Since the 1960s, numerous studies on sugarcane plant regeneration have been reported. Essentially, successful culture and regeneration of plants from protoplasts, cells, callus, and various tissue and organs, have been achieved in this crop. Although plant regeneration from callus cultures had been reported since the 1960s, definitive proof of somatic embryo development was not available until 1983. Since then, considerable progress has been made in understanding and refining somatic embryogenesis and plant regeneration in sugarcane, for which development of an efficient embryogenic system was critical for the application of transgenic technology. Recent research in Australia and South Africa has led to the development of direct somatic embryogenic systems, which may improve transgenesis in sugarcane.

In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.

This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.

A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTII) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTII fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and eliminating potential promoter–promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 g l⁻¹ of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48 h co-cultivation period combined with 75 mg l⁻¹ kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome; 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.

Release of inorganic mercury pollutants into shallow aquatic environments has resulted in the bacterial production of a more toxic organic mercury species, methylmercury. The bacterial organomercurial lyase (MerB) catalyses the protonolysis of the carbon–mercury bond and releases Hg(II), a less toxic, non-biomagnified form of mercury. Our objective was to engineer eastern cottonwood (Populus deltoides), a fast-growing tree adapted to growth in riparian environments, with the merB gene to explore its potential for phytoremediation of mercury. We produced multiple eastern cottonwood clones expressing a modified bacterial merB gene, confirmed that the gene was expressed in the transclones and tested the regenerated plants for their ability to tolerate exposure to an organic mercury source, phenylmercuric acetate (PMA), in vitro and in hydroponic culture, compared to wild-type control trees. Transgenic merB plants expressed high levels of MerB protein and showed some evidence of higher resistance to the organic mercury than wild-type plants, producing longer roots under exposure to PMA in vitro, although hydroponic culture results were inconclusive. Our results indicate that in order for merB to be useful in eastern cottonwood trees designed to degrade methylmercury at mercury-contaminated aquatic sites, it will probably need to be combined with other genes such as merA.

Adventitious bud regeneration from leaf and internode explants of Aloysia polystachya was achieved. Shoots from nodal segments grown in vitro were cut into pieces and used as sources of explants. Organogenesis was induced from both explants cultured on quarter-strength Murashige and Skoog (MS) semisolid medium (plus sucrose 5 g l⁻¹) containing different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 116 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD), 14-h photoperiod, and at a temperature of 27 ± 2°C. The type of explant markedly influenced organogenesis and growth of the regenerated shoots. The regeneration frequencies were higher with leaf explants, while the number of shoots formed per responsive explant was greater with internode explants. However, the growth of regenerated shoots from internodes was seriously affected by vitrification. The number of shoots produced per responsive leaf explant increased from one to seven as the percentage of leaf explants producing shoots increased from 20 to more than 80%. NAA at 0.05 μM in combination with BA at 0.5 μM induced the highest regeneration rate (87 ± 8.8%) after 20 d of culture, yielding 5.9 ± 0.8 shoots per responsive leaf explant. Histological examination confirmed the occurrence of direct organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium without plant growth regulators for 30 d. Finally, the elongated shoots were rooted by pre-treatment in an aqueous solution of NAA at 500 μM for 2 h and transferred to 1/4 MS. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 2-yr-old in vitro-regenerated plants was established.

In vitro seedlings of Lilium×formolongi Hort. cvs. Norikula, RaiZen No. 1, RaiZen No. 3, RaiZen Early, and Bailansa were used to induce callus by variously modified Murashige and Skoog (MS) media, using protocols for flask culture and bioreactor culture. Green embryogenic callus proliferated from roots near the base of bulblets of five varieties on media containing 0.53–5.3 μM α-naphthaleneacetic acid (NAA), and 28 cell lines were obtained by subcultures on the same medium. For flask culture, the fresh weight (FW) of embryogenic cell clumps doubled every 4 wk on MS basal salts supplemented with 0.53 μM NAA and 30 g l⁻¹ sucrose. The maximum frequency of somatic embryos that developed into plantlets was 76.67 ± 17% when plated onto solid MS basal medium without plant growth regulators (PGRs). Among the treatments using four types of bioreactors, the best cell growth and regeneration rate (74 ± 0.14%) of somatic embryos was in a modified 2-l bioreactor. Cells incubated in the other three bioreactors turned brown and died. Histological study revealed that regeneration was by somatic embryogenesis. The regenerants showed normal growth and flowering after 8–9 mo. in the field. A cell line of cv. Norikula has been subcultured in MS basal salts containing 0.53 μM NAA every 2 mo. for 6 yr. The cell aggregates became more synchronous and many typical embryogenic cells with dense cytoplasm were observed under a light microscope. The long-term embryogenic cells plated on MS basal medium still gave rise to numerous somatic embryos and converted into plantlets.

