucrose culture step (32–67%), indicating sensitivity to osmotic stress, which predisposed these genotypes to poor survival after LN exposure (12–26%). The effectiveness of the modified protocol for conserving a wider range of Ribes genotypes was further ascertained by screening 22 genotypes derived from nine Ribes species. The procedure was successful for 18 of the 22 genotypes in the gene bank in Scotland. Screening genotype responses at the time of storage demonstrated regrowth ≥60% for 15 genotypes, and only four genotypes had regrowth of 0–28%. Additional genotypes were also added to the USDA cryopreserved Ribes collection.

The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP.1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12 410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.

The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.

Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N⁶-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62 μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ. However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is suitable for further studies on embryo development and genetic transformation of Phalaenopsis.

Effects of genotype and explant orientation on shoot regeneration from cotyledonary explants of tomato were studied using 10 commercially important cultivars. The explant orientation affected shoot regeneration in all the tested genotypes. Cotyledons placed in abaxial (lower surface facing down) orientation consistently produced better shoot regenerative response and produced greater numbers and taller shoots compared to those inoculated in adaxial (upper surface facing down) orientation. Genotypic variation in terms of shoot regeneration response, number of shoots, and shoot height was apparent. Wounding of cotyledonary explants increased shoot regeneration response. However, shoot height was much lower in shoots regenerated from wounded explants compared to those that originated from intact cotyledons. Shoots produced from wounded cotyledons were abnormal in their form and structure.

The present study discusses the results of cytological studies of two kinds of sugar beet callus, i.e., embryogenic and non-embryogenic tissues. The calluses were produced through culture of secondary leaves on Murashige and Skoog medium containing two hormone combinations. One week after transfer of calluses onto fresh medium, their cells were viewed using electron microscopy and an image analyzer. Observations showed that cells of the two callus types had considerable differences in cell structure and various organelles. Of note were the high amount of polyploidization, rough endoplasmic reticulum, polysome, poly-nucleolus, and incomplete cell wall together with abnormal partitioning in non-embryogenic cells, as compared to embryogenic cells. In contrast, vacuolation of cytoplasm, perfect cell wall and partitioning structure, and the high proportion of nucleus/cytoplasm area were recognized in embryogenic cells.

Capsicum regeneration is often obtained through direct (adventitious) regeneration and only a few reports claim indirect regeneration (through callus) as an option to obtain complete plants. A possible reason for this may be because Capsicum cultures offer a narrow window in time for the regeneration process to occur, and after that, the ability to regenerate plants rapidly diminishes. In this study, the C. baccatum radicle-side half-seed was the explant of choice to induce the formation of callus on semisolid Murashige and Skoog (1962) medium supplemented with 5 mg l⁻¹ (22.2 μM) 6-benzylaminopurine, 1 mg l⁻¹ (5.7 μM) indole-3-acetic acid, and 2 mg l⁻¹ (6.1 μM) gibberellic acid. Organogenic calluses developed within 4–5 mo. and were subcultured every 2 mo. thereafter. Eventual bud elongation occurred and these shoots developed into complete plants after transfer to medium without plant growth regulators. The organogenic callus type retained its organogenic ability for more than 3 yr.

Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) seeds were germinated on modified Murashige and Skoog (MS) basal medium alone or supplemented with 2.3, 4.5, 11.3, 22.5, and 45.0 μM thidiazuron (TDZ), or 4.5, 13.6, 22.6, and 45.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 4.1, 12.4, 20.7, and 41.3 μM picloram. Both auxins delayed seed germination by approximately 5 d. Maximum germination was observed on MS medium supplemented with 45.0 μM TDZ. Callus induction occurred for seed germinated on 2,4-D and picloram-containing media, but not on TDZ medium. The greatest amount of callus (554 ± 198 mg per seed) was produced on 4.1 μM picloram. For shoot initiation, calluses were transferred to MS medium alone or supplemented with 2.5 μM TDZ. The highest number of shoots was recorded on TDZ medium from callus that had been obtained originally from media containing either 4.1 or 12.4 μM picloram or 13.6 μM 2,4-D (∼500). All shoots developed roots and grew to maturity on medium with 24.6 μM indolebutyric acid.

Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration response, line FC607 also had the highest mean number (8.3 ± 1.1) of shoots per explant. Individual plants within each line exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs.

Adventitious shoot induction and elongation was compared between root and petiole explants of Kentucky coffeetree (Gymnocladus dioicus L.) explants treated with a factorial combination of benzylaminopurine (BA) and thidiazuron (TDZ). Petiole explants initiated more adventitious shoots compared to root explants. Up to 83% of petiole explants initiated shoots compared to 67% of root explants. Maximal shoot induction was approximately 12 or five shoots per responding explant for petiole and root explants, respectively. For both explant types, TDZ was more effective than BA for shoot induction. There was an interaction between BA and TDZ on shoot induction in petiole explants, with the greatest percentage of explants forming shoots and the highest number of shoots initiated on the combination of 0.5 μM TDZ plus 10 μM BA and 1.0 μM TDZ plus 5 or 10 μM BA. In contrast, increasing concentrations of BA inhibited shoot initiation in root explants with and without TDZ. While BA inhibited shoot initiation in root explants, it promoted shoot initiation in petiole explants. In contrast, TDZ was equally effective at inducing shoots in root and petiole explants. This suggests that root and petiole explants of Kentucky coffeetree could be a useful model system for studying the differences in apparent mode of action between TDZ and BA on adventitious shoot initiation.

Embryonal-suspensor mass (ESM) lines characterized by a spiky morphotype (i.e., developed early embryos escaping from the ESM periphery) were recently shown to produce the best maturation yields in Maritime pine. How to select or preserve such a valuable morphotype during ESM maintenance (prior to maturation treatment) is still unknown. Several maintenance procedures were tested; 2400 ESM from 10 lines were subcultured each 7 or 14 d on maltose- or sucrose-containing medium without plant growth regulator (PGR) or supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine or a low concentration of abscisic acid. Multiple components analyses based on growth rate, macromorphology data, and micromorphology data, collected for 6 mo., allowed for the association of the spiky morphotype with additional traits (defining the phenotype I) such as whitish aspect, high vigor, high growth rate, and complex cellular organization (resulting from high early embryogenic ability). Since a gradual increase in growth rate as well as a decrease in cellular organization were concomitantly observed during the 6 mo. experiment, we concluded that ESM growth and early somatic embryo development were disconnected. In some lines, the progressive loss of early embryogenic ability (aging process) could be decreased using maltose-based and PGR-free medium. For most lines, the aging effect was minimized using a weekly subculture. An improved procedure for ESM maintenance prior to the maturation step is thus proposed.

An efficient system for the regeneration of plants from protoplasts was developed in Alstroemeria. Friable embryogenic callus (FEC) proved to be the best source for protoplast isolation and culture when compared with leaf tissue and compact embryogenic callus. Protoplast isolation was most efficient when FEC was cultured under vacuum for 5 min in an enzyme solution consisting of 4% cellulase, 0.5% Driselase and 0.2% Macerozyme, followed by culture for 12–16 h in the dark at 24°C. Cell wall formation and colony formation were better in a liquid medium than on a semi-solid agarose medium. Micro-calluses were formed after 4 wk of culture. Ninety percent of the micro-calluses developed into FEC after 12 wk of culture on proliferation medium. FEC cultures produced somatic embryos on a regeneration medium and half of these somatic embryos developed shoots. Protoplast-derived plants showed more somaclonal variation than vegetatively propagated control plants.

A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid 2.5 μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5 μM TDZ. Individual elongated shoots were rooted best on half-strength MS medium containing 0.5 μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150 μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.

An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9 μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9 μM BA and 1.34 μM NAA. The elongated shoots rooted within 7–8 d in half-strength MS basal salts supplemented with 1.34 μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.

