Forage and turf grasses are critical to sustainable agriculture and contribute extensively to the world economy. Tremendous progress has been made in genetic transformation of forage and turf grasses in the past decade. The rapid advancement of cellular and molecular biology and transgenic technology provides novel methods to accelerate and complement conventional breeding efforts. This review summarizes the latest developments in genetic transformation methods and the applications of molecular techniques for the improvement of forage and turf grasses.

Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing. At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.

Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repetitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean.

Anther-derived rice (Oryza sativa L. ssp. japonica variety Yerua P.A.) plants were obtained after cryopreservation by an encapsulation/dehydration technique. Immature anthers, excised from spikelets pretreated at 8°C for 8 d, were encapsulated in calcium alginate beads. The beads were cultured on N6 medium with 11.5 μM naphthaleneacetic acid (NAA) and 2.3 μM 6-furfurylaminopurine (KIN). Fifteen percent of the encapsulated anthers formed calluses when pretreated with sucrose for 3 d in liquid medium, desiccated on silica gel, slowly cooled to −30°C, immersed in liquid nitrogen (LN), thawed, and recultured. The cryopreserved encapsulated anthers produced 1.67 shoots/callus, in contrast to the control (non-cooled encapsulated anthers), which produced 6 shoots/callus. Eighty percent of the plantlets developed into normal plants after being transferred to greenhouse conditions. Histological observations showed that the origin of the plants was not modified by the cryopreservation process.

Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embryogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenic callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.

In an attempt to increase productivity, the effects of the elicitors methyl jasmonate (MJ) and salicylic acid (SA) on the production of bilobalide (B), ginkgolide A (GA), and ginkgolide B (GB) were studied in cell suspension cultures of Ginkgo biloba. MJ treatments increased the amounts of B, GA, and GB, concomitant with a slight decrease in cell growth. After treatment of 0.01 mM MJ, levels of GA and GB increased 4.3- and 8.2-fold over controls by 12 h and declined after 24 h. The 1.0 mM MJ treatment produced a maximal release of B after 12 h of exposure and increased the concentration of B in the culture medium up to 6.25-fold compared with the controls. Treatment with 1.0 mM SA transiently enhanced GA and GB production up to 3.1- and 6.1-fold, respectively, compared with the control. However, treatment 1.0 mM SA did not have a significant effect on B production. When treated with 0.01 mM SA, the level of B in the cells was increased 5.4-fold over controls by 12 h and declined after 24 h. The concentrations and exposure times of both MJ and SA were factors that strongly affected the production of B, GA, and GB. The results from this study suggest that MJ and SA directly or indirectly increased the production of B, GA, and GB in cells, and stimulated the release of these metabolites into the culture medium.

Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52 μM) and KIN (0.93 μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6 mg g⁻¹ DW, valtrate (VAL) 10.2 mg g⁻¹ DW, and didrovaltrate (DID) 2.9 mg g⁻¹ DW were observed in root cultures after 7–8 wk of culture.

Bactris major and Desmoncus orthacanthos are native palms from the Yucatan Peninsula which could be used as substitutes for rattan. When their seeds were germinated in vivo and in vitro they proved to be highly recalcitrant. Therefore, the culture of isolated embryos was studied as an alternative means of producing planting material for nurseries. It was found that the in vitro germination of the isolated embryos was gradually reduced by storage, falling to zero by 5 wk. However, isolated embryos from freshly collected seeds germinated at ∼100% frequency. The presence of the endosperm, whether still attached to the embryos or separated from them but in direct contact with the nutrient medium, greatly reduced germination in both species. High concentrations of abscisic acid (ABA, 100 μM) only slightly diminished it, suggesting a different cause for the observed endosperm-induced inhibition. This embryo rescue method permits the production of sufficient plants for in vitro micropropagation and the establishment of experimental plots to evaluate the full potential of these materials.

