Induction and growth characteristics of embryogenic avocado cultures

Embryogenic cultures were induced from immature avocado zygotic embryos representing different botanical races and complex hybrids. The optimum induction medium consisted of B5 major salts, MS minor salts, 0.4 mg l⁻¹ thiamine HCl, 100 mg l⁻¹ myo-inositol, 30 g l⁻¹ sucrose, 0.41 μM picloram and 8 g l⁻¹ TC agar. Somatic embryogenesis occurred directly from the explants on induction medium, and secondary embryos and proembryonic masses proliferated in liquid and on semisolid maintenance medium. Embryogenic culture maintainance was optimized in liquid, filter-sterilized MS medium, supplemented with 30–50 mg l⁻¹ sucrose, 4 mg l⁻¹ thiamine HCl and 0.41 μM picloram. Two types of embryogenic cultures were recognized: –genotypes that proliferated as proembryonic masses in the presence of auxin (PEM-type) and; –genotypes in which the heart stage and later stages of somatic embryos developed in the presence of auxin(SE-type). Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

Avocado shoot culture, plantlet development and net CO2 assimilation in an ambient and CO2 enhanced environment

Summary The proliferation and survival of avocado nodal cultures of juvenile origin were affected by the form and concentration of nitrogen. Optimum growth was achieved on modified Murashige and Skoog medium containing 67% KNO₃ and 33% NH₄NO₃ with total N of 40 mM supplemented with 100 mg l⁻¹ myo-inositol, 1 mg l⁻¹ thiamine HCl, 30 g l⁻¹ sucrose, and 4.44 μM BA with a 16-h photoperiod (120–150 μmol m⁻² s⁻¹). Proliferating shoots and plantlets were photosynthetically active. Better shoot growth and accumulation of higher biomass occurred in a CO₂-enriched environment than under ambient CO₂ conditions. CO₂ assimilation efficiency, however, was higher under the latter conditions than in a CO₂-enhanced environment, e.g., 31±7 and 17±2 μmol CO₂ m⁻² s⁻¹, respectively. The net CO₂ assimilation rates of in vitro grown plantlets were comparable to those of seedlings ex vitro.     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

Experimental Modelling of the Consequences of Brief Late Gestation Asphyxia on Newborn Lamb Behaviour and Brain Structure

Brief but severe asphyxia in late gestation or at the time of birth may lead to neonatal hypoxic ischemic encephalopathy and is associated with long-term neurodevelopmental impairment. We undertook this study to examine the consequences of transient in utero asphyxia in late gestation fetal sheep, on the newborn lamb after birth. Surgery was undertaken at 125 days gestation for implantation of fetal catheters and placement of a silastic cuff around the umbilical cord. At 132 days gestation (0.89 term), the cuff was inflated to induce umbilical cord occlusion (UCO), or sham (control). Fetal arterial blood samples were collected for assessment of fetal wellbeing and the pregnancy continued until birth. At birth, behavioral milestones for newborn lambs were recorded over 24 h, after which the lambs were euthanased for brain collection and histopathology assessments. After birth, UCO lambs displayed significant latencies to (i) use all four legs, (ii) attain a standing position, (iii) find the udder, and (iv) successfully suckle - compared to control lambs. Brains of UCO lambs showed widespread pathologies including cell death, white matter disruption, intra-parenchymal hemorrhage and inflammation, which were not observed in full term control brains. UCO resulted in some preterm births, but comparison with age-matched preterm non-UCO control lambs showed that prematurity per se was not responsible for the behavioral delays and brain structural abnormalities resulting from the in utero asphyxia. These results demonstrate that a single, brief fetal asphyxic episode in late gestation results in significant grey and white matter disruption in the developing brain, and causes significant behavioral delay in newborn lambs. These data are consistent with clinical observations that antenatal asphyxia is causal in the development of neonatal encephalopathy and provide an experimental model to advance our understanding of neuroprotective therapies.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Agrobacterium tumefaciens– mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos

Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l^–1 picloram and 50 mg l^–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l^–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively. Received: 2 June 1997 / Revision received: 26 September 1997 / Accepted: 11 October 1997     

Maturation of avocado somatic embryos and plant recovery

Avocado proembryonic masses from suspension cultures were used to develop a protocol for somatic embryo development and maturation. Avocado somatic embryos could develop from proembryonic masses both in liquid and on semisolid medium but only the latter could develop to maturity. Size and number of opaque somatic embryos were affected by gellan gum concentration, with the optimum response obtained on medium supplemented with 6–7 g l⁻¹ gellan gum. The optimum sucrose concentration for recovery of opaque somatic embryos was 90 g l⁻¹; however, the development of embryos was suppressed at this concentration. Consequently, recovery of cotyledonary, opaque somatic embryos was achieved on medium with 30 g l⁻¹ sucrose. Somatic embryo development from dedifferentiating proembryonic masses required media with a high ratio of NO ₃ ⁻ :NH ₄ ⁺ (1:0 and 3:1) as opposed to the standard ratio (2:1) of MS medium. Germination of somatic embryos was sporadic. In order to increase the frequency of plant recovery, shoots that developed from somatic embryos were micropropagated using standard protocols. This revised version was published online in June 2006 with corrections to the Cover Date.