A procedure for regenerating Zizyphus jujuba Mill. (Chinese date) plants through repetitive somatic embryogenesis (RSE) was developed. Primary somatic embryos were produced from cotyledon-derived cultures of germinated plants in vitro. The highest induction frequency of primary somatic embryogenesis (PSE) was obtained with a combination of 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.49 μM indole-3-butyric acid (IBA), and 0.44 μM benzyladenine (BA) (17.4%). These primary somatic embryos were multiplied by RSE on media with different plant growth regulator (PGR) combinations. The highest RSE frequency (51.3%) was obtained with 0.58 μM gibberellic acid (GA₃). However, the highest number (4.4 per primary somatic embryo) of repetitive somatic embryos was obtained with 0.98 μM 6-r-r-dimethylallylaminopurine (2-iP). For germination of somatic embryos, different PGRs, cold and desiccation treatments were tested. Desiccation of somatic embryos at 25 ± 1°C for 2 wk was the best treatment for germination with epicotyl elongation and root development. Of over 256 plants regenerated, 237 (92.6%) survived.

The regeneration of lily protoplasts isolated from suspension cells of Lilium japonicum was achieved by using the nurse culture method. The protoplasts divided only under the nurse culture application. The divided protoplasts grew into colonies and developed into visible calluses on a medium containing picloram. After the calluses were transferred to a hormone-free medium, plantlets formed from them. The highest frequency of plant regeneration was obtained on a medium containing 1 μM gibberellin 4 (GA₄). The cleaved amplified polymorphic sequences (CAPS) method was used to confirm that the regenerants were not plants that had escaped from nurse cells. We were able to transplant the plantlets to soil in pots without acclimatization, and they showed normal growth.

Immature seeds of Garcinia indica Choiss. were excised from immature fruits and cultured on Lloyd and McCown (1980), woody plant medium (WPM) with different combinations of auxins and cytokinins. Somatic embryos were obtained on the media supplemented with 6-benzylaminopurine (BA; 2.2–22.1 μM) alone or in combination with α-naphthalene acetic acid (NAA; 2.6 μM) with 80% frequency within a period of 2–3 wk. Subculture of embryos on medium containing BA (16.0 μM) supplemented with indole-3-acetic acid (IAA; 2.8–5.7 μM) and/or kinetin (4.6 μM) gave rise to clusters of secondary somatic embryos along with maturation of primary embryos. In subsequent subculture on hormone-free half-strength WPM, the embryo clusters germinated with an increase in the number of secondary somatic embryos. About 70% of somatic embryos germinated into complete plantlets, which were successfully established under greenhouse conditions.

Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shoot formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indole-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventitious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacetic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, i.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.

Plants were successfully regenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertn. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants. Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subcultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1, 2, and 3 wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3 wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5 ± 1.6, 52.5 ± 18.5, and 41.6 ± 8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.

Successful lettuce (Lactuca sativa L.) breeding requires assessment, selection, and rescue of germplasm from field-grown plants during a limited period of time. Tissue culture methods have proved successful in rescuing selected lettuce genotypes and producing seeds in a disease-free environment. The present study was conducted to determine the response of the apical and axillary shoot-tips of lettuce to storage duration prior to in vitro culture. The study focused on core length as a source of variation between maternal explants. Advanced maturity was obtained by applying an agrotextile row cover over lettuce plants. Apical and axillary buds were derived from covered lettuce with long cores (46 ± 0.8 mm) and from non-covered lettuce with short cores (30 ± 0.7 mm). The cores were stored at 2°C for 0, 2, or 13 d. Apical and axillary shoot-tips were removed from the cores and placed onto MS medium. After 3 mo. of growth culture, twice as many leaf primordia (6.6 leaves) developed from the apical shoot-tip as compared to those from the axillary shoot-tip (3.0 leaves). More shoot-tips survived when excised from short cores (83%) than when excised from long cores (71%). Root formation was affected by the source of shoot-tip and the size of donor explant. Three times more explants from apical shoot-tips and short cores rooted (71%) than did explants from apical shoot-tips and long cores (27%) or explants from axillary shoot-tips (long cores, 14%; short cores, 19%). Storage of cores for up to 13 d had no effect on the survival rate of shoot-tips, nor on shoot or root formation, suggesting that cores of lettuce plants can be stored prior to in vitro micropropagation without significant loss of regeneration efficiency.

Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many researchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21 d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplanting ex vitro compared with those cultured under Pm conditions.

