Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> Swartz)

Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical, and subtropical regions. A callus induction and plant regeneration protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg l⁻¹ 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.1 or 1.0 mg l⁻¹ benzylaminopurine (BA). A multifactorial experiment demonstrated the combination of 3 mg l⁻¹ dicamba and 1.0 mg l⁻¹ BA for induction of callus resulted in 12 times higher plant regeneration frequency compared to 3 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) alone or ten times higher plant regeneration frequency than the combination of 3 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ BA. These results are expected to support the development of a genetic transformation protocol for seashore paspalum.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Ovule rescue efficiency of Gossypium hirsutum × G. arboreum progeny from field-grown fruit is affected by media composition and antimicrobial compounds

Upland cotton (Gossypium hirsutum L.) is reproductively isolated from G. arboreum L. via post-zygotic breeding barriers. Literature on somatic embryogenesis of cotton suggests a number of media modifications that might also prove useful for ovule rescue of interspecific crosses. Additionally, endogenous microbes are common in field grown cotton and these potential contaminants must be controlled if interspecific progeny are to be obtained via large-scale field crossing followed by ovule or embryo culture. This study compared nine tissue culture media and two antimicrobial overlay treatments in a factorial design. The overlay treatments were: a 2 ml overlay of 250 mg l⁻¹ cefotaxime, 50 mg l⁻¹ tetracycline HCl, 2.5 mg l⁻¹ amphotericin B, and 50 mg l⁻¹ benomyl applied when the ovules were plated, and no overlay. All of the media in the factorial also contained 250 mg l⁻¹ cefotaxime. Crosses were made in a field at Stoneville, MS between the upland cultivar DeltaPine 90 and the G. arboreum accession A₂-190. Antimicrobial compounds greatly improved the efficiency of obtaining interspecific cotton progenies from field-grown fruit. Germination was not affected by the overlay nor did overlay treatment interact with media. Media significantly affected germination. Of the media studied, the highest frequency of germination was observed for MSB with 1.9 g l⁻¹ additional KNO₃. The addition of 0.5 g l⁻¹ asparagine and 1 g l⁻¹ glutamine did not affect the number of seedlings obtained. A filter paper growing surface or the addition of 0.5 mg l⁻¹ NAA and 0.05 mg l⁻¹ kinetin were disadvantageous.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

Salt lakes of the northern agricultural region,Western Australia

A survey of 11 sites covering three large (>10 km long, 6 sites) playa lakes and four pans (<1 km², 5 sites) of the Yarra Yarra salt lake system in the Northern Agricultural Region of Western Australia commenced in 2001. These salt lakes are shallow and ephemeral, with inundation being more regular following winter rainfall, but summer inundation also occurred in 2001. Salinity was generally higher in playas (156–368 g l⁻¹) than pans (30–284 g l⁻¹), but salinity responded noticeably to heavy rainfall events, especially in pans. pH values in the playa lakes (6.68–7.82) were less variable than in the pans (6.81–8.08). The range of dissolved oxygen concentrations was greater in pans (3.7–14.4 mg l⁻¹) than in playas (3.9–8.2 mg l⁻¹). Cationic concentrations generally followed the pattern of sea water cation dominance. Benthic microbial communities comprised either cohesive to loosely mucilaginous mats, or thin films of diatoms. Five genera of diatom and two species of filamentous cyanobacteria were recorded. Guest Editor: John M. Melack Saline Waters and their Biota     

Microphytoplankton assemblages in shallow waters at Admiralty Bay (King George Island,Antarctica) during the summer 2002–2003

Microphytoplankton populations were studied in shallow coastal water (<60 m) near the Brazilian Antarctic Station Comandante Ferraz (EACF) and three reference areas in Admiralty Bay in early and late summer (2002–2003). Phytoplankton was diverse (113 taxa), but not abundant (10³ cells l⁻¹). The highest abundances (>10⁴ cells l⁻¹) were caused by pennate benthic diatoms (Fragilaria striatula Lyngbye) that occurred mainly in early summer, associated with the presence of ice. In late summer, when the water temperature (−0.4 to 1.5°C), salinity (34 to 35), and phosphate (2.6 to 4.5 μmol l⁻¹) were highest and the dissolved oxygen was lowest (6.4 to 2.9 ml l⁻¹), centric diatoms (Thalassiosira spp.) were more abundant, suggesting an influence of oceanic waters. Phytoplankton abundance (≤10² cells l⁻¹) and chlorophyll a concentrations (0.22 μg l⁻¹) were lowest close to EACF. Pennate diatoms were dominant close to shore and in surface waters elsewhere, probably because of ice melting or sediment resuspension caused by water mixing.     

