Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7±7.8% and 20.1±4.7%, respectively, and the numbers of buds per explant were 5.9±1.6 and 3.2±0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.

An efficient plant regeneration protocol was established for castor (Ricinus communis L.). Hypocotyl tissue from zygotic embryo axis produced adventitious shoots when treated with either thidiazuron (TDZ, 1 μM) or 6-benzylaminopurine (BA, 20 μM). TDZ resulted in more than a threefold higher rate of shoot induction (a maximum of 24.2 shoots per explant) than BA (6.8 shoots). Our results also showed that the pretreatment of explants in the dark increased the number of shoots regenerated per explant by 82% and 36% with TDZ and BA, respectively. The elongation of hypocotyl tissue in the dark appears to be the primary cause of the increase. Comparable rates of rooting were achieved on the media supplemented with either indole-3-butyric acid (IBA, 84.3%) or 1-naphthalene-acetic acid (NAA, 87.4%) at 5 μM. However, IBA was more efficient in promoting root and shoot development, resulting in a higher rate of establishment (93.5%) in the soil, compared to the rate with NAA (39.5%). Histological analysis showed the adventitious induction of the shoot buds originated from the cortex of the hypocotyl tissue.

Burma reed (Neyraudia arundinacea Henr.) is a C₄ grass native to Southeast Asia and Indomalaya that grows quickly, exhibits strong resistance to environmental stresses, and is extremely adaptable. It can be widely utilized as a bioenergy crop for biomass conversion. In vitro multiple shoots were first established from axillary buds and then subcultured on propagation medium containing 10 μM 6-benzylaminopurine (BA) and 2.0 μM naphthaleneacetic acid (NAA). Multishoot clumps were used as explants to induce somatic embryogenesis and adventitious shoot formation. The results showed that auxin 2,4-dichlorophenoxyacetic acid or NAA play a key role for the induction of somatic embryogenesis and adventitious shoot formation, whereas cytokinin BA or kineatin enhance shoot proliferation and plant regeneration from callus and somatic embryos. Efficient somatic embryogenesis, mass propagation, and plant regeneration systems in Burma reed were established.

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.

Induction of somatic embryogenesis in Pinus armandii var. amamiana, an endemic and endangered species in Japan, was initiated from megagametophytes containing immature zygotic embryos on both media with and without plant growth regulators. Across nine open-pollinated families initiation frequency ranged from 0 to 20%, with an average of 1.5%. Embryogenic cultures were maintained and proliferated on a medium supplemented with 2,4-dichlorophenoxyacetic acid (3 μM) and 6-benzy-laminopurine (1 μM). Maturation of somatic embryos occurred on medium containing maltose (50 g l⁻¹), activated charcoal (2 g l⁻¹), abscisic acid (100 μM), and polyethylene glycol (100 g l⁻¹). The frequencies of germination and plant conversion of somatic embryos differed among the embryogenic lines from 16 to 51% and from 12 to 40%, respectively. Growth of regenerated somatic plants has been monitored in the field.

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Piclo-ram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single-and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P=0.0242) but the zone of explant origin (P=0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P=0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.

Up to 73% decrease in cost of media for plant regeneration and in vitro conservation was achieved in Curcuma longa cv Prathibha by using inexpensive carbon source and gelling agent. Laboratory reagent-grade sucrose was replaced by locally available commercial sugar (market sugar or sugar cubes) as carbon source and bacteriological grade agar by isabgol (also named isubgol) as gelling agent. No adverse effects on shoot regeneration and conservation on isabgol-gelled low-cost media were observed as compared to that on agar-gelled control medium (CM). Some 33–56% cultures of C. longa survived up to 12 mo. on isabgol-gelled medium in comparison to only 16% on CM. Genetic stability of 12-month-old in vitro-conserved plants was assessed using 25 random amplified polymorphic DNA (RAPD) primers; no significant variation was observed in RAPD profiles of mother plants and in vitro-conserved plantlets on CM and low-cost media.

An efficient protocol was developed for micro-propagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2±0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4±0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4±0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.

This paper addresses the assumed autonomy of vascular plants by revealing the presence of an obligate fungus structurally integrated with leaf anatomy of C4 grasses. We examined leaf surfaces of 26 species representing 14 genera of C4 grasses. In all species, we found similarities between leaf surface microhair-like structures and Uredomycete teliospores. These bicellular structures produced hyphae and spores, confirming they were fungal, rather than plant tissue. The plant-fungus structural morphology was also observed in Bouteloua eriopoda plants regenerated from embryonic meristem cells. The conserved symbiosis between fungi and C4 grasses suggests a lichenous association with evolutionary significance. The structural integration of endosymbiotic fungi with cells and tissues offers novel and unexplored approaches to developing physiological, ecological, and systematic models of C4 grasses.

The primary utilization of carbohydrates by cell suspension cultures of Rudgea jasminoides, a native woody Rubiaceae from tropical forests, was investigated. Sucrose, glucose fructose, glucose, or fructose were supplied as carbon sources. The growth curves of R. jasminoides cultured in glucose fructose, glucose, or fructose showed similar patterns to that observed when sucrose was supplied to the cells, except that an increase in dry mass was observed at the beginning of the stationary growth phase in the media containing only one monosaccharide. The increase in hexose levels in the media during the early stages of the cultures indicated extracellular hydrolysis of sucrose, which was further supported by the increase in the activity of acid invertase bound to the cell wall. Glucose was preferentially taken up, whereas uptake of fructose was delayed until glucose was nearly depleted from the medium. Measurements of intracellular sucrose content and cytoplasmatic and vacuolar invertases indicate that the enzymatic activity seems to be correlated with a decrease in the hexose flux into the cells of R. jasminoides. Our results indicate that the behavior of cell suspension cultures of R. jasminoides regarding sugar utilization seems to be similar to other dicotyledonous undifferentiated cell suspension cultures.

Agapanthus (Agapanthus praecox ssp. orientalis), a liliaceous perennial, is cultivated as an ornamental plant because of its beautiful, blue-violet to white flowers. We have previously produced transgenic plants containing the ß-glucuronidase (GUS) reporter gene under the control of cauliflower mosaic virus (CaMV) 35S promoter in an experimental strain of Agapanthus via Agrobacterium-mediated genetic transformation. These transgenic plants have been cultivated for 5 yr after transplantation to pots. In the present study, we carried out morphological characterization, and examination of the ploidy level, pollen fertility and stability of GUS gene expression in 12 transgenic plants derived from five independent lines at the flowering stage. Flow cytometry (FCM) analysis indicated that transgenic plants of four lines kept the diploid level, but those of the remaining one line were tetraploid. For all of the 12 transgenic plants, some morphological variations were observed both in vegetative and floral organs such as decreased number of leaves per inflorescence, smaller leaves, shorter inflorescence stalks, decreased number of florets per inflorescence and smaller florets. Pollen fertility of all the transgenic plants was below 5% as determined by acetocarmine staining. All the 12 transgenic plants showed stable expression of the GUS gene in leaves, roots, tepals, pistils, and stamens, as indicated by histochemical GUS assay, fluorometric GUS assay, and/or real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. No apparent GUS gene silencing was observed in transgenic agapanthus plants even after 5 yr of cultivation.