Production Of Somatic Embryos Research Articles

English

The first objective of this study was to assess and optimize somatic embryo production in a genetically diverse range of cacao genotypes. The primary and secondary somatic embryogenesis response of eight promising cacao clones and a positive control was evaluated using modified versions of standard protocols. The second objective was to optimize the efficiency of primary somatic embryogenesis for a commercially important cacao clone, CCN 51, which has proven to be quite recalcitrant to standard protocols, relative to CCN 10, a clone also included in our analysis. The efficiency of the overall process was assessed by determining the number of somatic embryos produced per starting somatic tissue explant, as well as the quality of embryos (normal vs. abnormal) produced. Donor floral explants were subjected to five tissue culture steps, each 15-25 days in duration. Although all studied genotypes produced primary somatic embryos, most of them originated only from brown or brown-white callus. Overall, flower petals performed better than staminodes, and our best performing genotype yielded an average of 7-10 embryos produced in brown callus explants with embryogenic response during primary somatic embryogenesis procedures. In conclusions our analysis from a pilot in a small-scale are: 1) it is possible to achieve a high production of plants by somatic embryogenesis, although the efficiency is highly genotype-dependent; it is therefore necessary to optimize hormone balance and hormone type, as well as the explant type for each genotype, 2) through the use of secondary somatic embryogenesis, it is possible to increase somatic embryogenesis production at least ten-fold, and 3) the observed response variation between genotypes may reflect differences in endogenous and exogenously-supplied hormones. The importance of adapting the tissue culture protocol to the genotype is discussed. Key words: Somatic embryogenesis, 2,4-D, cellular competence, propagation, abnormalities, ethylene, cacao.

Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata)

Dioscorea rotundata, commonly known as white yam, is an important staple food crop widely cultivated in West Africa and provides food security to millions of people. Genetic improvements of this crop using the advanced biotechnology tools have been hampered hitherto by the recalcitrant nature of regeneration through somatic embryogenesis. Therefore, we have developed an efficient and reproducible system for plant regeneration via somatic embryogenesis. Explants of different types (immature leaf, node, stem internode, root segment, petiole, and axillary bud) of D. rotundata accession TDr 2436 were tested for their embryogenic potentials on Murashige and Skoog (MS) medium supplemented with various auxins (2,4-D, NAA, and picloram). Among all explants tested, axillary bud explants cultured on MS medium supplemented with picloram (0.5–12 mg/l) favored the production of calli. Maximum proliferation of calli (526 mg fresh weight/explant) was achieved on MS medium supplemented with picloram (0.5 mg/l), casein hydrolysate (600 mg/l), and proline (1 g/l). Histology analysis confirmed that the embryogenic calli produced on this medium were mixed with non-embryogenic calli. Transfer of calli on MS basal medium supplemented with activated charcoal (1 %) changed the color of calli to purple and promoted the production of somatic embryos (87 embryos/callus) as well as adventitious shoot buds. Furthermore, upon transfer to MS medium supplemented with BAP (0.4 mg/l), the embryos continued their differentiation and maturation and germinated into complete plantlets. The adventitious shoot buds produced multiple shoots on MS medium supplemented with BAP (0.4 mg/l). Well-developed germinated plantlets were acclimatized in the screen house with 90 % survivability. Histology studies confirmed that the regeneration of D. rotundata reported here followed dual regeneration pathways. The embryogenic calli regenerated through development of somatic embryos and germinated into complete plantlets, however non-embryogenic calli regenerated through organogenesis and developed multiple shoots. The developed protocol has potential for somatic hybridization, mass clonal propagation, and genetic transformation applications.

Large-scale, high-efficiency production of coffee somatic embryos

The study aims to compare the efficiency of previously used liquid media for coffee, evaluating NAA concentrations in liquid medium and proline in semi-solid medium in the regeneration of somatic embryos and asses concentrations of BA and IAA in the maturation of embryos in temporary immersion bioreactors. For the regeneration of globular embryos from embryogenic aggregates and calli, we tested five concentrations (0.00, 0.25, 0.50, 1.00 and 2.00 mg L-1) of NAA in liquid medium and five concentrations (0.0, 0.5, 1.0, 2.0 and 4.0 g L-1) of proline in semisolid medium. The multiplication of embryogenic aggregates was highest in culture medium MM, reaching a density 7.5 times greater than that of the initial density. NAA promoted a linear increase in embryo regeneration. The medium containing 2.0 mg L-1 BA and 0.0 mg L-1 IAA yielded the highest percentage of large cotyledonary embryos.

Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin.

Previous studies have shown that the beneficial effect of suppression of the Arabidopsis phytoglobin 2 gene, PGB2, on somatic embryogenesis occurs through the accumulation of nitric oxide (NO) within the embryogenic cells originating from the cultured explant. NO activates the expression of Allene oxide synthase (AOS) and Lipoxygenase 2 (LOX2), genes encoding two key enzymes of the jasmonic acid (JA) biosynthetic pathway, elevating JA content within the embryogenic tissue. The number of embryos in the single aos1-1 mutant and pgb2-aos1-1 double mutant declined, and was not rescued by increasing levels of NO stimulating embryogenesis in wild-type tissue. NO also influenced JA responses by up-regulating PLANT DEFENSIN 1 (PDF1) and JASMONATE-ZIM-PROTEIN (JAZ1), as well as down-regulating MYC2. The NO and JA modulation of MYC2 and JAZ1 controlled embryogenesis. Ectopic expression of JAZ1 or suppression of MYC2 promoted the formation of somatic embryos, while repression of JAZ1 and up-regulation of MYC2 reduced the embryogenic performance. Sustained expression of JAZ1 induced the transcription of several indole acetic acid (IAA) biosynthetic genes, resulting in higher IAA levels in the embryogenic cells. Collectively these data fit a model integrating JA in the PGB2 regulation of Arabidopsis embryogenesis. Suppression of PGB2 increases JA through NO. Elevated levels of JA repress MYC2 and induce JAZ1, favoring the accumulation of IAA in the explants and the subsequent production of somatic embryos.

Effects of abscisic acid and an osmoticum on the maturation, starch accumulation and germination of Picea spp. somatic embryos

The aim of this study was to investigate whether ABA, sucrose or Phytagel applied to the maturation medium at different concentrations affected the growth, development and starch content of somatic embryos of Picea abies and Picea omorika. Embryogenic tissues of both spruce species were placed on maturation medium supplemented with various doses of ABA (10, 20, 40 and 80 µM), sucrose (17, 34 and 68 g/L) or Phytagel (4, 6 and 8 g/L). Our results showed that ABA and the osmoticum had a significant effect on the production and maturation of somatic embryos of both spruce species. Supplementing the medium with low concentrations of ABA (10 µM) or sucrose (17 g/L) resulted in the precocious germination of embryos. Adding sucrose to the maturation medium at the highest tested concentration (68 g/L) improved the growth of the radicles of the embryos of both spruce species during the germination stage. Moreover, the intensity of the growth of the hypocotyls and radicles of P. omorika germinating embryos depended on the Phytagel concentration applied to the maturation medium. The starch content and the starch accumulation pattern of Picea abies and P. omorika somatic embryos were dependent on the concentration of ABA, sucrose or Phytagel in the maturation medium. In general, during development, the P. abies somatic embryos accumulated a much greater amount of starch than P. omorika embryos did. The effects of ABA and osmoticum on the starch accumulation pattern of Picea abies and P. omorika somatic embryos are first reported in this paper.

Moderate stress responses and specific changes in polyamine metabolism characterize Scots pine somatic embryogenesis.

