Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with α-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett®, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett® system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia.

Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture.

Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method.

High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers

Excised tissues from transverse young stem sections of Lilium longiflorum were cultured on Murashige and Skoog medium supplemented with growth regulators at various concentrations. After 45 d in culture, the presence of α-naphthaleneacetic acid (NAA) in the culture medium at 5.4 μM resulted in bulblet formation while 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.2 μM resulted in root formation. The presence of IBA (indole-3-butyric acid) in the culture medium at 1.0 μM resulted in shoot formation while plantlet formation occurred when IBA was added at a concentration of 2.0 μM. When 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) was added to the culture medium at 1.1 μM, protocorm-like bodies (PLBs) formed, while 2.2 μM resulted in shoot formation (on abaxial and adaxial surfaces). The presence of NAA and TDZ in the culture medium at 5.4 μM and 0.4, 1.1 or 2.2 μM, respectively, resulted in somatic embryo formation while NAA- and 6-benzylaminopurine-(BA) containing culture medium formed callus or bulblets. The establishment of different regeneration systems when explants are exposed to various growth regulators demonstrates that the choice of growth regulator combinations and concentrations are of significance in determining the morphogenetic response and plant regeneration capacity.

Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin

Lilies are one of the most valuable bulbous ornamental cut flower crops and are commercially propagated by conventional scaling, which results in varied bulb quality. This article demonstrates the effectiveness of using specific organ sources in conjunction with the thin cell layer (TCL) system to establish successful organogenic and somatic embryogenic pathways in Lilium longiflorum. TCLs derived from different explant sources, such as receptacle, leaf, young stem, stem node, bulblets, pseudo-bulblets, shoot and somatic embryos, respectively, can be manipulated to form different organs. Furthermore, the choice of plant growth regulator and medium additives such as activated charcoal and sucrose strongly affect the success of the process. This article further highlights that transient transgene expression derived from either particle bombardment or Agro-infection is both strong and repeatable, as a result of the direct exposure of competent cells to the gene introduction method, resulting in a reduction of escapes. The ability to manipulate and control organogenesis through a TCL system, coupled with repeatable, efficient transformation opens up the possibility of micropropagating members of this genus with new and available characteristics, producing superior plants of high quality.

Thin cell layer culture system in Lilium : Regeneration and transformation perspectives

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [Eleusine indica (L) Gaertn] and its dinitroaniline-resistant biotypes The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1–2 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l–1 glycine, 100 mg l–1 asparagine, 100 mg l–1 casein hydrolysate, 30 g l–1 sucrose and 06% agar, pH 57 The presence of organogenic and embryogenic structures in these calli was histologically documented Cell suspension cultures derived from young calli were established in a liquid medium with the same composition Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l–1 kinetin (Kn) and 01 mg l–1 indole-3-acetic acid (IAA), 3% sucrose, 06% agar, pH 57 Calli derived from the R-biotype of E indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants Embryogenic cell suspension culture was a better source of E indica protoplasts than callus or mesophyll tissue The enzyme solution containing 15% cellulase Onozuka R-10, 05% driselase, 1% pectolyase Y-23, 05% hemicellulase and N6 mineral salts with an additional 02 M KCl and 01 M CaCl2 (pH 54–55) was used for protoplast isolation The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l–1 2,4-D and 02 mg l–1 Kn

Efficient callus formation and plant regeneration of goosegrass [Eleusine indica (L.) Gaertn.].

Callus induction, somatic embryogenesis and plant regeneration were obtained in two cultivars of Sorghum bicolor (L.) Moench. Transverse thin cell layers from roots/epicotyls of 15-day-old seedlings or of regenerated plantlets were used. Callus response depended on the genotype, the size of transverse thin cell layers, the level at which transverse thin cell layers were excised on the epicotyl, the composition of growth substances and the number of in vitro regeneration cycles undergone by the donor plant. Somatic embryos were differentiated under a defined dark/light sequence, from epidermised compact calluses (i.e having already differentiated an epidermis), obtained directly with dicamba or from friable callus initiated with kinetin and 2,4 dichlorophenoxyacetic acid. The importance of kinetin and dicamba on the induction of embryogenic potential is reported.

Van Le B

Papers

Somatic embryogenesis and plant regeneration in Sorghum bicolor (L.) Moench.