Shoot cultures of apple cv. Pinova were contaminated with faint pink pigmented yeast. Yeast isolates were identified as Rhodotorula slooffiae with standard physiological methods and molecular analysis. Growth of isolated yeasts was tested against different fungicides. The following fungicides inhibited the growth of yeast isolates, and were not phytotoxic to apple shoots at concentrations lower than the minimal phytotoxic concentrations (MPC): ProClin^® 300, mancozeb, triforine, myclobutanil, thiabendazole, mancozeb zoxamid, and silver nitrate. Some fungicides inhibited growth of yeasts, but were phytotoxic. These included miconazole, PPM™, copper sulfate, potassium sorbate, and cycloheximide. Benomyl was not phytotoxic, but was effective only at high doses. Decontamination of shoots was achieved using a combination of two treatments. Shoots were first soaked in half-strength Murashige and Skoog (MS) liquid medium containing silver nitrate (588 μM) and Silvet 77 (0.01%) for 1–2 h, and then transferred to a solidified MS medium containing both mancozeb (15 mg l⁻¹) and thiabendazole (40 mg l⁻¹).

Heterotrophic plantlets obtained by in vitro propagation are biochemically different compared to autotrophic plantlets. When heterotrophic plantlets are transferred to ex vitro conditions, higher irradiance levels are generally applied. Irradiance levels higher than those used in vitro lead to oxidative stress symptoms that can be counteracted by CO₂ concentrations above normal. We analyzed the stability and activity of Rubisco and leaf-soluble sugars and starch contents in chestnut plantlets transferred from in vitro to ex vitro conditions under four treatments obtained by associating two irradiances of 150 (low light, LL) and 300 (high light, HL) μmol m⁻² s⁻¹, respectively three and six times in vitro irradiance, with two CO₂ levels of 350 (low CO₂, LCO₂) and 700 (high CO₂, HCO₂) μl l⁻¹. In in vitro plantlets it was possible to immunodetect apparent products of degradation of Rubisco large subunit (LSU). In ex vitro plantlets, these degradation products were no longer detected except under LL associated with LCO₂. The decrease in soluble sugars and starch in plantlets under HL HCO₂ gave an indication of a faster acquisition of autotrophic characteristics. However, under the same treatment, a down-regulation of Rubisco activity was observed. From the results taken as a whole, two aspects seem to be confirmed: HL HCO₂ is more efficient in inducing an autotrophic behavior in chestnut ex vitro plantlets; actively growing systems as ex vitro plantlets reflect the down-regulation of Rubisco by HCO₂ without accumulation of carbohydrates.

An efficient rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Eclipta alba (Asteraceae) by enhanced axillary shoot proliferation in cotyledonary node segments was designed. The medium type, various carbon sources, plant growth regulators, and coconut water markedly influenced in vitro propagation of Eclipta alba. An in vitro plantlet production system has been investigated on Murashige and Skoog (MS) medium with the synergistic combination of benzyladenine (4.4 μM), kinetin (4.6 μM), 2-isopentenyladenine (4.9 μM), gibberellic acid (1.4 μM), 5% coconut water, and 3% sucrose which promoted the maximum number of shoots as well as beneficial shoot length. Subculturing of cotyledonary node segments on a similar medium enabled continuous production of healthy shoots with similar frequency. Rooting was highest (94.3%) on full strength MS medium containing 9.8 μM indolebutyric acid. Micropropagated plants established in garden soil, farmyard soil, and sand (2:1:1) were uniform and identical to the donor plant with respect to growth characteristics as well as floral features. These plants grew normally without showing any morphological variation.

In vitro propagation systems by means of areole activation were developed for Turbinicarpus laui, T. lophophoroides, T. pseudopectinatus, T. schmiedickeanus subsp. flaviflorus, T. schmiedickeanus subsp. klinkerianus, T. schmiedickeanus subsp. schmiedickeanus, T. subterraneus, and T. valdezianus. In vitro-germinated seedlings were used as a primary source of explants. Multiple shoot formation from areoles was achieved for three explant types (apical, lateral, and transverse), cultured on Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 10 g l⁻¹ agar and several treatments with cytokinins. Efficiencies were in the range from 7.8 shoots per explant in T. valdezianus up to 19.7 shoots per explant in T. pseudopectinatus, using the best treatment for each species and in a single proliferation cycle. Four of the studied species responded best when 6-benzylaminopurine (3.3–8.8 μM) was used, while 6-(γ,γ-dimethylallylamino)purine (19.7–24.6 μM) showed better results in two species. The two remaining species showed no significant differences in their response to both cytokinins. Regarding explant type, the best results were obtained with transverse cuts for five species, with apical explants for one species, and the two remaining species showed no significant differences among the explants tested. Rooting of the in vitro-generated shoots was achieved most efficiently on half- or full-strength MS basal medium. Rooting frequencies were in the range from 54.2 to 94.2%, and the frequency of survival of the plants once transferred to soil was 91.6% on average.

