An esterase is involved in geraniol production during palmarosa inflorescence development

Total incorporation of exogenously administered [2-14C]acetate into essential oil of palmarosa (Cymbopogon martinii) was found to be relatively higher than that of either [U-14C]sucrose or [U-14C]glucose during inflorescence development. Among the major essential oil constituents, biogenesis of geranyl acetate was much higher than that of geraniol. Alkaline hydrolysis of [14C]labeled geranyl acetate revealed that the majority of the label incorporated into geranyl acetate was present in the geraniol moiety, indicating that only newly synthesized geraniol gets acetylated to form geranyl acetate. Geranyl acetate cleaving esterase (GAE) activity followed a similar pattern during both in vivo and in vitro inflorescence development, with maximum activity at immature inflorescence stages, suggesting the involvement of GAE in geraniol production during inflorescence development. Five esterase isozymes (Est-A to E) were detected in the enzymic fraction of palmarosa inflorescence and all showed GAE activity, with Est-B being significantly increased during inflorescence development. The role of GAE in geraniol production and improving the palmarosa oil quality is discussed.     

Biotransformation of geranyl acetate to geraniol during palmarosa (Cymbopogon martinii, Roxb. wats. var. motia) inflorescence development

Only immature palmarosa (Cymbopogon martinii, Roxb. wats. var. motia) inflorescence with unopened spikelets accumulated essential oil substantially. Geraniol and geranyl acetate together constituted about 90% of the palmarosa oil. The proportion of geranyl acetate in the oil decreased significantly with a corresponding increase of geraniol, during inflorescence development. An esterase enzyme activity, involved in the transformation of geranyl acetate to geraniol, was detected from the immature inflorescence using a gas chromatographic procedure. The enzyme, termed as geranyl acetate cleaving esterase (GAE), was found to be active in the alkaline pH range with the optimum at pH 8.5. The catalysis of geranyl acetate was linear up to 6 h, and after 24 h of incubation, 75% of the geranyl acetate incubated was hydrolyzed. The GAE enzymic preparation, when stored at 4 degrees C for a week, was quite stable with only 40% loss of activity. The physiological role of GAE in the production of geraniol during palmarosa inflorescence development has been discussed.     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Ameliorative Effect of Chlormequat Chloride and IAA on Drought Stressed Plants of <Emphasis Type="Italic">Cymbopogon Martinii</Emphasis> and <Emphasis Type="Italic">C. winterianus</Emphasis>

Responses of Cymbopogon martinii and C. winterianus to drought stress and chlormequat chloride and IAA application are compared. These two species are important source of essential oil production in drought regions. For both species and their cultivars relative water content (RWC), herbage yield and oil amount decreased under drought, while oil biosynthesis increased. Oil concentration increased significantly under drought in C. winterianus while peroxidase activity increased in C. martinii. Amount of geraniol increased under drought stress in C. martinii while citronellal and geraniol accumulation decreased in C. winterianus. Ameliorative effects of chlormequat chloride and IAA were observed in drought stressed plants of both species. Herbage yield increased significantly in chlormequat chloride and IAA treated stressed plants of C. winterianus, while oil concentration increased in C. martinii. Ameliorative effect of IAA in increasing oil yield was significant in drought stressed plants of both the species. Changes in various morpho-physiological traits indicated that chlormequat chloride and IAA can partially alleviate the detrimental effect of drought in these aromatic grasses.     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Propagation of <Emphasis Type="Italic">Haemaria discolor</Emphasis> via <Emphasis Type="Italic">in vitro</Emphasis> seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm⁻³ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm⁻³ α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.This research was supported by grant (91AS-3.1.1-CI-C3) from the Council of Agriculture, Executive Yuan of Taiwan and grants (NSC-89-2317-B055-002 and NSC-91-2317-B324-001) from the National Science Council of Taiwan. This paper is Agricultural Research Institute Contribution No. 2158.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Functional characterization of a geraniol synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its application in production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geraniol synthase (GES) catalyzes the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries. Here we report the functional characterization of CaGES from Camptotheca acuminata, a camptothecin-producing plant, and its application in production of geraniol in Escherichia coli. The full-length cDNA of CaGES was obtained from overlap extension PCR amplification. The intact and N-terminus-truncated CaGESs were overexpressed in E. coli and purified to homogeneity. Recombinant CaGES showed the conversion activity from GPP to geraniol. To produce geraniol in E. coli using tCaGES, the biosynthetic precursor GPP should be supplied and transferred to the catalytic pocket of tCaGES. Thus, ispA(S80F), a mutant of farnesyl diphosphate (FPP) synthase, was prepared to produce GPP via the head-to-tail condensation of isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A slight increase of geraniol production was observed in the fermentation broth of the recombinant E. coli harboring tCaGES and ispA(S80F). To enhance the supply of IPP and DMAPP, the encoding genes involved in the whole mevalonic acid biosynthetic pathway were introduced to the E. coli harboring tCaGES and the ispA(S80F) and a significant increase of geraniol yield was observed. The geraniol production was enhanced to 5.85 ± 0.46 mg L^?1 when another copy of ispA(S80F) was introduced to the above recombinant strain. The following optimization of medium composition, fermentation time, and addition of metal ions led to the geraniol production of 48.5 ± 0.9 mg L^?1. The present study will be helpful to uncover the biosynthetic enigma of camptothecin and tCaGES will be an alternative to selectively produce geraniol in E. coli with other metabolic engineering approaches.     

