Potential antioxidant and anti-inflammatory action of Hypericum hookerianum extracts in a liposome system evaluated with zebrafish embryos.

The purpose of this study was to assess the antioxidant and anti-inflammatory potential of liposomal formulations enriched with Hypericum hookerianum (Hyp) aqueous extracts. Cotyledon segments derived from protocorms of H. hookerianum were cultured on Murashige and Skoog (MS) media supplemented with Kinetin (KN, 1 mgl-1) and Naphthalene acetic acid (NAA, 0.1 mgl-1) to induce hypericin-rich red shoots (HypR, 0.87 mg/G DW). Highly stable liposomes (-29.4 mV) were successfully developed which encapsulated 63 ± 0.8% Hyp extracts, respectively. MTT assay subsequently confirmed the biocompatibility of liposome compositions using fibroblast cell lines. This work also evaluated acute toxicity of L-HypR and L-HypG formulations using Danio rerio (Zebrafish) embryos for 96 hpf. The expression of antioxidant and anti-inflammatory genes were found to be upregulated for L-HypR than L-HypG (green shoots without hypericin) formulations. These properties of L-HypR may be extremely useful for incorporating lipophilic substances into the food or pharmaceutical industry.

Utilization of zygotic embryos of an economic rattan palm Calamus thwaitesii Becc. (Arecaceae) for somaplant regeneration and cryobanking.

Zygotic embryos excised from immature green fruits of the rattan palm, Calamus thwaitesii and cultured for 16 weeks under optimum culture conditions in Murashige and Skoog (MS) medium supplemented with 31.67 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 35.23 µM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) produced mixed (compact and friable) calli at 70 and 92 % rates. The semi-friable part of the callus (~500 mg) separated and subcultured in medium containing 2.22 µM 6-benzyladenine and 1.07 µM α-naphthalene acetic acid produced groups of 10.37 ± 0.60-21.52 ± 0.48 discrete globular embryoids of varied size in 6-8 weeks. Calli raised in presence of 2,4,5-T were relatively more prolific, friable and embryogenic than those induced by 2,4-D. Embryoids (2.0-3.0 mm) isolated and cultured in basal medium germinated into plantlets at 65 % efficiency while the immature (0.5-2.0 mm) ones produced calloid structures. Approximately 15 % of the in vitro plantlets raised from the 2,4-D-induced embryogenic calli produced secondary immature embryoids on the sheath and lamina parts of leaves which were isolated and cultured in basal medium developed into rooted plantlets at 62 % rate in 12-16 weeks. The continued growth of the embryo-derived callus through successive subcultures together with differentiation of embryoids into plantlets, and the formation of immature embryoids on in vitro plantlets in MS basal nutrient medium reports for the first time a reliable method of producing at least 116 plants from a single embryo in a year. Rooted plantlets treated with 50 % glycerin survived at 78 % rate after hardening and 82.7 % of the hardened plants reintroduced into forest segments showed uniform growth free of morphological abnormalities after 3 years of observation. In addition to embryogenesis, cryopreservation of the zygotic embryos through simple drying and encapsulation-dehydration methods resulting 60-70 % recovery rates also offers another option for long-term conservation and sustainable utilization of this plant genetic resource.

A protocol for high frequency regeneration through nodal explant cultures and ex vitro rooting of Plumbago rosea L.

