Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview.

Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.

Recovery of Oil Palm (Elaeis guineensis Jacq.) Somatic Embryos Cryostored For 20 Years.

　　BACKGROUND: A cryopreservation protocol has been established for oil palm somatic embryos (SEs), the efficiency of which must be evaluated, both in terms of regeneration and of long-term storage capacity, before its large-scale routine use. OBJECTIVE: To test the survival and recovery of 29 clones of oil palm somatic embryos cryostored for 20 years. MATERIALS AND METHODS: Clumps of SEs were pregrown for 7 days on medium containing 0.75 M sucrose, dehydrated in air-tight containers containing silica gel to moisture contents between 19-35% fresh weight, and then immersed directly in liquid nitrogen and stored in cryotanks for 20 years. RESULTS: Survival of SEs cryopreserved and rewarmed immediately displayed an average value of 19.1% for the 29 clones tested while survival of SEs rewarmed after 20 years of cryostorage was significantly higher, with an average of 33.2% for the 28 surviving clones. Out of these 28 surviving clones, three were lost due to contamination or regrowth decline, six produced only shoots and the rest proliferated. CONCLUSION: It is possible to cryostore oil palm SEs for extended periods and to regenerate proliferating cultures and plantlets from the cryopreserved material. The cryopreservation protocol established can thus be efficiently used to store oil palm germplasm and to manage large-scale production in industrial laboratories.

Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.).

In the present study we have identified and characterized the proteins expressed during different developmental stages of Elaeis guineensis calli obtained from zygotic embryos. We were interested in the possible proteomic changes that would occur during the acquisition of somatic embryogenesis and therefore samples were collected from zygotic embryos (E1), swollen explants 14days (E2) in induction medium, primary callus (E3), and pro-embryogenic callus (E4). The samples were grinded in liquid nitrogen, followed by total protein extraction using phenol and extraction buffer. Proteins were analyzed by two-dimensional electrophoresis (2-DE) and the differentially expressed protein spots were analyzed by MALDI-TOF mass spectrometry (MS and MS/MS). Interestingly, we have identified proteins, which can be used as potential candidates for future studies aiming at the development of biomarkers for embryogenesis acquisition and for the different stages leading to pro-embryogenic callus formation such as type IIIa membrane protein cp-wap13, fructokinase and PR proteins. The results obtained shed some light on the biochemical events involved in the process of somatic embryogenesis of E. guineensis obtained from zygotic embryos. The use of stage-specific protein markers can help monitor cell differentiation and contribute to improve the protocols for successfully cloning the species. BIOLOGICAL SIGNIFICANCE: Understanding the fate and dynamics of cells and tissues during callus formation is essential to understand totipotency and the mechanisms involved during acquisition of somatic embryogenesis (SE). In this study we have investigated the early stages of somatic embryogenesis induction in oil palm and have identified potential markers as well as proteins potentially involved in embryogenic competence acquisition. The use of these proteins can help improve tissue culture protocols in order to increase regeneration rates. This article is part of a Special Issue entitled: Environmental and structural proteomics.

In vitro rescue of interspecific embryos from Elaeis guineensis x E. oleifera (Arecaceae)

The African oil palm (Elaeis guineensis) is the most effective oil producer in tons per hectare. Nevertheless, its increasing cultivation in Latin America is harmed by the “lethal yellowing”. Genetic resistance to this anomaly can be found in the germplasm of American oil palm or caiaué (E. oleifera), a native species from the Amazon rainforest. However, the procedures adopted to induce seeds of E. guineensis to germination frequently result mild for interespecific hybrids. Embryo in vitro cultivation can be a viable option. This work was aimed initially to test liquid MS medium supplemented with different glucose or sucrose concentrations for the in vitro cultivation of zygotic embryos from E. guineensis x E. oleifera controlled pollinations. Additionally we investigated different compost mixtures to acclimatize the regenerated hybrid plantlets. Concentrations of 10, 20 and 30g/L of both sugars were tested on flasks containing five mature zygotic embryos, with 15 repetitions per treatment in a total of 450 explants. The number of embryos displaying shoots and radicles at least 2mm in length per experimental unit was evaluated during phase one of in vitro cultivation. Plantlets displaying shoots and radicles were transferred to phase two of in vitro cultivation and subsequently to acclimatization, under 70% shading with manual water supply. The experiments of acclimatization were conducted with 130 plantlets randomly distributed in pure horticultural compost, 3:1 or 1:1 compost:sand mixtures and each plantlet was defined as an experimental unit. Data were submitted to ANOVA, t test and analyzes of correlation (p≤0.05). Highest emergence rates were 97% for shoots and 73% for radicles, observed in MS medium supplemented with 20g/L (110mM) of glucose. This sugar in concentrations of 20 or 30g/L provided balanced shoot/root development, and this was considered one of the reasons for the higher frequency of plantlet establishment. The survival percentage was 55% after the first 43 days of acclimatization and by the fourth month, 66 plants developed simultaneously longer shoot and root systems in pure horticultural compost. in conclusion, radicle development was an impairment to plantlet establishment and was overcame under media with glucose above 110mM. Acclimatization could benefit from an extended period of in vitro development. Rev. Biol. Trop. 59 (3): 1081-1088. Epub 2011 September 01.

