Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hosta tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% \[both with and without 1 μM benzyladenine (BA)\]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7× and 2.3× for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1× for agar and 3.4× for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1 μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.

Pathogens of plants produce effector proteins necessary for successful parasitism. The effectors enhance pathogen virulence by manipulating signaling in the plant. Plants produce resistance (R) proteins that mediate recognition of specific effectors and respond by initiating plant defenses. In many cases, R-proteins perceive effectors indirectly; virulence signaling initiated by the effector is shunted, via the R-protein, into a resistance response. Therefore, by understanding how effectors manipulate virulence targets we will concurrently gain insight into how this signaling elicits R-protein-mediated defense responses.

This article reviews techniques for gene identification and cloning in allohexaploid bread wheat (Triticum aestivum L.). Gene identification and cloning in wheat are complicated by the large size and high redundancy of the genome. Both classical mutagenesis and transposon tagging are important tools for the study of grain dormancy and plant hormone signaling in wheat. While classical mutagenesis can be used to identify wheat mutants with altered hormone sensitivity, it can be difficult to clone the corresponding genes. We review the techniques available for gene identification in wheat, and propose that transposon-based activation tagging will be an important tool for wheat genetics.

Embryogenic soybean \[Glycine max (L.) Merrill\] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration, inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48 kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.

Creeping bentgrass is a very important turfgrass species used extensively on golf course greens, fairways, and tees. One of the challenges of creeping bentgrass management is the control of grassy weeds, most of which respond to herbicides in a similar manner to that of creeping bentgrass. As part of a weed management program for golf courses, Roundup^®-tolerant creeping bentgrass will be simple to employ and more effective in controlling problem weeds than currently available methods. The goal of this research was to evaluate fitness-related reproductive traits in four transgenic creeping bentgrass events modified to express a Roundup^®-tolerant gene, cp4 epsps, to determine if these creeping bentgrass events had gained an unexpected reproductive fitness advantage. We compared transgenic events ASR333, ASR801 with their non-transformed tissue culture line, C99056L and transgenic events ASR365, ASR368 with their non-transformed tissue culture line, B99061R. Populations of plants from three conventional cultivars were also included for comparison to determine whether significant variations, if present in transgenic events, were novel to the non-transformed organism, Agrostis stolonifera L. Our results showed that none of the four transgenic events surveyed were significantly different from the respective non-transformed tissue culture line plants for the following characteristics: first heading date, anthesis duration, inflorescence length, number of florets per inflorescence, pollen size, and seed-set capacity through open-pollination. One of the transgenic events, ASR333, needed significantly more days for anthesis initiation than the non-transformed tissue culture line, C99056L; while another transgenic event, ASR801, exhibited significantly shorter pollen longevity than plants of the tissue culture line, C99056L. However, ASR801 was not significantly different from the conventional cultivars ‘Penn A-4’ and ‘Penncross’ for pollen longevity. Plants of both transgenic events ASR365 and ASR368 did not differ significantly from plants of the tissue culture line, B99061R, for all characters measured.

The liliaceous perennial plants, Tricyrtis spp., are cultivated as ornamental plants in Japan. Natural populations of several Japanese Tricyrtis spp. are severely threatened by indiscriminate collection and habitat destruction. In this study, a plant regeneration system based on somatic embryogenesis has been developed for efficient clonal propagation of T. hirta, T. hirta var. albescens, T. formosana, T. formosana cv. Fujimusume, T. flava ssp. ohsumiensis, and T. macrantha ssp. macranthopsis. Flower tepal explants of these genotypes were cultured on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) alone or in combination with N-(1,2,3-thiadiazol-5-yl)-N′-phenylurea (thidiazuron, TDZ). Calluses induced on media containing 2,4-D produced somatic embryos following their transfer to a plant growth regulator-free medium, indicating that these calluses were embryogenic. A combination of 4.5 μM 2,4-D and 0.45 μM TDZ was most effective for inducing embryogenic calluses from tepal explants. Among various explant sources, filaments were most suitable for inducing embryogenic calluses on a medium containing 4.5 μM 2,4-D and 0.45 μM TDZ. Embryogenic calluses were only obtained from filament explants for T. macrantha ssp. macranthopsis. Embryogenic calluses could be maintained by subculturing monthly onto the same medium, and a 1.5–3.5-fold increase in fresh weight was obtained after 1 mo. of subculture. Depending on the plant genotype, 50–500 somatic embryos per 0.5 g fresh weight of embryogenic callus was obtained 1 mo. after transfer to a plant growth regulator-free medium. Most of the embryos developed into plantlets, and they were successfully acclimatized to greenhouse conditions. Regenerated plants showed no alteration in the ploidy level as indicated by chromosome observation and flow cytometric analysis.

