Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Production of haploids of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.) by anther culture

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.     

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer

Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.     

Morphogenesis in Isolated Microspore Cultures of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility.     

PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

Metal accumulation in two contiguous eutrophic peri-urban lakes,Chivero and Manyame,Zimbabwe

Concentrations of aluminium, cadmium, chromium, cobalt, copper, iron, lead, nickel and zinc were determined in surface water, benthic sediments, and the gills, liver and stomach muscle tissues of Oreochromis niloticus and Clarias gariepinus in peri-urban lakes Chivero and Manyame, Zimbabwe. Five sites were sampled in each lake once per month in November 2015, February, May, August and November 2016. Pollution load index detected no metal contamination, whereas the geo-accumulation index reflected heavy to extreme sediment pollution, with Fe, Cd, Zn, Cr, Ni and Cu present in both lakes. Significant spatial temporal variations were detected for Al, Cr, Cu and Pb across sites within and between the two lakes. High Fe, Al and Cr concentrations in water and sediments in lakes Chivero and Manyame derive from geogenic background sources in addition to anthropogenic loads and intensity. Elevated concentrations of Al, Pb, Cu, Cd, Fe and Zn detected in gills, liver and stomach tissue of catfish corroborate concentrations in water and sediments, and pose the highest ecological and health risk for hydrobionts in lakes Chivero and Manyame. Contiguity of peri-urban lakes exposes them to similar threats, necessitating creative water management strategies, which ensure ecological continuity.