Elaeis guineensis es el productor de aceite más eficaz en toneladas por hectárea, su cultivo, cada vez mayor en América Latina, se ha visto perjudicado por el “amarilleamiento letal”. La resistencia genética a esta anomalía se puede encontrar en el germoplasma de la palma aceitera americana o caiaué (E. oleifera), una especie nativa de la selva amazónica. Sin embargo, los procedimientos adoptados para inducir la germinación de las semillas de E. guineensis frecuentemente produce resultados modestos para híbridos interespecíficos. El cultivo de embriones in vitro puede ser una opción viable. En este trabajo se probó el medio líquido MS complementado con diferentes concentraciones de glucosa o sacarosa en el cultivo in vitro de embriones cigóticos de E. guineensis x E. oleifera originados de polinización controlada. Además se investigaron diferentes mezclas de compost para aclimatar los híbridos regenerados. Las concentraciones de 10, 20 y 30 g/L de ambos azúcares se probaron en frascos que contenían cinco embriones cigóticos maduros, con 15 repeticiones por tratamiento y un total de 450 explantes. El número de embriones que muestran brotes y radículas de al menos 2mm de longitud por unidad experimental se evaluó durante la primera fase de cultivo in vitro. Las plántulas que mostraron brotes y radículas fueron trasladadas a la segunda fase de cultivo in vitro y, posteriormente, se aclimataron, por debajo de 70% de sombra con el suministro manual de agua. Los experimentos de aclimatación se llevaron a cabo con 130 plántulas distribuidas al azar en el compost hortícola puro, compost 3:1 o 1:1: mezclas de arena y cada plántula se definió como una unidad experimental. Los datos fueron sometidos a un análisis de varianza, prueba t y análisis de correlación (p≤0.05). Las tasas más altas de emergencia fueron 97% y 73% para brotes y radículas respectivamente, en el medio MS complementado con 20g/L (110mM) de glucosa. Este azúcar en concentraciones de 20 o 30g/L permitió un desarrollo balanceado de brotes/desarrollo de raíces, que fue considerado como una de las razones de la alta frecuencia de establecimiento de las plántulas. El porcentaje de supervivencia fue de un 55% después de los primeros 43 días de aclimatación y por el cuarto mes, 66 plantas desarrollaron simultáneamente hojas largas y un sistema radical en el compost hortícola puro. En conclusión, el desarrollo radicular fue un impedimento para el establecimiento de plántulas y se superó en el medio con glucosa por encima de 110mM. La aclimatación podría beneficiarse con un largo período de desarrollo in vitro.

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos.

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.

A novel transcript of oil palm (Elaeis guineensis Jacq.), Eg707, is specifically upregulated in tissues related to totipotency.

In this study, we report the molecular characterization of clone Eg707 isolated from cell suspension culture of the oil palm. The deduced polypeptide of clone Eg707 is highly similar to an unknown protein from Arabidopsis thaliana. The presence of an Ald-Xan-dh-C2 superfamily domain in the deduced protein sequence suggested that Eg707 protein might be involved in abscisic acid biosynthesis. Eg707 might be present as a single copy gene in the oil palm genome. This gene is highly expressed in tissue cultured materials compared to vegetative and reproductive tissues, suggesting a role of this gene during oil palm somatic embryogenesis or at the early stages of embryo development. Expression analysis of Eg707 by RNA in situ hybridization showed that Eg707 transcripts were present throughout somatic embryo development starting from proembryo formation at the embryogenic callus stages till the maturing embryo stages. Since proembryo formation within the embryogenic callus is one of the first key factors in oil palm somatic embryo development, it is suggested that Eg707 could be used as a reliable molecular marker for detecting early stage of oil palm somatic embryogenesis.

Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins.

Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.