Somatic embryogenesis (SE) is one of the methods with the highest potential for the vegetative propagation of commercially important coniferous species. However, many conifers, including Scots pine (Pinus sylvestris L.), are recalcitrant to SE and a better understanding of the mechanisms behind the SE process is needed. In Scots pine SE cultures, embryo production is commonly induced by the removal of auxin, addition of abscisic acid (ABA) and the desiccation of cell masses by polyethylene glycol (PEG). In the present study, we focus on the possible link between the induction of somatic embryo formation and cellular stress responses such as hydrogen peroxide protection, DNA repair, changes in polyamine (PA) metabolism and autophagy. Cellular PA contents and the expression of the PA metabolism genes arginine decarboxylase (ADC), spermidine synthase (SPDS), thermospermine synthase (ACL5) and diamine oxidase (DAO) were analyzed, as well as the expression of catalase (CAT), DNA repair genes (RAD51, KU80) and autophagy-related genes (ATG5, ATG8) throughout the induction of somatic embryo formation in Scots pine SE cultures. Among the embryo-producing SE lines, the expression of ADC, SPDS, ACL5, DAO, CAT, RAD51, KU80 and ATG8 showed consistent profiles. Furthermore, the overall low expression of the stress-related genes suggests that cells in those SE lines were not stressed but recognized the ABA+PEG treatment as a signal to trigger the embryogenic pathway. In those SE lines that were unable to produce embryos, cells seemed to experience the ABA+PEG treatment mostly as osmotic stress and activated a wide range of stress defense mechanisms. Altogether, our results suggest that the direction to the embryogenic pathway is connected with cellular stress responses in Scots pine SE cultures. Thus, the manipulation of stress response pathways may provide a way to enhance somatic embryo production in recalcitrant Scots pine SE lines.

Development of tobacco (Nicotiana tabacumL.) doubled haploid lines resistant topotato virus Y0

Viral diseases such as Potato Virus Y (PVY) in tobacco plants can cause a lot of damage annually. In order to obtain PVY resistant doubled haploids (DH) of Nicotiana tabacum with desirable traits. Two varieties of tobacco VAM resistant to PVY and K326 of recognized high quality of leaves were crossed. The F1 hybrids were grown in pots and the anthers were cultured on PGR-free MS medium. After production of somatic embryos, colchicine treatment (0.4%) was applied for 8 hours. Thereafter, 184 plantlets were transferred to greenhouse and then moved to the field and self-pollinated. The derivatives of colchicine treated and fertile plants were inoculated with PVY0. DAS-ELISA technique and rating of symptoms were used to select resistant plants. From a total of seventeen DH, seven plants were selected as pure lines and evaluated under field condition. The DH lines showed significant differences between them for morphological and chemical traits. Some of the DHs had longer and wider leaves than K326 cultivar as control. The content of sugars was not affected by genotype. Finally, three doubled haploid lines (TDH-03, TDH-09, TDH-11) with desirable characteristics were selected as PVY resistance.

Somatic Embryogenesis in Lisianthus (Eustoma russellianum Griseb.).

Somatic embryogenesis is, for the main floricultural crops, a promising system for commercial scale-up, providing cloned material to be traded as seedlings. Somatic embryos, having the contemporary presence of root apical meristem and shoot apical meristem, can be readily acclimatized. For Lisianthus it is possible to induce embryogenic callus from leaf fragments of selected genotypes and to obtain embryos either in agarized substrate or in liquid suspension culture. The production of somatic embryos in liquid medium is high and can be modulated in order to synchronize the cycle and the size of the neoformed structures. The possibility to use the liquid substrate with high propagation rates reduces labor costs and could support the costs of eventual automation. In this paper we report a stepwise protocol for somatic embryogenesis in the species Eustoma russellianum.

<i>In Vitro</i> Propagation Potential via Somatic Embryogenesis of the Two Maturing Early Cultivars of European Chestnut (<i>Castanea sativa</i> Mill.)

Turkey ranks the third in the production of chestnuts in the world having an important place both in domestic and global markets. However, the chestnut production and the number of trees have been diminishing in recent years. Therefore, in vitro propagation of the chestnut, in addition to the classical propagation techniques, should be applied. Especially the propogation of the early maturing cultivars and production of the quality chestnuts will provide a better income to the producer. Here, somatic embryo production and regeneration from the immature cotyledons of the early maturing cultivars of the European chestnut (Castanea sativa Mill), Haciibis and Karamehmet, were studied using the somatic embryogenesis, one of the in vitro propagation techniques. To induce the somatic embryogenesis, 168 different combinations were applied to both cultivars. The somatic embryogenesis rate in Haciibis cultivar, in which the interactions were observed among the applications, was found to be 9.9% while it was 11.1% for the Karamehmet cultivar. Dessication, cold treatment, gibberellic acid (GA3) and benzyladenine (BA) + naphthaleneacetic acid (NAA) applications were performed on the regeneration of the somatic embryos, and 40% conversion to plant was obtained with desiccation together with BA + NAA supplementation to the medium.