Field and experimental studies on the combined impacts of cyanobacterial blooms and small algae on crustacean zooplankton in a large,eutrophic, subtropical,Chinese lake

Field and experimental studies were conducted to evaluate the combined impacts of cyanobacterial blooms and small algae on seasonal and long-term changes in the abundance and community structure of crustacean zooplankton in a large, eutrophic, Chinese lake, Lake Chaohu. Seasonal changes of the crustacean zooplankton from 22 sampling stations were investigated during September 2002 and August 2003, and 23 species belonging to 20 genera were recorded. Daphnia spp. dominated in spring but disappeared in mid-summer, while Bosmina coregoni and Ceriodaphnia cornuta dominated in summer and autumn. Both maximum cladoceran density (310 ind. l⁻¹) and biomass (5.2 mg l⁻¹) appeared in autumn. Limnoithona sinensis, Sinocalanus dorrii and Schmackeria inopinus were the main species of copepods. Microcystis spp. were the dominant phytoplankton species and formed dense blooms in the warm seasons. In the laboratory, inhibitory effects of small colonial Microcystis on growth and reproduction of Daphnia carinata were more remarkable than those of large ones, and population size of D. carinata was negatively correlated with density of fresh large colonial Microcystis within a density range of 0–100 mg l⁻¹ (r = −0.82, P< 0.05). Both field and experimental results suggested that seasonal and long-term changes in the community structure of crustacean zooplankton in the lake were shaped by cyanobacterial blooms and biomass of the small algae, respectively, i.e., colonial and filamentous cyanobacteria contributed to the summer replacement of dominant crustacean zooplankton from large Daphnia spp. to small B. coregoni and C. cornuta, while increased small algae might be responsible for the increased abundance of crustacean zooplankton during the past decades.     

Taxane Production in Suspension Culture of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">Media</Emphasis> var. <Emphasis Type="Italic">Hicksii</Emphasis> Carried Out in Flasks and Bioreactor

Paclitaxel and 10-deacetylbaccatin III (10-DAB III) were produced in suspension cultures of Taxus × media var. Hicksii grown in shake-flasks and in a 7-l bioreactor reaching, in the bioreactor, 4.4 mg l⁻¹ (on day 14) and 37.5 mg l⁻¹ (on day 11). In shake-flasks the highest total content of paclitaxel and 10-DAB III was 7.3 mg l⁻¹ (on day 4) and 8.8 mg l⁻¹ (on day 18). Phenylalanine, at 0.05 mM, increased paclitaxel accumulation in cells cultivated in bioreactor and flasks 30-fold and 9-fold (from 0.02 mg l⁻¹ to 0.6 mg l⁻¹ and to 0.2 mg l⁻¹, respectively). The 10-DAB III content in cells from flasks was increased from 0.4 mg l⁻¹ to 1.6 mg l⁻¹.     

Nitrate loss from a restored floodplain in the Lower Cosumnes River, California

Floodplain restoration has been advocated as a means to restore several ecological services associated with floodplains: water quality improvement, fish rearing habitat, wildlife habitat, flood control, and groundwater recharge. A history of agricultural encroachment on the lower Cosumnes River has resulted in extensive channelization and levee construction. In fall 1998, an experimental floodplain was established by breaching a levee in order to restore the connection between the main channel and its historic floodplain. In this study, we examined how effective this newly restored floodplain was at processing nitrate (NO ₃ ⁻ ) before reentering the main channel downstream. Two methods were used to examine nitrate loss. In December 2001, we determined denitrification potentials of the floodplain soils before seasonal flooding inundated the floodplain. Next, we conducted a series of field soil column (mesocosm) experiments from March to June 2002 to study NO ₃ ⁻ -N loss from the overlying water in both sandy and clayey soils and at three levels of NO ₃ ⁻ -N (ambient, +1 mg N l⁻¹, +5 mg N l⁻¹). In addition, we examined NO ₃ ⁻ -N loss from mesocosms with water only to determine if loss was related primarily to soil or water column processes. Denitrification potentials were highly variable ranging from 1.6 to 769 ng N₂O–N cm⁻³ h⁻¹. In addition, the denitrification potential was highly correlated with the amount of bioavailable carbon indicating that carbon was a limiting factor for denitrification. Nitrate-N loss rates from mesocosms ranged from 2.9 to 21.0 μg N l⁻¹ h⁻¹ over all treatments and all 3 months examined. Significant loss of NO ₃ ⁻ -N (60–93%) from the water only treatment only occurred in June when warmer temperatures and solar radiation most likely increased NO ₃ ⁻ -N uptake by phytoplankton. When scaled up, potential NO ₃ ⁻ -N loss from the restored floodplain represented 0.6–4.4% of the annual N load from the Lower Cosumnes River during a typical wet year and ~24% during a dry year. During dry water years, the effectiveness of the floodplain for reducing nitrate is limited by the amount of N supplied to the floodplain. Results from this study suggest that restored floodplains can be an effective NO ₃ ⁻ sink.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

Micropropagation of bromeliad <Emphasis Type="Italic">Aechmea fasciata</Emphasis> via floral organ segments and effects of acclimatization on plantlet growth

Although suckers and seedlings can be used for the propagation of bromeliads, the low number of propagules and cross-variation limit their uniformity and mass cultivation. In this study, high-efficiency shoot organogenesis and plant regeneration were achieved on callus derived from petal and ovary explants of Aechmea fasciata (Bromeliaceae). Calluses were induced on half-strength Murashige and Skoog inorganic salts (1/2MS) supplemented with 1.0–1.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid in combination with 1.0 or 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and shoots regenerated after transfer to 1/2MS basal medium containing the combination of 1.0 mg l⁻¹ NAA + 0.5 mg l⁻¹ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea. Those plantlets grown under a middle light intensity (50 μmol m⁻² s⁻¹) showed a dramatic increase in survival percentage (up to 95%) and the maximum number of newly developing roots. The plantlets that were transplanted onto pots were successfully grown in the greenhouse.     