Rapid propagation of Pogostemon heyneanus Benth. (Lamiaceae) was accomplished through culture of node explants on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA). Random amplified polymorphic DNA (RAPD) and gas chromatographic (GC) analysis of in vitro-derived progenies were used to determine the true-to-type nature of in vitro-derived plantlets. At the optimum level of BA (2.22 μM), the axillary buds underwent a degree of dedifferentiation to become small globular green masses from which a mean of 17.1 shoots were developed within 40 d. Retaining the culture without subculture enhanced the number of shoots (>30 shoots). Increased callus proliferation was observed at higher concentrations of BA in concomitance with a reduction in number of shoots. However, prolonged culture without subculture (more than 60 d) initiated 25–30 shoot buds from the callus. Culture of node segments excised from in vitro shoots on fresh medium with optimal BA (2.22 μM) exhibited a similar response, but with an increase of shoots (mean of 26.3 shoots per node) within 40 d. Subculture of shoot clumps on half-strength MS basal medium resulted in elongation (more than 4 cm) of most of the shoots along with the development of new shoots. Shoots developed were rooted most successfully on half-strength MS medium with 4.9 μM indole-3-butyric acid (IBA). Plantlets derived from the best rooting medium established in small cups exhibited 95% survival. Plantlets successfully established in field conditions exhibited morphological characteristics identical to the source plant. The RAPD profile of the in vitro-derived plants and source plant, using 10 random primers, was similar. The gas chromatogram of the extracted oils from in vitro-derived plants and the source plant showed similar patterns.

The influence of partial substitution of agar by galactomannans (GMs) in culture media was studied in pear (Pyrus communis L. cv. ‘Durondeau’) micropropagation. GMs, extracted from seeds of Cassia fastuosa (cassia) or Cyamopsis tetragonolobus (guar gum, a commercial GM), were mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent. The production of multiple shoots and the formation of roots from shoots were compared with the control solidified with agar alone at a concentration of 0.6% (w/v). In the media solidified with the mixtures of agar/guar and agar/cassia GMs, an increase of 32 and 17%, respectively, was obtained in the number of regenerated shoots. The modified media promoted a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM and containing 9.80 μM indole-3-butyric acid. Less callus formation at the base of the shoot was also observed on this medium. The improved in vitro performance of shoot formation and rooting, combined with a significantly lower cost, suggests a potential use of agar/GM gels in plant tissue culture.

A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid, NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 g l⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acclimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.

In vitro plants of lemon grass were established, starting from shoot apices derived from plants cultivated under field conditions. The effect of the immersion frequency (two, four, and six immersions per day) on the production of biomass in temporary immersion systems (TIS) of 1 liter capacity was studied. The highest multiplication coefficient (12.3) was obtained when six immersions per day were used. The maximum values of fresh weight (FW; 62.2 and 66.2 g) were obtained with a frequency of four and six immersions per day, respectively. However, the values for dry weight (DW; 6.4 g) and height (8.97 cm) were greater in the treatment with four immersions per day. The TIS used in this work for the production of lemon grass biomass may offer the possibility of manipulating the culture parameters, which can influence the production of biomass and the accumulation of secondary metabolites. We describe for the first time the in vitro production of Cymbopogon citratus biomass in TIS.

Shoot cultures of three Hibiscus moscheutos (L.) cultivars were infested with micro-arthropods (mites). Nodal segments (1 cm long) were excised from these cultures and encapsulated in a sodium alginate gelled Driver and Kuniyuki Walnut DKW medium containing 10, 50, or 100 mg l⁻¹ acephate (insecticide) or 10 mg l⁻¹ acephate plus 0, 50, or 100 mg l⁻¹ benomyl (fungicide), then placed in refrigerated (5°C) darkness for 4 wk. Acephate was tested alone if visible fungus was not touching the shoot masses and benomyl was tested if fungus was in contact with the proliferating shoots. Cold-stored encapsulated nodes were then placed on DKW medium with 0.1 μM thidiazuron for 6 wk for subsequent shoot development. The presence of acephate in the encapsulation medium completely eradicated or killed the mites, with 38–69% of cultures fungus-free; 12% of the fungal-contaminated nodes encapsulated with 100 mg l⁻¹ benomyl were fungus-free.

A butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one, the phyto-active compound isolated from smoke) was tested for its effect on somatic embryogenesis with an important species for commercial horticulture, Baloskion tetraphyllum. When somatic embryos of B. tetraphyllum were transferred to basal medium supplemented with butenolide, enhanced development of growth-competent somatic embryos was observed. This study demonstrates that butenolide exhibits promotive effects on both germination of seed (stimulation of zygotic embryos), and stimulation of development and maturation of somatic embryos with B. tetraphyllum, providing a potential new class of phyto-active compounds for in vitro culture.