Induction and development of anther-derived gametic embryos in cucumber (Cucumis sativus L.) by optimizing the macronutrient and agar concentrations in culture medium

Anther culture response of a cucumber cultivar (Beta Alpha) and three Iranian landraces of cucumber (Basmenj, Isfahani, and Korki) on MS culture media with an altered macronutrient strength (half, full, 1.5-fold, and double-strength) or various concentrations of agar (0, 3.5, 7, and 14 g L-1) was evaluated in two separate experiments. The effect of different concentrations of agar on the development of callus-derived embryos was also assessed. The androgenic potentials of various cucumbers were significantly (P < 0.01) influenced by the different macronutrient strengths and agar concentrations, although anther culture responses could vary according to the cucumber type. A doubled strength of macronutrients in culture medium resulted in the highest percentage of callogenesis (100%) in Isfahani cucumber, while use of full strength of macronutrients produced the highest percentage of embryogenic calli (83.33%) in the mentioned landrace compared to other tested forms. The maximum number of gametic embryos (1.26 and 1.23 embryos/anther) differentiated from Isfahani anther cultures on full- and half-strength MS medium, respectively. In the second experiment, the highest percentages of callogenesis were obtained in MS culture media solidified with 14 g L-1 and 7 g L-1 agar for Basmenj, with 3.5 g L-1 agar or liquid medium for Isfahani, with 7 g L-1 agar for Beta Alpha, and with 14 g L-1 agar for Korki. Culture media solidified with 7 g L-1 agar produced the highest percentage of embryogenic calli compared to other treatments. Anther culture of Korki cucumber on culture medium with 14 g L-1 agar produced the maximum number of embryos per anther compared to other cucumbers. Improved production of mature somatic embryos (torpedo and cotyledonary embryos) was achieved with higher agar concentrations (7 and 14 g L-1) for the cucumber cultivar and all landraces.

Ultrastructural observations in somatic embryogenesis ofnatural tetraploid Trifolium pratense L.

Previous reports of plant regeneration of natural tetraploid T. pratense L. 'Elci' could be realized only through the apical meristem calli. In order to proceed to the production stage, other regeneration methods need to be tried. Aseptic seedlings were used for the production of somatic embryos through various 2,4-D and kinetin trials. Nonuniform external callus cells with translucent cytoplasm were observed in various developmental stages of somatic embryos. Beneath these cells, there were uniformly aligned, dark- stained embryo cells with dense cytoplasm. Despite the similar developmental stages and cell characteristics of zygotic and somatic embryos, the walls of somatic embryo cells revealed a highly wavy pattern. The nucleus generally contained only one nucleolus, which was spherical, dark stained, and electron-dense. Electron-dense droplets were seen in vacuoles. The cytoplasm consisted of starch- containing amyloplasts, mitochondria, plastids, ribosomes, endoplasmic reticulum, dictyosomes, lipid, and protein bodies. In some of the somatic embryos at the globular and heart stages, vacuole or electron-translucent zones were observed in the nucleolus. Additionally, a few embryo degenerations were recorded during developmental stages of the zygotic embryo. For the first time, the somatic embryos of natural tetraploid T. pratense were produced from hypocotyl (85%), cotyledon (75%), and apical meristem (60%) explants in 0.3 mg/L 2,4-D and 2 mg/L kinetin-containing MS medium. Our study developed an effective and efficient in vitro production method for using natural tetraploid T. pratense in biotechnological studies.

Advances in Conifer Somatic Embryogenesis Since Year 2000.

