High irradiance increases organogenesis in friable callus of <Emphasis Type="Italic">Caustis blakei</Emphasis> Kük. (Cyperaceae)

Summary Caustis blakei is an attractive cut foliage plant harvested from the wild in Australia and marketed under the name of koala fern. Previous attempts to propagate large numbers of this plant have been unsuccessful. The effect of four light irradiances on organogenesis from compact and friable callus of C. blakei was studied for 21 wk. Both callus types produced numerous primordial shoots but many failed to develop into green plantlets. However, significantly more primordial shoots and green plantlets developed on the friable callus than on the compact callus, and significantly more green plantlets were regenerated under the higher photon irradiances of 200 and 300 μmol m⁻²s⁻¹ than under the lower irradiances of 100 and 150 μmol m⁻²s⁻¹. The compact callus produced its maximum number of green plantlets early in the experiment (after 9 wk), while the friable callus continued to produce primordial shoots and green plantelets throughout the period of the experiment, and reached its maximum production of green plantlets at 21 wk under the irradiance of 300 μmol m⁻²s⁻¹. Organogenesis from friable callus under high irradiance (300 μmol m⁻²s⁻¹) offers an efficient propagation method for C. blakei.     

High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Regeneration of almond from immature seed cotyledons

Adventitious shoots were regenerated from immature cotyledons of four almond cultivars (`Ne Plus Ultra', `Nonpareil', `Carmel' and `Parkinson'). Open-pollinated fruit were collected from orchard-grown trees 100–115 days after full bloom. The proximal ends of the cotyledons were excised and the embryonic axes discarded. The effects of different concentrations of thidiazuron (TDZ) and indole-3-butyric acid (IBA) and the presence or absence of light for the first 7 days of culture were determined. Shoot regeneration rates were highest for cotyledons cultured for 8 weeks on Murashige and Skoog (MS) basal medium containing TDZ (10.0 M), followed by 4 weeks on medium without plant growth regulators. Regeneration levels were further improved if cotyledons were maintained in darkness for the first 7 days. IBA (0.5 M) significantly reduced the development of adventitious shoots. The frequency of cotyledons that developed adventitious shoots under the optimum tested conditions for `Ne Plus Ultra', `Nonpareil', `Carmel', and `Parkinson' were 80.0%, 73.3%, 100.0% and 86.7%, respectively.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

Factors affecting regeneration of pigeonpea (Cajanus cajan L. Millsp) from mature embryonal axes

Regeneration of viable plants was obtained via organogenesis from mature embryonal axes explants of pigeonpea. Shoots were produced from the apical region of embryonal axes after 20 days of dark incubation on modified Murashige and Skoog (1962) medium containing 8.86 M benzylaminopurine (BAP) and 1.07 M -naphthalene acetic acid (NAA). When the explants were cultured under light-dark (16--8 hrs) conditions, shoots were initiated only after 65 days of culture initiation. The explants lost their ability to regenerate plantlets when they were cultured in continuous light. Regenerated shoots elongated either in the same medium or in MS basal medium. About 40% of the elongated shoots sequentially produced simple, bifoliate and finally trifoliate leaves instead of producing trifoliate leaves directly. The elongated shoots were rooted efficiently upon transferring them to half strength MS medium supplemented with 2.41 M indole-3-butyric acid (IBA). Rooted plantlets were successfully transferred to soil after hardening.     

Influence of bombardment with BA-coated microprojectiles on shoot organogenesis fromPhlox paniculata L. andPinus pinea L. tissues

Summary Leaves ofPhlox paniculata L. were bombarded with BA-coated gold microprojectiles with the Biolistic PDS/1000-He gene gun. Increasing the concentration of BA from 0.01 to 0.1 mg/μl increased the mean number of adventitious shoots from 7 to 16 shoots per leaf. Bombardment of cotyledons ofPinus pinea L. (Italian stone pine) with BA-coated gold particles significantly reduced the frequency of shoot organogenesis by about 40%. However, induced shoot regenerants from bombarded explants of bothP. paniculata andP. pinea were longer (>1 cm) than those derived from control explants after 3 and 5 wk following bombardment, respectively.     

Shoot regeneration in response to carbon source on internodal explants of Annona muricata L.

Adventitious shoot regeneration from internodal explants of mature plants of Annona muricata L. was obtained on Nitsch media. Meristems were induced with sorbitol as the sole carbon source supplemented with 2 mg l^–1 of benzylaminopurine and 0.5 mg l^–1 naphthaleneacetic acid. Adventitious shoots were developed only when the explants were transferred onto growth regulator-free media containing sucrose, galactose, or glucose. A hypothesis is proposed for the involvement of sorbitol in the induction and development of de novo shoots from internodal explants of mature trees of A. muricata.     

Cyclin‐dependent kinase activity maintains the shoot apical meristem cells in an undifferentiated state

As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration between cell division rates and fate specification is essential for normal organ formation and plant development. However, the inter‐dependence of cell‐cycle machinery and meristem‐organizing genes is still poorly understood. To investigate this mechanism, we specifically inhibited the cell‐cycle machinery in the shoot apex by expression of a dominant negative allele of the A‐type cyclin‐dependent kinase (CDK) CDKA;1 in meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven into the differentiation pathway, as indicated by premature cell expansion and onset of endo‐reduplication. Although the meristem structure and expression patterns of the meristem identity genes were maintained in most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a threshold model in which the effect of the cell‐cycle machinery on meristem organization is determined by the level of CDK activity.