Acute toxic effects of 8‐epidiosbulbin E,a 19‐norclerodane diterpene from yam Dioscorea antaly,on medaka Oryzias latipes embryos

The fractionation of an aqueous extract of yam Dioscorea antaly from Madagascar led to the isolation of terpenoids and flavonoids. Compounds were identified on the basis of modern mass spectrometry and two‐dimensional nuclear magnetic resonance (2D‐NMR). Toxicological effects of the most abundant isolated compound, 8‐epidiosbulbin E were studied on medaka Oryzias latipes embryo‐larval development. The lethal concentration (killing 50%; LC₅₀) to embryos treated 24 h before hatching and for 3 days after hatching was estimated to be 0·56 mg ml^?1 (P< 0·05). No mortality was observed with O. latipes larvae exposed after hatching until day 4. Anatomo‐pathological studies of embryos exposed to 0·56 mg ml^?1 showed development anomalies of the central nervous system, liver, muscle and intestine. The present data thus extend the model of O. latipes embryos as a useful animal model to analyse the effects of food toxins.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

Effect of age and environmental conditions on the movement characteristics of Pacific oyster (Crassostrea gigas) trochophores

The movement characteristics of Pacific oyster trochophores have received very little coverage in the scientific literature. Described here are not only changes in the swimming characteristics of Crassostrea gigas trochophores (size: 53–77 μm) in relation to time after fertilization and to inter‐female variation but also the effects of salinity and pH on movement characteristics. The percentage of motile trochophores was measured on images obtained through a dissecting microscope and the Velocity Average Path (VAP) was assessed using a Computer Assisted Sperm Analysis (CASA) system. At 20°C, the first movements of the trochophores were observed at 6.5 h post‐fertilization. The mean (± SD) percentage of swimming trochophores and the VAP observed in seawater at 11.5 h post‐fertilization were 85 ± 10% and 146 ± 75 μm s^?1, respectively (n = 8 females). Significant inter‐female variation in the percentage of motile trochophores (range: 66 ± 16–93 ± 3%) and in the VAP (55 ± 47–180 ± 90 μm s^?1) was detected. Most of the trochophores were motile in a 9‰ salinity solution. Adjusting the pH of the seawater to values from 5.10 to 9.08 had no effect on swimming characteristics. The present study shows that the movement of oyster trochophores exhibits high plasticity in relation to environmental conditions because the highest percentages of swimming trochophores and optimal velocity values were recorded within large ranges of salinity and pH. Further research is required to determine whether the swimming performances of trochophores can be used to assess embryo quality in commercial hatcheries.     

Humic substances and the water calcium content change the toxicity of malachite green

Laboratory experiments were conducted to test interactive effects of calcium (Ca²⁺) content and the presence of humic substance (HS) on malachite green (MAG)‐induced toxicity in fish embryos and larvae by means of a semistatic 144‐h‐embryo‐larval‐test with zebrafish (Danio rerio). Two kinds of reconstituted water samples were used to produce the test media by mixing salts into deionized water resulting in either hard water (↑Ca ? HS), or soft water (↓Ca ? HS). By adding HS two additional test media were produced (↑Ca + HS, ↓Ca + HS). MAG was tested in concentrations of 0.05, 0.10, 0.15, 0.20, 0.25 mg L^?1. The toxicity ranking of MAG (mg L^?1) to embryos based on 96‐h‐LC₅₀ in the different test water samples is: ↑Ca ? HS (0.061) > ↑Ca + HS (0.123) = ↓Ca ? HS (0.12) ≥ ↓Ca + HS (0.134) and on 144‐h‐LC₅₀ to larvae is: ↑Ca ? HS (0.038) > ↑Ca + HS (0.06) > ↓Ca ? HS (0.077) = ↓Ca + HS (0.077). Mortality of all the groups was significantly different (P < 0.05). Increased Ca²⁺ concentrations did not protect zebrafish embryos and larvae from MAG‐induced toxicity. At high Ca²⁺ conditions, the mortality of the embryos as well as of the larvae is reduced in the ↑Ca + HS group relative to the ↑Ca ? HS group. Thus, at high Ca²⁺ conditions the HS does affect the MAG‐induced mortality. The mechanism which causes the higher toxicity of MAG in the presence of higher Ca²⁺ concentrations is poorly understood. A probable explanation could be the stimulation of the calcium‐binding protein calmodulin as well as the calmodulin kinase II in cell membranes in the presence of high Ca²⁺ concentrations.     

Effects of growth regulator concentrations and explant size on shoot organogenesis from callus derived from zygotic embryos of sunflower (Helianthus annuus L.)