This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

Effect of tissue proliferation and somatic embryo induction inLarix kaempferifollowing treatment with organic nitrogen sources and plant growth regulators

This study was conducted to evaluate the effects of different types and concentrations of organic nitrogen sources (-Glutamine and casein hydrolysate, CH) and plant growth regulators (auxins and cytokinins) on embryogenic tissue proliferation and somatic embryo production in L. kaempferi. Overall, the highest tissue fresh weight was obtained at either 2 or 4 weeks in culture when 1,000 mg/L -Glutamine was added to the culture medium, which showed similar results with other treatments. In experiments with different types and concentrations of plant growth regulators on somatic embryo production, the highest production (426.3/90 mg tissue) was found when 0.2 mg/L IBA was added; however, no somatic embryos were induced following treatment with 0.2 mg/L BA or Kinetin. The effect of various concentrations of IBA on somatic embryo production was also tested. The best result (303/90 mg tissue) was obtained when plants were treated with 0.2 mg/L IBA; 1.0 mg/L IBA was also effective (281/90 mg tissue). The lowest result (109.3/90 mg tissue) was obtained with 5.0 mg/L IBA.

Environmental conditions at the initial stages of Pinus radiata somatic embryogenesis affect the production of somatic embryos

Physicochemical conditions present during the initiation of somatic embryogenesis in Pinus radiata determine the rate of embryogenic cell lines generated as well as the final number of somatic embryos. The effect of physical and chemical conditions during the initiation and proliferation stages of somatic embryogenesis in radiata pine was investigated. The first objective was to assess if different temperatures and/or water availability during the initiation or proliferation of embryogenic tissue impacted the success rate during each stage of somatic embryogenesis. The second objective was to determine what stage has a greater influence on subsequent stages of development. Some treatments (18 °C, 4 g L−1 gellan gum) resulted in a higher percentage of initiation whereas others (28 °C, 2 g L−1 gellan gum) gave rise to lower initiation. Our results indicated that initiation at different temperatures affected the subsequent stages of somatic embryogenesis. Thus, these embryogenic tissues were influenced by the environmental conditions present at initiation. We were able to successfully regenerate somatic embryos from cell lines initiated under all the environmental conditions tested.

Acidogenic growth model of embryogenic cell suspension culture and qualitative mass production of somatic embryos from triploid bananas

A young male flower-derived embryogenic suspension cell population of AAA ‘Pei Chiao’, ‘Dwarf Cavendish’, and AAB ‘Raja’ was used for developing an acidogenic growth model . We hypothesized that a close relationship exists between the self-regulated pH medium and the corresponding changes in the growth phases. Studies have reported that a pH below 4.6 may prevent the embryogenic cells from undergoing polar growth. Controlling pH up to a level 4.6 within 2 days during the changes of pre-embryogenic cells (PECs) and proembryogenic masses into embryogenic determined cells (EDCs) uniformly resulted in unequal cell division. The hydrogen ion buffer 2-N-morpholino-ethanesulfonic acid at 10 g L−1 was added to MA2 and MA3 media, showing the medium pH of MA3 up to 5.0, thus maintaining a relatively stable pH in AAA ‘Pei-Chiao’ and AAB ‘Raja’ cells that autoregulate acidification, significantly increasing the number of somatic embryos. When the proliferation and globularization phases were acidified to pH 3.5 ± 0.2, cells were released to free single cells of PECs and EDCs after 21 days. This study provides possible explanation that PECs deposit callose on their cell walls as a possible protector from strong acidic condition. Regulation at pH 5.0 ± 0.2 resulted the production efficiency achieved was 0.9 million somatic embryos per 1 mL of the settled cell volume.

Retraction Note to: Plant regeneration in Chlorophytum borivilianum Sant. et Fernand. from embryogenic callus and cell suspension culture and assessment of genetic fidelity of plants derived through somatic embryogenesis.

[This retracts the article DOI: 10.1007/s12298-012-0113-y.].