Tamarind, a multipurpose tropical tree species, is economically important for sustainable development of wasteland due to its hardy nature and adaptability to various agroclimatic conditions. Reports on in vitro morphogenesis in this species are limited, due to its recalcitrant and callogenic nature. To overcome these limitations, an attempt was made to induce meristematic activity in seedling explants. Seedlings were germinated in medium with or without thidiazuron (4.54, 9.08, 13.12, 18.16 μM). This growth regulator restricted the differentiation of the apical meristem to form shoots. It triggered proliferation of the meristematic tissue at the cotyledonary node and a large number of meristematic buds appeared in a radial pattern around the node. The meristematic activity extended to the junction of the epicotyl and hypocotyl, giving rise to buds in the form of protuberances in all sides of the junction. These buds differentiated to form shoot primordia and subsequently to shoots in medium devoid of growth regulators. Plants developed by micrografting of these shoots on seedling-derived rootstocks survived in soil.

In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary–torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ l-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.

An in vitro culture procedure was established for repetitive embryogenesis and plant regeneration from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu (Orchidaceae). Seed-derived protocorms were cultured on modified half-strength Murashige and Skoog (1962) basal medium (½MS) devoid of plant growth regulators. After 45 d, 28.1% of protocorms formed embryos from their posterior regions. 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45, 4.54, and 13.62 μM) promoted direct embryo formation. The best response was at 13.62 μM TDZ, and 100% of the protocorms formed a mean number of 13.5 embryos after 45 d of culture. By contrast, naphthaleneacetic acid (NAA) at 0.54 and 5.37 μM inhibited direct embryo formation. On basal medium devoid of plant growth regulators, 18.8% of primary proliferating embryos could form more embryos. TDZ (0.45, 4.54, and 13.62 μM) also promoted this process. Proliferating embryos/protocorms were transferred to basal medium devoid of plant growth regulators for plantlet formation. Plantlets were successfully obtained from the embryos after 4–6 wk. Following subculture every 6 wk for three passages, the plantlets were transferred to sphagnum moss in a container for acclimatization in the greenhouse. The survival rate was 100%.

Despite high commercial interest, the success of biotechnological applications in cotton (Gossypium hirsutum) has been limited due to difficulties in genetic transformation. Major problems have been genotype dependence and low frequency of somatic embryogenesis, making it difficult to regenerate plants from transgenic tissue. This study reports an increase in somatic embryogenesis efficiency and the induction of developmental synchrony in embryogenic callus cultures of cotton by a single cycle of myo-inositol depletion in liquid culture. Calluses were initiated on hypocotyl or cotyledon explants of cultivar Coker 312 by culturing these explants on callus-inducing solid medium \[Murashige and Skoog salts plus vitamins of Gamborg's B₅ medium, 30 g l⁻¹ glucose, 100 mg l⁻¹ myo-inositol, 2.2 μM 2,4-dichlorophenoxyacetic acid, and 0.88 μM 6-benzyladenine\]. The calluses were transferred to an identical liquid basal medium devoid of plant growth regulators. This induced the development of embryogenic cells. Friable clumps of cells formed after 20 d in the medium were selectively collected over filter mesh 40 and subjected to one cycle of myo-inositol starvation. This induced a highly synchronized embryogenesis in the culture. The optimized protocol gave 100% embryos at the globular stage, out of which more than 80% developed into bipolar torpedo-stage embryos. About 68% of these were converted to plantlets by subculturing onto a simplified solid medium, and finally grown into healthy, fertile plants.