A rapid clonal multiplication scheme comprising direct multiple shoot initiation and downsizing of the node with buds proliferated upon during subculture was developed for Plumbago rosea. Sixty five per cent of the nodes (approximately 2.0 cm) dissected out of young shoots from field grown plants and cultured in MS agar medium containing 3% sucrose and 15.4 microM BAP remained contamination free and responded at 95% rate with callusing at basal cut end and axillary bud break in 5 days followed by the formation of 2.41 +/- 0.14 shoots of 0.87 +/- 0.14 cm length in 3 weeks. Though differences in frequency and number of buds formed between nodes of 1-5 positions from the young shoots was negligible, the shoots emanated from the youngest node were shorter (0.92 +/- 0.19 cm) than those (2.3 +/- 0.50 cm) of the mature 5th node. Synergistic influence of BAP and auxins on caulogenesis was absent. Bud emergence in shorter (approximately 0.5 cm) nodes was delayed up to 3 weeks and extensive callus proliferation from the cut basal end overlapped the 8.2 +/- 0.37 axillary shoots/buds formed after 7 weeks. Reduction in the size (downsized) of the 2.0 cm node with buds to 1.0 cm by dissecting out the basal internodal segment having the callus and subculture of them (approximately 1.0 cm) with buds in contact with the medium for 3 weeks contributed to maximum multiplication of 42.1 +/- 5.40 shoot buds. Division of the shoot cluster and transfer of 2-3 shoots (0.5-1.5 cm) in a clump to MS basal liquid medium induced elongation of the shoots to 4.1 +/- 0.18 cm in 2 weeks. Shoots of 3.0-4.2 cm length were rooted within 3 weeks at 100% efficiency in vitro or ex vitro without hardening. In vitro rhizogenesis in presence of 0.49 microM IBA is recommended for enhanced rooting and high yield of commercially important tuberous roots during cultivation in the field.

In vitro cloning and homestead cultivation of primitive Musa cultivars.

Two primitive diploid Musa cultivars, Matti and Chemmatti from the extreme southern part of the Western Ghats were multiplied by in vitro culture of sucker-derived shoot apices. Decontaminated corm explants (1 cm x 1 cm) having shoot apex (approximately 0.3 cm) cultured for 1 month in Murashige and Skoog basal agar medium was cut vertically into eight segments and each segment having a part of shoot meristem was cultured in presence of 6-benzylaminopurine (BAP) and combinations of BAP and indole-3-acetic acid (IAA) or indole-3-butyricacid (IBA) to produce multiple shoots. After 12 weeks of culture, maximum number of shoots (32) in both the cultivars were produced in approximate 60% of the explants in presence of BAP and IAA each at 1.5 mg/l(-1) (Matti) and 40% of the explants in 2.5 mg/l(-1) of BAP and 1.5 mg/l(-1) of IAA (Chemmatti). Buds were formed from the base of the subcultured shoots and somewhat more number (34) of shoots were obtained in Matti than in Chemmatti (31) after 8 weeks. Difference in the concentration of cytokinin required for shoot initiation and multiplication, persistence of exudation through the subculture and red colouration of the early formed sheathing leaf bases in the shoots in Chemmatti indicated possible genotypic differences between the two cultivars. Multiple shoot proliferation achieved through five subcultures of the isolated shoots without any decline. Transfer of shoots (4-5 cm) into MS basal medium favoured rooting in 4 weeks and rooted plants (9 cm) were hardened and established (80-95%). Mericlones of Matti cultivated in homesteads produced bunches of uniform characters in 13 months.

Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant.

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2-3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l-1 and 0.1 mg alpha-naphthaleneacetic acid l-1 produced more ajmaline (0.01 mg g-1 dry wt) and ajmalicine (0.006 mg g-1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g-1 dry wt) and ajmalicine (0.0007 mg g-1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.

In vitro mass multiplication and production of roots in Plumbago rosea.

Mass multiplication of Plumbago rosea was achieved by indirect organogenesis in young stem, leaf and root explant cultures of 6-9-month-old plants. All the explants responded similarly in a hormonal regime of 2.5 mg/L BA and 1.5 mg/L NAA with the formation of nodular callus in 4 weeks; the callus was divided and subcultured at 4-week intervals in the presence of 3.0 mg/L BA to produce up to 23.5 +/- 1.6 shoots in 18 weeks and then at 2.0 mg/L BA to produce up to 79.6 +/- 1.5 shoots in 23 weeks. The shoots of 2.0-3.5 cm length were rooted easily in half-strength MS agar medium supplemented with 0.1 mg/L IBA and rooted plants established within 4 weeks at a 95-98 % rate without hardening. Eight weeks after establishment, the micropropagated plants were transferred to experimental plots and cultivated for 10 months to obtain a significantly higher number (18.0 +/- 0.5) of larger tuberous roots (137.4 +/- 3.4 g fw/plant) compared to conventional rooted cuttings (14.0 +/- 1.7, 47.9 +/- 1.6 g fw/plant). During this period, the concentration of the root-specific compound, plumbagin recorded per g dw (1.5 %), was higher than that of conventionally propagated plants (0.9-1.0 %). The early formation of plumbagin-rich tuberous roots holds significant potential for the commercial cultivation of the micropropagated plants.