Emergência de plântulas de Astrocaryum aculeatum G. May. em função da temperatura e do período de embebição das sementes / Emergence of Astrocaryum aculeatum seedlings according temperature and soaking period of seeds

O presente trabalho teve por objetivo avaliar a emergência de plântulas de Astrocaryum aculeatum a partir de sementes submetidas a diferentes temperaturas e períodos de embebição. O delineamento experimental foi inteiramente casualizado, em esquema fatorial 4 (temperaturas de embebição em água: 25ºC, 30ºC, 35ºC e 40ºC) X 3 (períodos de embebição: 2, 4 e 6 dias), com testemunha (sem embebição) e com quatro repetições. As semeaduras foram realizadas em viveiro. A emergência e o índice de velocidade de emergência só diferiram entre a testemunha e os tratamentos aplicados, independentes do período e da temperatura, com resultados favoráveis para a embebição das sementes. O tempo médio de emergência apresentou efeito de interação significativo, destacando-se a utilização da temperatura de embebição de 40ºC, associada ao período de 4 dias, que proporcionou um menor tempo médio (163 dias). O tempo inicial de emergência foi menor na temperatura de 35ºC (80 dias), enquanto o tempo final de emergência não apresentou diferença entre as médias. Sementes embebidas por 2 dias apresentaram 50 por cento de sementes mortas ao final do experimento, enquanto as embebidas por 4 dias, apenas 38 por cento. A emergência de plântulas de A. aculeatum foi favorecida pela embebição, independente da temperatura e do período utilizados.

This study evaluated the seedling emergence of Astrocaryum aculetum seeds soaked in water for different periods at different temperatures. The experimental design was entirely randomized, in factorial 4 (temperatures of soaking in water: 25ºC, 30ºC, 35ºC e 40ºC) X 3 (period of soaking: 2, 4 and 6 days), additional treatment (control, without soaking), with four replications. Before (control) and after the soaking periods in different temperatures, the seeds were planted in nursery. The emergence and its velocity differed only in the comparison of the control with the applied treatments, with favorable results of all soak treatments, independent of temperature and duration. The mean time of emergence presented a significant interaction effect, with the four day 40ºC soaking temperature period, presenting a lower mean time (163 days). The initial emergence time was lower in 35ºC temperature (80 days), while the final time didn't show differences among means. Seeds soaked for two days had 50 percent dead seeds, while seeds soaked for four days had just 38 percent. Seedling emergence was favored by soaking, independent of temperature and duration.

In vitro conservation of oil palm somatic embryos for 20 years on a hormone-free culture medium: characteristics of the embryogenic cultures, derived plantlets and adult palms.

This study was conducted over a period of 20 years, to assess the problems involved in developing subcultures over a very long period, of oil palm (Elaeis guineensis Jacq.) somatic embryos which were maintained in vitro on a Murashige and Skoog mineral-based culture medium, without growth regulators. Analysis of the proliferation rate of the embryogenic cultures, along with the survivability of the regenerated plantlets after their transfer into soil and of the flowering of the derived adult palms has been conducted for cultures maintained in vitro during 1 to 20 years. From the ninth year of maintenance, the tissue quality of the somatic embryos gradually began to decline. However, after more than 20 years, 30% of the 20 clones tested still continued to proliferate satisfactorily on the same maintenance medium, keeping their multiplication potential intact. Even though a depressive effect of the age of the lines has been observed on the survival capacity of plants under natural conditions, it is noteworthy that among the clones originating from 20-year-old cultures only eight of them (40%) have exhibited the "mantled" floral abnormality. Different hypotheses concerning the origin of the disruptions observed on the in vitro cultures, plantlets and adult palms that occur over a very long period of in vitro conservation are discussed.

Biolistic mediated production of transgenic oil palm.

Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.

Contrasting globulin and cysteine proteinase gene expression patterns reveal fundamental developmental differences between zygotic and somatic embryos of oil palm.

Oil palm (Elaeis guineensis Jacq.) somatic embryos differ from zygotic embryos in that they accumulate only small amounts of storage proteins. We compared the balance between deposition and degradation of storage proteins during zygotic or somatic embryogenesis and germinative growth in the two types of embryos. During mid to late zygotic embryogenesis, storage proteins accumulated and globulin 7S (GLO7A) gene transcripts were detected, whereas neither protease activity nor cysteine proteinase (CPR) gene transcripts were detected. Globulin degradation occurred after 8 days of in vitro germination in zygotic embryos and was accompanied by a decrease in GLO7A transcripts. Transcripts of three cysteine proteinase genes of the papain family were detected as early as Day 2 of in vitro germination. Several proteolytically active protein bands were identified by zymography, and CPR-like proteins were detected with an antibody raised against the Vicia sativa L. cysteine proteinase CPR1. Protease activities and CPR-like proteins were observed from Day 8 onward when globulin degradation occurred. During somatic embryogenesis and subsequent germinative growth, only small amounts of storage proteins accumulated, even though GLO7A transcripts were detected. Two of the three cysteine proteinase genes were expressed throughout both somatic embryogenesis and germinative growth. Protease activities and CPR-like protein species were detected in somatic embryos at several developmental stages. In contrast to zygotic embryogenesis, the accumulation of globulins and their subsequent mobilization appear to be concomitant processes during somatic embryogenesis, which could explain the low accumulation of storage proteins in somatic embryos.

Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: identifying genes associated with callogenesis and embryogenesis.

BACKGROUND: Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. RESULTS: A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. CONCLUSION: This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as early markers for embryogenesis, the genes need to be tested on earlier stages of tissue culture and a wider range of genotypes. This collection of ESTs is an important resource for genetic and genome analyses of the oil palm, particularly during tissue culture development.

Somaclonal variation in micropropagated oil palm. Characterization of two novel genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin.

In vitro micropropagation based on somatic embryogenesis provides an efficient means to multiply selected genotypes of oil palm (Elaeis guineensis Jacq.). Despite its considerable potential, somatic embryogenesis can yield plants bearing a homeotic flowering abnormality known as mantled. Because the mantled abnormality is epigenetic, it cannot be detected with conventional structural molecular markers. Thus, to develop a means of discriminating among callus cultures carrying or lacking the mantled abnormality, we used a gene expression approach. We describe two novel oil palm genes, EgM39A and EgIAA1, both of which display increased transcript accumulation in epigenetically abnormal calli. EgIAA1 codes for an oil palm relative of the Arabidopsis thaliana (L.) Heynh. AXR3/IAA17 protein involved in early auxin response and EgM39A codes for a protein of unknown function sharing sequence similarities with asparagine synthetases. In addition to their enhanced expression in somaclonal variant callus lines, both genes displayed increased transcript accumulation in response to auxin treatment. Normal seed-derived zygotic embryos germinated in the presence of auxin accumulated increased amounts of EgIAA1 transcripts after a few hours of treatment, suggesting a role in auxin response similar to that demonstrated for IAA genes in other species. The EgM39A gene also displayed enhanced transcript accumulation in auxin-treated zygotic embryos. Although only a small increase was seen after 24 h, greater changes were observed after 15 days. Both genes show potential as early markers of clonal conformity and may help to elucidate the nature of the epigenetic changes causing the mantled abnormality.

Immature embryo: a useful tool for oil palm (Elaeis guineensis Jacq. ) genetic transformation studies

Oil palm (Elaeis guineensis Jacq.) is the highest yielding oil-bearing crop. However, being a perennial crop, genetic improvement of oil palm is extremely slow. Indeed, compared to other annual oil crops such as soybean and rapeseed, genetic manipulations remained less important. Therefore, to remain competitive, oil palm growers and breeders need new and novel approaches. In this report, the potential of immature embryos (IE) as a useful tool for oil palm genetic transformation studies was evaluated. It was evident that IEs were amenable to both direct and Agrobacterium-mediated gene transfer. Due to the abundant supply of IE, optimization of biolistic and Agrobacterium-mediated gene transfer into IEs were easily carried out. Transient transformation frequencies were comparable to other plant systems reported, with as high as 97.4 percent recorded for biolistic and 64.4 percent for Agrobacterium-mediated gene transfer. Like most moncots, oil palm tissues were less sensitive to kanamycin, geneticin and chloramphenicol. Instead, both hygromycin and phosphinotrycin were toxic 20 mg/l, making both suitable candidates for selecting putative transformants. IEs were also more responsive to in vitro manipulations as compared to other explants such as leaf and root tissues. Rapid in vitro response to callusing and embryogenesis or rapid and highly efficient direct germination resulted in a shorter culture period. This would minimize the production of abnormal clonal palms, which has been associated to chromosomal aberration due to prolonged time in culture. In addition, IEs also allows rapid and direct introduction of elite genes into breeding programs and in biclonal seed production.

Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inoculum size, and conditioned medium on biomass production.

We report the successful culture of oil palm (Elaeis guineensis Jacq.) suspension cells in a bioreactor. In vitro propagation of this perennial monocotyledonous tree is an important part of the oil palm industry's approach to clonal propagation of high-yielding accessions. During culture of oil palm cells in a batch bioreactor, nutrients and extracellular metabolites were monitored, and kinetic parameters and nutrient-to-biomass conversion yields were calculated. The biomass increased approximately 3.5-fold per month, consistent with values reported for shake flask cultures. Although the carbon source was completely depleted by the end of the run, nitrogen sources remained in large excess and the sugar-to-biomass conversion yield remained low. Linear growth indicated that the cells were limited. The results obtained from the bioreactor runs indicated that we should be able to improve biomass production by carrying out optimization studies. Therefore, we initiated multi-factorial analyses using response surface experimental designs to investigate the effects of different nitrogen sources, as well as inoculum size and conditioned medium, on biomass production in flask cultures. Whereas glutamine does not have a significant effect on biomass production, ammonia has a positive effect up to an optimum concentration. Both inoculum density and conditioned medium have positive, synergistic effects on biomass production.