Black grama (Bouteloua eriopoda) is an important forage grass in southwestern USA rangelands. Plants were regenerated by somatic embryogenesis. Surface-disinfested seeds were germinated and the embryonic shoots were excised and cultured on Murashige and Skoog (MS) medium gelled with agar. Callus was induced from apical meristems. Calluses were cultured on MS solid medium with six concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or Dicamba (6-dichloro-o-anisic acid) for 6 wk under light or dark conditions. Somatic embryo induction was greatest on 4.52 μM Dicamba, under light, after transferring to an auxin-free medium. Embryo development progressed from globular torpedo to mature embryos phenotypically identical to those naturally produced in seed. These germinated and grew into intact plants and were established in soil and grown to maturity. To our knowledge, this is the first report of somatic embryo induction and regeneration in black grama grass.

Baloskion tetraphyllum is a member of the Restionaceae and is an important species for the rehabilitation of disused mine sites and wetland areas, and is also highly prized as a cut flower. Its use for restoration of disturbed land is, however, severely limited, due to very poor propagation success by conventional methods. A study was conducted to evaluate the potential of somatic embryogenesis for the large-scale propagation of this species. A variety of auxins (at different concentrations) were investigated for their efficacy in stimulating somatic embryogenesis. Somatic embryos were successfully induced from excised coleoptiles of B. tetraphyllum on half-strength Murashige and Skoog (½MS) medium supplemented with 1 μM 2,4-dichlorophenoxyacetic (2,4-D). To scale up the production, proliferation of secondary somatic embryos was achieved using primary somatic embryos as the tissue source on ½MS 1 μM 2,4-D resulting in a 30-fold increase in somatic embryo numbers. Almost all the somatic embryos developed into plants and were established ex vitro. The other auxins investigated, including p-chlorophenoxyacetic acid, indole-3-acetic acid, α-naphthaleneacetic acid, and picloram, were not as effective as 2,4-D. The age of the explant material significantly influenced somatic embryogenesis with white, young coleoptiles (5–7 d) producing 50% more somatic embryos than green, more mature (8–14 d) coleoptiles. The protocol developed for B. tetraphyllum has the potential to be commercially viable, with an estimated 22 000 somatic embryos produced from 1 g of plant material. This research may also have a positive impact on the propagation of other important Restionaceae species.

Although pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore, we decided to modify the culture medium composition of this phase looking for an increase in protease excretion. Four independent experiments were performed to evaluate the effects of different levels of sucrose (0–350.4 mM), inorganic salts \[0–200% Murashige and Skoog (MS) salt strength\], inositol (0–2.20 mM), and thiamine (0–1.2 μM). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. Specific protease activity, the most important indicator recorded, was highest with 262.8 mM sucrose, 100% MS salt strength, 0.3 μM thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 mM sucrose, 100% MS salt strength, 0.55 mM inositol, 0.3 μM thiamine) are not always adequate for protease excretion.

We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum) I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4 wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1 ± 0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.

Chinese leymus is a perennial grass in the Gramineae family, which is widely distributed in Northern China and Mongolia. The breeding cycle in Chinese leymus is time-consuming because of its high level of seed dormancy. To improve the breeding process, we established a simple technique that shortens the breeding cycle by culturing immature embryos in vitro to produce plantlets immediately. This technique can potentially save 1 yr in each sexual breeding cycle. Factors affecting germination of immature embryos were evaluated. Immature embryos at different developmental stages were cultured on Murashige and Skoog basic medium. The immature embryos collected 11–16 d after pollination expressed the maximum germination rate (100%), but the maximum efficiency of recovery was observed at 15–16 d after pollination. The germination time of embryos increased and length of the germinated shoots decreased as the age of the embryos increased. All plantlets survived transplantation to the field. Two generations were obtained within 2 yr starting from immature embryo cultures, as compared to one generation using conventional breeding techniques.

Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6 μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.

Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 μM α-naphthaleneacetic acid (NAA) and 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants callused at a frequency of 18.2–100%, while somatic embryos were produced from these calluses at a frequency of 25–87.5% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 μM, respectively, and maintained by subcultures every 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1% activated charcoal, and roots >5 mm in length developed within 3 wk. A 7-d exposure to 3 μM gibberellic acid and 5 μM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4 yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.