A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng.

Leaf explants collected from flowering plants of Vanda spathulata were cultured in Mitra medium with combinations of 6-benzyladenine (BA; 13.2-88.8 microM) and indole-3-acetic acid (IAA; 0.0 -85.6 microM). Combination of BA (66.6 microM) and IAA (28.5 microM) induced maximum shoots (17.33) from foliar meristems (leaf base). BA individually did not induce caulogenesis in leaf explants. For optimized multiplication, BA:IAA (2:1 microM) was essential at 22.2- 88.8 microM of BA. Re-cultured leaf explants produced lesser number of shoots compared to original explants and were nearly equal at combinations of 22.2-44.4 microM of BA and 5.7-28.5 microM of IAA. Rooting of shoots (> 95%) occurred in medium containing banana pulp (75 gl(-1)) and IAA (5.7 microM) within 3-9 weeks. Plantlets with 2-5 roots of 2-5 cm length established easily in community pots at 80-90% rates without hardening.

Ammonium nitrate in the culture medium influences regeneration potential of cryopreserved shoot tips of Holostemma annulare.

Influence of NH4NO3 in the pre-freeze and post-freeze culture medium and 2 or 30 day preconditioning in the presence of 0.5 M sucrose on regeneration of shoot tips of Holostemma annulare following cryopreservation using an encapsulation-dehydration protocol was studied. A long preconditioning phase of 30 days significantly reduced tissue water and improved post-freeze recovery of shoot tips. Under the long preconditioning treatment, Murashige & Skoog (MS) medium free of NH4NO3 (MS-3) allowed maximum regeneration (59%) of liquid nitrogen (LN) exposed shoot tips with less frequency of callusing (10.4%) after 45 days of post-freeze culture. Corresponding desiccated control shoot tips showed 85-90% regeneration. A 3.75 mM NH4NO3 concentration (MS-4) favoured 72-89% and 43-47% regeneration after desiccation and LN exposure respectively. The standard MS medium with 20.6 mM NH4NO3 (MS-1) allowed poor regeneration after desiccation (39-53%) as well as LN exposure (8-23%). The study reveals the importance of reducing ammonium nitrate in the culture medium to get maximum recovery of cryopreserved shoot tips of Holostemma annulare.

Morphogenetic responses of six Philodendron cultivars in vitro.

In vitro morphogenic response of nodal explants from six cultivars of Philodendron viz, blue mistic, painted lady, pink prince, pluto, royal queen and green emperor was studied. Frequency and number of shoot formation depend on the cultivars and concentration of BAP. High frequency and number of shoot formation were obtained w hen the nodal explants were cultured in Nitsch medium supplemented with BAP (6.8-11.8 microM), sucrose (2%) and agar (0.45%), initially in the dark for 8-10 weeks followed by 16 hr photoperiod. Regenerated shoots were rooted on medium without growth regulators. After two weeks of hardening, rooted and rootless shoots were established in the soil with more than 90 and 65% survival rates respectively, while the unhardened plantlets showed a much lower percentage (20%) establishment. A standard protocol for the rapid multiplication of Philodendron is presented.

Rapid in vitro multiplication and restoration of Celastrus paniculatus Willd. sub sp. paniculatus (Celastraceae), a medicinal woody climber.