Leaf-tip necrosis of micropropagated statice plantlets is a serious problem in commercial laboratories in Taiwan. Endophytic bacteria were detected in plantlets obtained from commercial laboratories with a leaf-tip necrosis problem. Endophytic bacteria were detected in flower stalks collected from four different statice farms at frequencies ranging from 61 to 100%. All plantlets regenerated from flower-stalk explants that tested free of endophytic bacteria did not develop leaf-tip necrosis. The most frequently detected endophytic bacteria were Pasteurella multocida, Stenotrophomonas maltophilia, and Alcaligenes sp. Most endophytic bacteria in statice plantlets were eliminated by the subculture of plantlets on medium with augmentin, cefotaxime, or augmentin plus cefotaxime. Those plantlets freed from endophytic bacteria by subculture on antibiotic-amended medium did not develop leaf-tip necrosis. Our results show that leaf-tip necrosis of micropropagated statice plantlets is associated with endophytic bacteria, and that the disease can be controlled by using explants pre-tested to be free from endophytic bacteria or by the subculture of affected plantlets on antibiotic-amended medium.

The photosynthetic capacity changes and the main enzymatic systems related to carbon metabolism were investigated during the in vitro culture of plantain shoots (Musa AAB cv. CEMSA 3/4) in temporary immersion bioreactors (TIB) and their subsequent acclimatization. The maximal rate of photosynthesis (Pn), transpiration, and the activity of the carbon metabolism enzymes phosphoenolpyruvate carboxylase (PEPC), acid invertase (AI), pyruvate kinase (PK) and sucrose phosphate synthase (SPS) were measured every 7 d during the 21 d of elongation in TIB, and the following 42 d of acclimatization. Sucrose content in the liquid medium and in the leaves was also determined. The most significant changes in plant growth were observed during acclimatization. During the in vitro stage photosynthesis was limited (4–6 μmol CO₂ m⁻² s⁻¹); the photosynthetic rate however increases rapidly and significantly as soon as in vitro culture is over during acclimatization. PEPC activity increased during the whole evaluation period. The highest levels were achieved around days 42 and 56. PK and SPS activities were optimal during the first weeks in acclimatization (28–35 d), while AI increased at the beginning of the elongation phase (7 d), and later at the end of the acclimatization (49–63 d). The relationships between morphological parameters, photosynthetic capacity of the plantlets and the carbon metabolism enzymes during both phases of the culture are discussed.

Silicone tubing is frequently used for gas exchange in cell culture systems, due to its biocompatibility and high permeability to CO₂ and O₂. In cell culture chambers, medium pH and oxygen levels are often maintained by gas exchange through a coil of silicone tubing. Culture medium is recirculated between the gas exchanger and the culture chamber which contains a suspension of cells. We report that the type of agent used for silicone vulcanization (peroxide or platinum) can markedly affect its biocompatibility, and that tobacco cell culture represents a particularly sensitive indicator of tubing cytotoxicity. Under the conditions studied (cell suspension maintained with forward–reverse flow and stirring), peroxide-cured silicone tubing was toxic to the tobacco BY2 cell culture, in contrast to the platinum-cured silicone tubing that was completely biocompatible. Upon further investigation by mass spectrometry, it was determined that a component with a molecular mass of 288 Da, possibly a tetrachlorinated biphenyl, was present in culture medium in contact with peroxide-cured tubing but not in medium in contact with platinum-cured tubing. Additional curing of peroxide-cured tubing resulted in cell morphology and viability comparable to controls. These data suggest that improperly cured silicone tubing can release catalytic byproducts which can be toxic to plant cells, and that the BY2 tobacco cells represent a suitable model system for studies of materials biocompatibility.

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