Absence of turnover and futile cycling of sucrose in leaves of<Emphasis Type="Italic"> Lolium temulentum</Emphasis> L.: implications for metabolic compartmentation

To study the interdependence of sucrose accumulation and its hydrolyzing enzyme, soluble acid invertase (AI; EC 3.2.1.26), in fructan-accumulating temperate grasses and cereals, experiments were performed in which sucrose synthesis was abolished in leaves of Lolium temulentum by four independent inhibitory factors, each having a distinct mechanism of action. Trials in the light with mannose or vanadate and in the dark with anoxia or cyanide showed that previously accumulated sucrose was stable in the tissue over a 5- to 6-h period. Conversely, putatively vacuolar AI activity in tissue homogenates was sufficient to completely convert endogenous sucrose to monosaccharide within the same period. Continuous invertase-mediated breakdown of sucrose was thus not a feature of this tissue. It is concluded that AI and sucrose were not in metabolic contact in vivo, implying differential compartmentation. In darkness, in uninhibited leaves, sucrose concentrations fell linearly with respect to time at a rate of –0.6 mg g^–1 FW h^–1, over a 5- to 6-h period. This value is equivalent to rates of dark respiration measured by gas exchange. Dark-utilisation of sucrose was not accompanied by monosaccharide accumulation in the tissue. The rate of sucrose loss was 3-fold lower than rates of extractable AI activity. Hence, if AI was involved in dark-utilisation, then this implies at least a partial differential localisation of enzyme and substrate. However, the dark-consumption of sucrose was completely abolished by anoxia and by cyanide. It follows that dark-mobilisation (unlike invertase hydrolysis per se) was respiration-dependent and did not result from a simple co-localisation of sucrose and invertase. Taken together, the results show that sucrose and invertase do not share the same metabolic compartment in grass leaves. It is possible that invertase has no role in the mobilisation of stored sucrose in leaves of the fructan-accumulating grasses.Abbreviations AI Acid invertase - PAR Photosynthetically active radiation - TLC Thin-layer chromatography     

In vitro induction of inflorescence in <Emphasis Type="Italic">Dioscorea</Emphasis><Emphasis Type="Italic">zingiberensis</Emphasis>

Inflorescence induction and morphogenesis of regenerated flowers were investigated in vitro in Dioscorea zingiberensis C. H. Wright. Inflorescence induction was influenced by the type and concentration of phytohormones. When floral bud explants were incubated on a Murashige and Skoog medium containing a combination of 2.0 mg l⁻¹ 6-benzyladenine and 0.5 mg l⁻¹ indole-3-butyric acid, the highest frequency of inflorescence induction was observed. However, in the presence of gibberellic acid, induction efficiency was reduced although node length of inflorescence was increased. Ontogenetic studies revealed that the inflorescence primordia originated directly from axillary epidermal cells of the perianth and bract of the explants after 7 days. In vitro, male flowers developed normally and blossomed after 90–100 days. In addition, some bisexual flowers were observed. These results demonstrated that there were differences in sexual differentiation of floral buds in vitro compared with that in vivo.     

Biotransformation of menthol and geraniol by hairy root cultures of <Emphasis Type="Italic">Anethum graveolens</Emphasis>: effect on growth and volatile components

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l⁻¹), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC–MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17–52%), apiole (11–24%), palmitic acid (7–16%), linoleic acid (4–9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, α-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro efficacy of <Emphasis Type="Italic">Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

Physiological characterisation of <Emphasis Type="Italic">acuB</Emphasis> deletion in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the ∆acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.     

A simple method for <Emphasis Type="Italic">in vivo</Emphasis> labelling of lipid fractions in developing seeds of <Emphasis Type="Italic">Brassica campestris</Emphasis> L

An in vivo method of labelling lipid fractions in developing seeds of Brassica campestris using [1–¹⁴C] acetate has been developed. The “wick” method for introducing label into the intact plant is quite effective, safe and easy to use. The results obtained were reproducible and comparable to those reported earlier for seeds procured from greenhouse grown plants. The labelling pattern showed that rapid oil deposition began around 20 days after anthesis (DAA) and continued until about 45 DAA. The proportion of label in polar lipids declined and that in non-polar lipids increased during the phase of active oil synthesis. Among phospholipids, the label was incorporated mainly in phosphatidyl choline (PC), which was found to be the major fraction of phospholipids. During development, the two galactolipids i.e. monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) followed patterns exactly opposite to each other. The content of the label in MGDG decreased, while that in DGDG increased, indicating the conversion of MGDG to DGDG during maturation.