Nodes, shoot tips, internodes and leaf bases (approximately 1.0 cm) excised from young vines of the flowering woody climber, Celastrus paniculatus WilId. sub. sp. paniculatus (Celastraceae) were cultured in Murashige and Skoog (MS) medium containing agar (0.6%), sucrose (3%) and varied concentrations of 6-benzyl aminopurine (BAP) and kinetin. All the explant types were regenerative and maximum number (3.6) and frequency (94%) of axillary shoot formation of (5.08 cm long) was recorded in the nodes cultured in BAP (1 mg L(-1)) after 6 weeks. Combinations of BAP (1 mg L(-1)) and indole-3-acetic acid/l-naphthalene acetic acid (0.01-1 mg L(-1); IAA/NAA) tested with nodes induced formation of less number (3 and 2.2) of shoots at same frequency (94%). All the explant types viz. node, shoot tip, internode and leaf base of in vitro derived shoots responded earlier and better in lower concentrations of BAP (0.5-2 mg L(-1)) with formation of 8, 3.1, 6.4 and 1.8 shoots respectively during the same period. In spite of the advanced and increased caulogenic responses, differences in cytokinin requirements between different explants observed during culture initiation still persisted with the nodes, shoot tips, internodes and petiole segments responding best at 0.5, 1 and 2 mg L(-1) BAP, respectively. The repeated reculture up to 10 cycles of the nodes from the shoot cultures each at 6-week intervals enabled multiplication and stocking of shoots without decline. Rooting of 3-7 cm shoot cuttings was induced in half-strength MS liquid medium containing IAA (1 mg L(-1)) with formation of 7.25 roots of 2.41 cm length within 6 weeks. Rooted plants were established at 84-96% rate in community pots without hardening, the least value (84%) being obtained with NAA- induced thick and calloid rooted plants. Four month old community potted plants were reintroduced into native forest habitats at 95% efficiency and 8 months after restoration, the plants were uniform in morphological, growth, cytological and peroxidase and esterase isozyme characteristics.

In vitro multiplication of Nothapodites foetida (Wight.) Sleumer through seedling explant cultures.

In vitro multiplication of Nothapodites foetida (Wight.) Sleumer was achieved using axenic seedling explant cultures. Isolated nodes (1.0-1.2 cm) and shoot tips (1.0-1.5 cm) cultured in Murashige and Skoog's agar medium containing varying concentrations of TDZ, BA and combinations of 2iP and GA3. Single shoot (0.8-1.2 cm) was regenerated in each culture after 6 weeks. Axillary shoots were then excised and recultured for 8 weeks in medium containing TDZ (0.05 mgL-1) which formed shoots (about 4 in no.; 2 cm) from the basal node. Axillary branches (2) which formed on 60% of these shoots after 10-12 weeks of culture were separated and recultured in the same medium for 8 weeks. Three shoots (0.8-1.0 cm) per culture were regenerated. Shoots of 0.8-1.8 cm length were subcultured on a low cytokinin (0.01 mgL-1 TDZ) regime to induce shoot elongation (2.0-3.5 cm) in 4 weeks. Shoot cuttings were rooted (60%) in the medium containing IBA (1.5 mgL-1). Rooted plantlets established in pots (90%) after hardening resumed normal growth in 3 months.

Micropropagation of terrestrial orchids, Anoectochilus sikkimensis and Anoectochilus regalis.

Two horticulturally important jewel orchids of the genus Anoectochilus were successfully micropropagated. Isolated nodes of A. sikkimensis collected from Sikkim in Eastern Himalayas and subsequently reared under nursery conditions and A. regalis collected from Western Ghats in Southern India were cultured for 12 weeks on Woody Plant Medium (WPM) to produce a maximum of 4.8 and 5.6 callus--free axillary shoots respectively at 95 and 98% efficiency. During reculture of the explants from in vitro raised shoots under the same conditions, the total number of shoots obtained from the nodes (21.4) and shoot tips (8.2) of A. regalis were significantly higher than those hardy and slow growing shoots of A. sikkimensis (12.3 and 4.3) respectively. Shoots (4-6 cm) were rooted in medium containing NAA (2.70 microM) and activated charcoal (0.2%). The rooted plants established at 95-98% rate in community pots after hardening. After 6 months, green house adapted community potted plants of A. regalis were transferred to natural forest habitat locally with 95 and 70% survival respectively after 12 months. The plants, established in community pots and native forest habitat were free from any morphological and growth defects.

Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree.