Effects of medium growth regulator composition and embryo size on shoot organogenesis of callus derived from globular- to torpedo-shaped zygotic embryos of five sunflower (Helianthus annuus L.) genotypes were examined. Forty growth regulator combinations composed of 0 to 5 mgl^-1 naphthaleneacetic acid (NAA) and 0 to 1 mgl^-1 6-benzylaminopurine (BA) were tested. The frequency of zygotic embryos forming shoot-regenerating callus was analysed according to categorical data modelling using a maximum-likelihood approach. Both NAA and BA must be present to induce the formation of morphogenic callus from zygotic embryos, but each growth regulator effect varied with the genotype. For four genotypes, NAA and BA effects were neither linear nor quadratic; whereas, they were linear for the fifth one. Most effective concentrations across genotypes were 0.1 mgl^-1 NAA and 0.5 mgl^-1 or 0.2 mgl^-1 BA. However, the optimal growth regulator combination depended on the genotype and an interaction between the two growth regulators. The frequency of shoot-regenerating callus also varied with the size of the embryo explant. For all five genotypes, 0.4 to 1.2 mm long heart-shaped zygotic embryos formed morphogenic callus more frequently than smaller less-developed ones.     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid     

Picolinic acid-induced direct somatic embryogenesis in sweet potato

Somatic embryos are being considered as an alternative material for in vitro germplasm conservation of sweet potato [(Ipomoea batatas (L.) Lam.)]. Picolinic acid was tested for somatic embryo production in sweet potato apical meristem tip cultures. Low level (0.2 mgl^-1) of picolinic acid combined with kinetin or 6-benzylamino purine (6-BAP) (1.0 and 2.0 mgl^-1) suppressed shoot growth and induced callus proliferation. Increased amount of picolinic acid (2 and 3 mgl^-1) in combination with kinetin (0.25 and 1.0 mgl^-1) induced direct somatic embryogenesis from apical meristem tips of variety Regal but not in Jewel. The primary embryos matured and germinated bipolarly yielding whole plantlets and unipolarly producing embryogenic hyperhydrated-fasciated shoots. The hyperhydrated-fasciated shoots, when cultured in picolinic and kinetin-enriched medium, produced secondary embryos. The secondary embryos also germinated bipolarly and unipolarly, resulting in subsequent cycles of embryogenesis. This recurrent embryogenesis ensures maintenance and proliferation of embryogenic tissues. Somatic embryos were also formed in mannitol-induced hyperhydrated shoots in response to picolinic acid and kinetin or 6-BAP treatment. Embryogenesis did not occur in non-hyperhydrated leaf, petiole, and internode sections.     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^&#x2212;1 sucrose. A sucrose concentration lower or higher than 30 gl^&#x2212;1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^&#x2212;1 tyrosine in the medium. Seedlings germinated in the presence of 0.2&#x2013;0.4 mgl^&#x2212;1 &#x03B1;-naphthaleneacetic acid and 0.3&#x2013;0.5 mgl^&#x2212;1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60&#x2013;70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.     

Picloram induced somatic embryogenesis in Gasteria and Haworthia

Various leaf sections of Gasteria verrucosa Haw. and Haworthia fasciata Haw. were cultured on media to examine the effect of picloram (4-amino 3, 5, 6-trichloropicolinic acid) and 2, 4-D (2, 4-dichlorophenoxy acetic acid) on somatic embryogenesis. Picloram (0.5, 1.0, 2.0, 3.0 mgl^-1) outperformed 2, 4-D (0, 1.0, 2.0, 3.0 mgl^-1) as the auxin source of both earliness of callus and embryo induction and final yield of embryos produced at both kinetin levels examined (0.25, 1.0 mgl^-1). Embryos arose initially as a yellow, compact globular masses from the area just beneath the epidermis in linear pattern parallel with the main axis of the leaf and then developed a heartshaped appearance. Embryo formation was preceded by growth of callus almost crystalline in appearance on the cut surface. Subsequent shoot formation developed from green pigmented loci in crystalline callus derived from embryos. Shoot and root development in Gasteria was induced on a defined medium containing quarter strength MS or B5 salts with no hormonal supplementation.     

Somatic embryogenesis from seed explants of Abies alba

Megagametophytes of Abies alba containing the immature embryos were dissected from the seed coats and divided by longitudinal and transverse sections. They were placed with the cut surface down on modified Schenk & Hildebrandt medium containing 50 mgl^-1 myo-inositol and 2% sucrose, supplemented with 1 mgl^-1 N⁶-benzyladenine (BAP). An embryogenic type of callus proliferated after one month of culture. Closer examination revealed the presence of structures resembling early stages of embryogenesis as well as of single elongated, vacuolated cells and clusters of cells with dense cytoplasm. Under appropriate conditions, some of the somatic embryos elongated and formed cotyledons.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Developmental biology of medaka fish (Oryzias latipes) exposed to alkalinity stress

