Establishment, characterization and plant regeneration from highly chlorophyllous embryogenic cell cultures of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud.

 A finely dispersed, homogeneous and highly chlorophyllous cell suspension (TIANSJ98 cell line) was obtained from shoot apices of Bouteloua gracilis (H.B.K.) Lag. ex Steud. cultured on MPC medium containing MS salts supplemented with 2,4-D (1 mg/l), BAP (2 mg/l) and adenine (40 mg/l). When the TIANSJ98 cell line was grown in this medium with shaking at 180 rpm it had doubling times of 7.2 and 3.7 days in terms of fresh and dry weight, respectively. Total chlorophyll content in this cell culture ranged from 121.6 to 18.3 μg/g FW at 12 and 21 days following culture initiation. Plants regenerated from the TIANSJ98 cell line, via somatic embryogenesis, were grown to maturity and produced seeds. Although different cell culture systems have been described for cereals and grasses, to the best of our knowledge this is the first report of a highly chlorophyllous and regenerable cell suspension in Poaceae. Received: 18 May 2000 / Revision received: 18 September 2000 / Accepted: 21 September 2000     

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.     

Lipid class composition of bleached and recovering Porites compressa Dana, 1846 and Montipora capitata Dana, 1846 corals from Hawaii

Corals rely on stored reserves, especially lipids, to survive bleaching events. Lipid class composition reveals the lipid source, and provides evidence of metabolic changes (i.e., photoautotrophic or heterotrophic) during bleaching and recovery. Porites compressa Dana, 1846 and Montipora capitata Dana, 1846 corals were experimentally bleached in outdoor tanks with seawater temperature elevated to 30 °C (treatment corals). Additional control fragments were maintained in separate tanks at ambient temperatures (27 °C). After one month, all fragments were returned to the reef for 0, 1.5, 4, or 8 months. Lipid class composition was analyzed by Iatroscan (thin layer chromatography-flame ionization detection). In treatment P. compressa, triacylglycerol (TG) decreased at 0 and 1.5 months, phospholipid (PL) also decreased at 1.5 months, and both remained lower relative to controls along with wax esters (WE) after 8 months. Neither treatment nor control P. compressa had any detectable monoacylglycerol (MG) or diacylglycerol (DG). Overall, P. compressa first consumed available storage, then structural lipids, and all lipid classes remained low at the end of the study. In treatment M. capitata, TG and PL decreased, while MG increased relative to controls at 0 months. At 4 months, free fatty acid (FFA), sterol (ST), and PL in treatment M. capitata were two to ten times higher than controls. Treatment and control lipid class composition were not different from each other at 8 months. In contrast to P. compressa, M. capitata consumed some lipid classes and augmented others, probably due to sequential metabolism of storage lipids and increased heterotrophy. Overall, lipid class assimilation was more rapid in treatment M. capitata corals that switch between heterotrophy and photoautotrophy, than in treatment P. compressa corals that rely mostly on photoautotrophy.     

Developing a Photoautotrophic Micropropagation System for Woody Plants

in vitro including cotyledonary stage somatic embryos have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is due not to poor photosynthetic ability, but to the low CO₂ concentration in the air-tight culture vessel during the photoperiod. Furthermore, we have shown that the photoautotrophic growth of several woody plants in vitro can be significantly promoted by increasing the CO₂ concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. In this paper, the advantages of photoautotrophic micropropagation in a conventional, small culture vessel with a microporous gas filter for enhancing natural ventilation and in a large culture vessel with a forced ventilation unit are described for woody plants such as acacia (Acacia mangium), coffee (Coffea arabusta), eucalyptus (Eucalyptus camaldlensis), mangosteen (Garcinia mangostana), neem (Azadirachta indica), paulownia (Paulownia fortunei), and pine (Pinus radiata). Received 30 August 2001/ Accepted in revised form 27 September 2001     

The lipids in photoautotrophic and heterotrophic cell suspension cultures of Chenopodium rubrum

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.