Rapid clonal multiplication of Aegle marmelos (L.) Corr. (Rutaceae), a medicinal tree, was achieved by enhanced axillary bud proliferation in young single-node segments of a 25-year-old tree cultured in Murashige and Skoog (MS) nutrient medium. Bud break was dependent on cytokinin supply, but the synergistic combination of 2.5 mg l-1 6-benzylaminopurine (BAP) and 1.0 mg l-1 indole-3-acetic acid (IAA) induced the formation of 12.1 shoots of up to 5.2 cm length in 48% of the explants after 7 weeks of culture. Explants of in-vitro-grown shoots - node, whole leaf, shoot tip and internode - were subcultured in the presence of 0.05-2.5 mg l-1 BAP to produce 11.3, 18.4, 5.3 and 3.2 shoots and shoot buds at a 100%, 70%, 95% and 40% rate respectively, in 7 weeks. Different shoot nodes and leaves were equally regenerative and adventitious organogenesis in the latter was confined to cut petiolar ends. Nodal explants responded most favourably at low BAP (0.05-0.1 mg l-1) and produced uniform (3.8-5.3 cm) shoots facilitating their simultaneous harvest for rooting. Repeated subculturing through five cycles of nodes and leaves of shoot cultures enabled continuous production of healthy callus-free shoots without any sign of decline. Shoot cuttings (3.0-5.2 cm) were best rooted in half-strength MS medium with 0.5 mg l-1 IAA (70%) or 10.0 mg l-1 indole-3-butyric acid (90%). Eighty-eight percent of the rooted plants were established in polybags after hardening.

Rapid propagation through shoot tip culture of Trichopus zeylanicus Gaertn., a rare ethnomedicinal plant.

Rapid micropropagation of Trichopus zeylanicus Gaertn. subsp. travancoricus Burkil ex Narayanan, a rare ethnomedicinal herb endemic to the Western Ghats of southern India, was achieved by culturing shoot tips (0.3-0.5 cm) of 2-month-old axenic seedlings on Woody Plant Medium. Among the cytokinins tested, only BAP induced callus-free multiple shoot bud formation, with a maximum of 8.5±0.4 buds per explant being obtained with 2.0 mg.l(-1) BAP after 8 weeks of culture. Shoot tips containing proliferated buds were divided and subcultured on medium containing 0.2 mg.l(-1) BAP to produce 12.0±1.0 shoots per explant in 6 weeks. Excision of buds after culture initiation, with subculture of the debudded basal tissue in 2 successive passages yielded 20.0±1.0 and 13.5±0.5 buds per explant respectively. Each bud cultured in turn for 4 weeks on WPM with 1.0 mg.l(-1) BAP formed 3.8±0.4 secondary buds which were repeatedly recultured to increase bud production. Altogether this method enabled an estimated harvest of 7848 buds from a single shoot tip in 28 months. Shoots (3-5 cm) developed from bud cultures were rooted in half-strength WPM medium with 0.5 mg.l(-1) each of NAA and IBA, and 90-100% of the rooted plants were established in the field after hardening. Micropropagated plants were grown to maturity free of defects in growth, morphological, flowering and seed set characteristics.

In vitro multiplication and field establishment of Adhatoda beddomei C. B. Clarke, a rare medicinal plant.

Clonal propagation of Adhatoda beddomei C.B. Clarke (Acanthaceae), a rare medicinal shrub, was achieved through callus-free axillary meristem proliferation from stem node explants of field-grown plants cultured in SH medium. Shoot multiplication was a function of cytokinin activity but sustained growth of the shoots was dependent on the synergistic effect with the auxin, IAA. An optimum number of 5-10 shoots per explant were obtained in 6 weeks using 3.0 mg.l(-1) BAP, 0.5 mg.l(-1) 2-ip and 1.0 mg.l(-1) IAA, Upon subculture, vertical halves of the precultured node with the differentiated shoots yielded a larger aggregate number of shoots (23-27) than the uncut precultured node left intact (15-17). Shoot multiplication was rapid and consistent over prolonged periods when the hormonal concentrations were reduced to 1.0 mg.l(-1) BAP and 0.2 mg.l(-1) IAA during subculture, and reculture of the nodal explants derived from shoot cultures. Rooting of 3-5 cm shoots thus obtained was greatly accelerated in stationary liquid medium containing 0.2 mg.l(-1) IBA or IAA. Hardening of the rooted plantlets in the humidity chamber was essential for high frequency (95%) survival. Micropropagated plants established in the field flowered after fifteen months and were free from apparent defects in cytological, growth and flowering characteristics.