Alkalinity stress is common in cultured aquatic animals and considered to be one of the major stress factors for fishes when they are transferred to saline‐alkali waters. To evaluate potential effects of alkalinity on the developmental biology of Oryzias latipes, fertilized eggs, larvae and breeding fish were exposed to different carbonate alkalinity concentrations of 1.5–64.5 meq l^?1, for 9, 120, and 60 days, respectively. The mortality of embryos significantly increased when exposed to the high concentrations (16.5–64.5 meq l^?1). Although more than 50% of survived embryos hatched in 16.5 and 31.4 meq l^?1 concentrations of carbonate alkalinity, most were not able to swim up after hatching. Morphological abnormalities such as coagulated embryos, halted embryo development, and hatching failure were observed at stages 15, 29–33 and 38 in high concentrations (31.4, 64.5 meq l^?1). Almost all larvae in 16.5 and 31.4 meq l^?1 treatments died 70 d post‐hatch. Growth of juveniles exposed to carbonate alkalinity of 5.3 and 8.8 meq l^?1 was not significantly different at 70 d and 120 d post‐hatch. The number of eggs released by breeders, the fertilization rate and the hatching rate of eggs were significantly lower in the 31.4 meq l^?1 treatment than in other treatments. Although medaka are capable of surviving in high alkalinities (31.4, 64.5 meq l^?1) for an extended period of time, these conditions are stressful to the fish, especially at the embryonic and reproductive stages.     

ONTOGENY AND TAPHONOMY: AN EXPERIMENTAL TAPHONOMY STUDY OF THE DEVELOPMENT OF THE BRINE SHRIMP ARTEMIA SALINA

Abstract: Although the relationship between ontogeny and phylogeny has been of long‐standing interest to palaeontologists, the fossil record has provided little insight into the development of long extinct organisms. This has changed with the discovery of numerous assemblages of fossilized invertebrate embryos and larvae, but realising their evolutionary significance is hampered by a paucity of data on the relationship between ontogeny and taphonomy. We describe the results of an experimental taphonomy study of the development of the anostracan brine shrimp Artemia salina, which show that in conditions of aqueous aerobic and anaerobic autolysis and microbial decay, the developmental stages exhibit differential preservation potential. The most decay resistant developmental stage is the diapause cyst, encapulsating the gastrula, in which the gross morphology of the embryo can be maintained for 18 months or more in simple anaerobic conditions. Otherwise, the embryo shrinks within the cyst and cellular and tissue detail of breaks down as lipid droplets coalesce. Postembryonic excysted larvae decay more rapidly. The rate of decay is similar among all larval stages with the exception of the L₄ larva, which resists cuticle failure for longer than later developmental stages. The larvae decay leading to liquefaction of the muscles and viscera, leaving an intact but empty and progressively shrunken and distorted cuticle that eventually loses structural integrity and collapses. Our experimental results provide an explanatory model for the phenomenal abundance of putative diapause stage embryos, in the absence of postembryonic stages, as seen in the Ediacaran Doushantuo Formation of South China and the incompleteness of fossilized developmental sequences of embryos and larvae more generally. It also cautions against the association of developmental stages in fossil deposits without additional evidence. Finally, the pattern of decay seen in larvae provides an explanation for the preservation style of Orsten‐type Lagerstätten where preservation of cuticular detail can be astonishingly fine, but extends internally to muscles and viscera only rarely.     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Embryonic substances induce alarm response in adult zebrafish (Danio rerio)

Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10⁻⁶ g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

RETRACTED ARTICLE: Expression of a rice chitinase gene enhances antifungal response in transgenic litchi (cv. Bedana)

To enhance the antifungal response of litchi (Litchi chinensis Sonn.), transgenic plants were generated by transferring rice chitinase gene driven by a maize-ubiquitin promoter along with its first intron into the zygotic embryos via Agrobacterium tumefaciens-mediated transformation. After co-cultivation for 2 days, zygotic embryos were transferred onto Murashige and Skoog (MS) modified media supplemented with 25 mgl⁻¹ hygromycin and 400 mgl⁻¹ cefotaxime. Consequently embryos were selected and the antibiotic resistant transgenic plantlets were regenerated. The culture time from zygotic embryo to transgenic plants was 14 months. The integration of the transgene was confirmed by PCR, RT-PCR, Southern and western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed plants. The chitinase activity when examined using the native polyacrylamide in-gel assay, indicated that the foreign gene expression resulting in the protein of expected molecular weight that showed chitinase activity. The transgenic plants showed delayed onset of the disease and smaller lesions following in vitro inoculation of die-back, leaf spots and blight pathogen (Phomopsis sp.). The transgenic plants were adapted to the greenhouse and did not show any phenotypic alterations.