Indução e regeneração de embriões somáticos de cana-de-açúcar RB996961

Sugarcane is a crop widely planted in Brazil because of the economic importance of its products, like sugar and ethanol. Thus, the crop is often inserted in breeding programs. Therefor, the aim of this study was to evaluate the morphogenic response to somatic embryogenesis and somatic embryos regeneration for genotype RB996961. Transversal segments of young leave rolls with 2-3 mm size from plants with 6 to 9 months old were subcultured on solid MS medium supplemented with three concentrations of 2.4-dichlorophenoxyacetic acid (2.4-D) for embryogenic callus induction. Better results were obtained using 13.5 or 31.5 µM L -1 of 2.4-D in 45 or 60 days of incubation on induction medium, one to three subculture and 15 to 45 days of incubation on regeneration medium for genotype RB996961. This results show that the protocol used to genotype RB996961 was efficient to somatic embryos production (over 80%), nevertheless, it is necessary to adjust the regeneration approach (62.3 to 73.4%).

An unique system of somatic embryogenesis in the tree fern Cyathea delgadii Sternb.: the importance of explant type, and physical and chemical factors

The effect of explant type (etiolation, age, length), physical (photoperiod, darkness) and chemical (sucrose and mineral salt concentrations) factors on somatic embryogenesis efficiency and sporophyte development in Cyathea delgadii was evaluated. Five-month-old cultures of sporophytes, which had developed 4–5 leaves, were used as a source of explants. The percentage response of stipes and the average number of somatic embryos were calculated after 2 months of culture. Etiolation of sporophytes used for culture initiation, was a critical factor in SE induction. Other factors studied significantly affected the production of somatic embryos. Optimum results were obtained when explants measuring 2.5 mm in length were excised from the youngest leaf of an etiolated sporophyte. Maximum SE efficiency (84.0 % response in explants; 42.3 somatic embryos per explant) was obtained on half-strength Murashige and Skoog medium supplemented with 1 % sucrose, under photoperiod conditions. However, light stimulated two different morphogenetic responses in explant cells: SE and apospory. In darkness, the number of somatic embryos was reduced to 32.9 per explant, but this was the only morphogenetic response observed. The presence of light alone was sufficient for somatic embryos growing on plain agar gel to reach maturity, but further development stopped shortly thereafter. The presence of both light and sucrose was necessary for the development of an embryonic leaf and root. For further sporophyte growth, mineral salts were essential. The micropropagation system described here is the first investigation into factors responsible for SE in ferns.

Gene expression profiling of shoot-derived calli from adult radiata pine and zygotic embryo-derived embryonal masses.

BackgroundAlthough somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation.FindingsShoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from in vitro-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant’s origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1).ConclusionsWhile limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess some level of embryogenic character. Additionally, expression of vegetative meristem markers is consistent with their vegetative origin, as are differences in expression patterns as compared with EM.

Study on the presence and influence of phenolic compounds in callogenesis and somatic embryo development of cocoa (Theobroma cacao L.).

Cocoa (Theobroma cacao L.) like most tropical trees is recalcitrant in tissue culture. Somatic embryogenesis is generally efficient micropropagation technique to multiply elite material. However, Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. One of the factors often considered as a component of in vitro recalsitrance is a high phenolic content and oxidation of these compounds. In cocoa tissue culture accumulate large amounts of poliphenolics compounds which probably impair further development. This study was conducted to investigate the composition of phenolic compounds in cocoa flower and leaves, and their changes troughout the somatic embryogenesis process. Calli were induced in cacao floral and leaves explants on a half-strenght Murashige and Skoog medium containing 30 g/L Glucose and combination of 2,4 dichlorophenoxyacetic acid (2,4 D) with kinetin (kin). Total polyphenol content was observed on Sulawesi 1 cocoa clone. Embryogenic and non-embryogenic callus were also compared. The percentage of callus production from flower tissue is 85%, percentage of embryogenic callus 40 %, although the percentage of somatic embryo production from embryogenic callus callus is 70%. The conservation of callus into somatic embryos followed by decline in phenol content and an increase in peroxidase. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. So that, phenolic compound can influence the production of calli and an absence the phenolic compound can enhance production of somatic embryo.Kata kunci: Theobroma cacao L., polifenol, embrio somatik, kalus, flavonoid, katekin, in vitro recalcitance