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant.

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l(-1)) induced high frequency (88%) development of axillary shoot buds (3.2) in 4-5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26-35 shoots) was recorded when using culture initiation media with 0.5 mg.l(-1) each of BAP and NAA followed by subculture in 0.2 mg.l(-1) BAP. The shoot multiplication rate was further accelerated by reculturing 0.4-0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l(-1) BAP. Shoot cuttings (3.5-7.0 cm) were rooted in 0.2 mg.l(-1) IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.

Mechanical separation of palisade and spongy-parenchyma cells from the leaves of mesomorphic dicotyledons for photosynthetic studies.

Palisade and spongy-parenchyma cells were isolated from the leaves of a number of mesomorphic dicotyledons by simply brushing the upper and lower sides, respectively, of the leaves with nylon brushes. Cross-contamination with the opposite cell type was minimal and both cell types were photosynthetically as active as leaf discs. The rates and early products of CO2 incorporation in the two cell types isolated from Zinnia elegans Jacq. plants grown in full natural light were the same, indicating that the photosynthetic physiology of the two cell types is quite similar.

Growth of Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) in Still Nutrient Medium.

Cell suspension cultures were established from the callus proliferation of leaf explants of 10- to 12-day-old seedlings of the peanut (Arachis hypogaea L. var. TMV-3). The cells could be cultivated in both agitated and still media, the latter promoting more of chlorophyll (Chl) synthesis. High Chl content (210-240 micrograms Chl per gram fresh weight), yield of free and pipetable cells, presence of all the pigments in the same ratio as that of the leaf tissue, and high rates of O(2) evolution (140-170 micromoles O(2) per milligram Chl per hour) were some of the desirable features of the still-grown cell cultures. However, considerable variations with regard to the above characters were observed between the cell cultures of different varieties of the peanut.O(2) evolution by the cultured cells was dependent on exogenous supply of HCO(3) (-). A well-developed photosynthetic apparatus as evidenced from photosystem I and photosystem II activities of the isolated chloroplasts and variable fluorescence measurements with the cell cultures was further documented by electron microscopic evidence of distinct granal stackings in chloroplasts and sodium dodecyl sulfate-polyacrylamide gel separation of thylakoid membranes into P700 Chl a protein complex and light-harvesting Chl a/b complex. Evidence is presented for the relative increase in the Chl associated with P700 Chl a protein complex in contrast to the light-harvesting Chl a/b complex in the cultured cells as compared to intact leaf.

Carbon Assimilation in Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) Grown in Still Nutrient Medium.

The relative transport of photosynthetic and dark carboxylation products in photoheterotrophic cells of Arachis hypogaea L. var. TMV-3 at varied phases of growth were determined. Despite the presence of an equally competent photosynthetic apparatus as determined from (14)CO(2) incorporation rates in the dark and light, pulse-chase experiments revealed little or no change in the radioactivity of the C(3) intermediates but rapid disappearance of label from the dark carbon assimilates (malate and other tricarboxylic acid cycle intermediates) with a simultaneous increase in the aminoacid pool in early log-phase (10 days old) cells. However, significant flow of carbon through the photosynthetic intermediates resulting in the accumulation of sugars occurred in the late log-phase (34 days old) cells. Limitation of exogenous sugar in the nutrient milieu and depletion of reserve carbohydrates stored in starch of the chloroplasts of the cells were considered as the decisive factors in promoting transport of C(3) cycle intermediates through the reductive pentose phosphate pathway in photoheterotrophic cells. The observed drain of radioactivity even from the small amounts of tricarboxylic acid cycle intermediates synthesized during photosynthesis into glutamate indicated that the transport of carbon through the nonautotrophic pathway is not controlled by these factors.