The purpose of this study was to develop an efficient micropropagation system for Mucuna pruriens, an important medicinal plant in India. A range of cytokinins was investigated for multiple shoot regeneration with cotyledonary node explants from 7-d-old aseptic seedlings. Of all the cytokinins, 6-benzyladenine (BA), kinetin (KIN) and 2-isopentenyl adenine (2-iP) tested in Murashige and Skoog medium (MS), BA was the most effective and 5.0 μM was found to be optimum for inducing maximum shoots. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The highest efficiency of shoot proliferation was observed in 5.0 μM BA and 0.5 μM α-naphthalene acetic acid (NAA) in half-strength MS medium at pH 5.8. The best condition for rooting was half-strength MS medium solidified with agar and with 2.0 μM indole-3-butyric acid (IBA). After rooting, the plantlets were transferred to plastic pots filled with sterile soilrite where 90% grew and all exhibited normal development.

An efficient in vitro shoot propagation method for Vaccinium cylindraceum Smith by axillary bud proliferation was developed in this study. Cultures of V. cylindraceum were established on a Zimmerman and Broome medium supplemented with increasing concentrations of N⁶-[2-isopentyl] adenine (2-iP), using nodal and shoot-tip explants from three different islands. Best shoot production was obtained when nodal segments were cultured in the medium supplemented with 12.3 and 24.6 μM 2-iP. Shoots were rooted in vitro in the same media without 2-iP and cultured ex vitro in Jiffy 7^® pellets. About 99% of the rooted plants survived.

A rapid shoot multiplication protocol was established for an important medicinal plant, Vitex negundo L., belonging to the family Verbenaceae, using Murashige and Skoog medium, achieved by shoot multiplication as well as callus regeneration. Shoot multiplication was induced by different concentrations of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ), Benzyladenine and 6-furfuryl amino purine separately along with 10% (v/v) coconut water. Green organogenetic callus was obtained by the combined effect of 0.5–2.15 μM TDZ and 1.7 μM indole-3-acetic acid (IAA) along with 1% polyvinylpyrrolidone (PVP), and produced the maximum number of shoots when subcultured onto medium containing 2.7 μM TDZ alone. Elongation of in vitro shoots was observed in MS medium containing 2.4 μM gibberellic acid and rooting was induced by the combined effect of 1.71 μM IAA and 1.62 μM α-naphthalene acetic acid.

We have investigated the interaction of Gluconacetobacter diazotrophicus, a non-nodulating endophytic nitrogen-fixing bacterium isolated from the intercellular spaces of sugarcane, with Arabidopsis thaliana and the crop plants maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), oilseed rape (Brassica napus), tomato (Lycopersicon esculentum), and white clover (Trifolium repens). Using seedlings grown aseptically in sucrose-containing culture media, we have shown that inoculation with very low numbers of G. diazotrophicus results in extensive intracellular colonization of root meristems and progressive systemic intracellular root colonization. Light microscopic examination of thin sections of resin-embedded root tips of Arabidopsis and these crop plants inoculated with β-glucuronidase (GUS)-labeled and with NifH promoter–GUS-labeled G. diazotrophicus showed blue-stained G. diazotrophicus within the cytoplasm of root cells, indicating that intracellular conditions were suitable for nitrogenase gene expression. Electron microscopy confirmed that these blue-stained intracellular G. diazotrophicus were within membrane-bounded vesicles. We discuss whether these novel inoculations with G. diazotrophicus are likely to enable non-nodular endosymbiotic nitrogen fixation and whether these inoculations can also provide a plant system to investigate the endosymbiotic theory of the origin of eukaryotic organelles.

To pursue the causal factors of ploidy variation in tissue culture of asparagus (Asparagus officinalis L.), ploidy status of stem explants and the calluses induced from these explants were examined using flow cytometry (FCM). The frequency of higher ploidy (>8C) cells in the stem explants increased with increasing distance from the shoot apex. Calluses derived from sections far from the apex also showed higher ploidy distributions, and also higher ploidy (16C) in general than was observed in the explant tissue. Detailed studies using FCM and charge-coupled device (CCD)-equipped fluorescence microscopy showed that 16C cells appeared in the explant tissues within 1 wk of culture, possibly induced by the plant growth regulators (PGRs), especially 1-naphthaleneacetic acid (NAA), in the culture medium.