Molluscan cellulolytic activity responses to zinc exposure in laboratory and field stream comparisons

Changes in cellulolytic activity of Asiatic clams (Corbicula fluminea) and snails (Mudalia dilatata) were monitored throughout 30-d exposures to constant additions (0.0, 0.025, 0.05, 0.50, and 1.0 mg l⁻¹) of zinc (Zn). All exposures of 0.05 mg Zn 1⁻¹ or greater significantly reduced enzyme activity (exo- and endocellulase) in both molluscs as early as 10 d following exposures in outdoor laboratory streams incorporating New River water as diluent. More sterile laboratory stream exposures were less consistent in yielding quantifiable differences that could be attributed to metal induced stress apart from effects of nutritional stress. Tests conducted under natural field conditions during all seasons did not differ significantly with respect to changes in annual energetics of either clams or snails. However, evidence of differing uptake routes, with respect to two ecologically and physiologically distinct molluscs, was apparent in bioaccumulation, growth, and enzyme activity throughout exposure and following 60-d recovery.     

Rapid in vitro multiplication and ex vitro rooting of Malus zumi (Matsumura) Rehd

Micropropagation system of Malus zumi was optimized by studying the influence of plant growth regulators and culture conditions. The axillary buds were used for mutiplication of in vitro shoot culture on agar Murashige and Skoog (1962) (MS) medium with combination of 1 mg l⁻¹ BAP, 0.5 mg l⁻¹ NAA or 0.5 mg l⁻¹ IAA or 0.5 mg l⁻¹ IBA under 16 h photoperiod. The shoot growth in culture was not significantly affected within a broad range (5.0–7.0) of initial medium pH. The highest shoot (13) was obtained on medium containing 1.0 mg l⁻¹ BAP and 0.5 mg l⁻¹ IAA. Well-developed shoots, 35–50 mm in length, were successfully rooted ex vitro at 86.3% by a 2-h-treatment with aqueous solution containing MS salts and 100 mg l⁻¹ IBA prior to their planting in growing substrate composed of soil and vermiculite (1:1 v/v). The survival rate of transplantation reached 88.0% when transferred to field condition.     

Callus induction and shoot regeneration of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L. cv. Wonder Bush and cv. Pole Sieva

We report the first protocol for callus induction and shoot regeneration for Phaseolus lunatus L. cv. Wonder Bush and cv. Pole Sieva. We used different explants viz., epicotyls, cotyledons and hypocotyls. The medium used was MS basal medium with thidiazuron (0.5 mg l⁻¹) and IAA (0.05 mg l⁻¹) for the induction of callus followed by BAP (1.0 mg l⁻¹) for the induction of shoots. Epicotyl explants showed the fastest response and the highest percentage of shoot regeneration. This protocol opens new biotechnological strategies to transfer economically important genes to this important crop species.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.     

Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.)

Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l⁻¹ combined with thiamine at 0.1, 0.5, 2, or 5 mg l⁻¹. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l⁻¹ thiamine and 2 mg l⁻¹ biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l⁻¹ thiamine combined with 1 mg l⁻¹ biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.     

Active calcium ion transport in Xenopuslaevis skin

The skin of intact, free-swimming Xenopus laevis transports Ca²⁺ inwardly in a manner that is proportional to the external [Ca²⁺] up to about 0.3 mmol · l⁻¹, saturates above 0.3 mmol · l⁻¹, and is opposed to the electrochemical gradient. Efflux is relatively constant at external concentrations between 0.016 and 0.6 mmol · l⁻¹; net flux which is negative below 0.125 mmol · l⁻¹ becomes positive above this external [Ca²⁺]. Allometric analysis suggests that both Ca²⁺ influx and efflux scale to the 2/3 power approximately like surface area. There were no significant differences in influx between summer and fall animals; however, efflux was greater in the fall and this resulted in a change from positive balance in the summer to negative balance in the fall. Isolated skins were shown to support a Ca²⁺ uptake rate of nearly 30 nmol · cm⁻² · h⁻¹. The phenylalkylamine verapamil in the apical bathing solution significantly inhibited this at 25 μmol · l⁻¹. The benzothiazepine diltiazem was also effective at 50 μmol · l⁻¹ while the dihydropyradine nifedipine was ineffective up to 100 μmol · l⁻¹. The inorganic ion La³⁺ was effective at blocking Ca²⁺ uptake at 300 μmol · l⁻¹; Ni²⁺ was also effective at 500 μmol · l⁻¹ but Co²⁺ was ineffective up to 500 μmol · l⁻¹. These results suggest that apical calcium channels in Xenopuslaevis skin have properties similar to mammalian L-channels and fish gill Ca²⁺ channels. Accepted: 23 January 1997     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.