Non-embryogenic Callus Research Articles

Extracellular matrix as the early structural marker for Centella asiatica embryogenic tissues

Embryogenic and non-embryogenic calli were induced from the Centella asiatica leaf explants on Murashige and Skoog medium supplemented with kinetin and 2,4-dichlorophenophenoxyacetic acid. The extracellular matrix (ECM) layer was seen on the surface of embryogenic cells but not on the non-embryogenic cells. The ECM formed bridges with net-like material between the embryogenic cells. This network like structure was believed to play an important role in plant morphogenesis and can serve as an early structural marker of embryogenic competence in Centella asiatica calli culture.

Somatic embryogenesis and organogenesis in apomictic and sexual Brachiaria brizantha

Brachiaria brizantha (syn. Urochloa brizantha) is an important tropical forage grass widely cultivated in Brazil. In order to optimize tissue culture conditions for B. brizantha, in vitro culture of mature seeds, basal segments and leaf segments from in vitro plants of an apomictic and a sexual genotype of B. brizantha was performed. When cultured on different media, leaf segments yielded non-embryogenic calluses which formed several roots. Friable calluses from mature seeds and basal segments explants incubated on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine yielded 80% compact and nodular embryogenic structures. Calluses with such compact embryogenic structures were highly regenerable upon transfer to medium supplemented with kinetin and naphthalene acetic acid. They produced isolated somatic embryos, multiple fused scutelli or isolated scutellum with polyembryos that germinated into isolated or multiple shoots. Green and morphologically normal plants were obtained for the two genotypes. Changing the media from pH 5.8 to pH 4.0 increased the number of explants that formed calluses as well as the number of shoots per explant. When embryogenic calluses from mature seeds were successively sub-cultured for 4 months, aiming at repetitive somatic embryogenesis, all the regenerated plants were albinos. The embryogenic nature of the compact structure was confirmed by scanning electron microscopy.

The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC. A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC. In this research, a vector pA5 containing a chitinase signal sequence fused to gfp (green fluorescent protein) and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L. cv. Chardonnay NEC. It was determined that NEC 10 d after subculture was the best target tissue; 30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L −1 acetosyringone (AS) was optimized as protocol. The use of bacterial densities as 1.0 at OD 600 did not result in serious tissue hypersensitive reaction and it had higher efficiency. Kanamycin at 200 mg L −1 was picked for positive expression selection. The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques (RT-PCR) and fluorescent microscopy after three sub-cultures of the selected cell line. Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC. This transformation platform could facilitate the verification of candidate somatic embryogenesis (SE) decisive genes, and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products, as NEC not only proliferates fast, but also keeps in a rather stable condition.

LEAFY COTYLEDON1, FUSCA3 expression and auxin treatment in relation to somatic embryogenesis induction in Arabidopsis

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.

Somatic embryogenesis efficiently eliminates viroid infections from grapevines

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral explants used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even 3 years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which has been only an assumption so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow the availability of virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.

Anti-oxidant enzyme responses during in vitro embryogenesis in Catharanthus roseus

SummaryThe present study was carried out to analyse the activities of several anti-oxidant enzymes at various stages of somatic embryogenesis in Catharanthus roseus. The hypothesis was that anti-oxidant enzymes accumulated as part of a cellular defence mechanism in response to stress. We therefore measured superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities in various in vitro-grown tissues such as embryogenic and non-embryogenic calli, and in embryos at various stages. SOD activity increased gradually from the early embryogenic stage to heart-shaped stage embryos, but declined in the later stages (i.e., torpedo-shaped and cotyledonary embryos). In contrast, APX activity was high in non-embryogenic callus and decreased rapidly during the stage of embryo initiation. This pattern was the same for CAT. The maximum CAT activity was observed in non-embryogenic callus, then it declined almost linearly at the embryonic and post-embryonic developmental stages. The effect of exogenous hydrogen peroxide (H2O2) on in vitro embryogenesis was also evaluated. Lower H2O2 levels (0.025 mM) promoted embryo formation, whereas higher levels (0.10 mM) inhibited embryogenesis in C. roseus. Finally, higher soluble protein, free amino acid, and proline contents were found in embryogenic calli compared to non-embryogenic calli.

Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli

Late embryogenesis abundant 14 (LEA14) cDNA was isolated from an EST library prepared from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Quantitative RT-PCR revealed a variety of different IbLEA14 expression patterns under various abiotic stress conditions. IbLEA14 expression was strongly induced by dehydration, NaCl and abscisic acid treatments in sweetpotato plants. Transgenic sweetpotato non-embryogenic calli harboring IbLEA14 overexpression or RNAi vectors under the control of CaMV 35S promoter were generated. Transgenic calli overexpressing IbLEA14 showed enhanced tolerance to drought and salt stress, whereas RNAi calli exhibited increased stress sensitivity. Under normal culture conditions, lignin contents increased in IbLEA14-overexpressing calli because of the increased expression of a variety of monolignol biosynthesis-related genes. Stress treatments elicited higher expression levels of the gene encoding cinnamyl alcohol dehydrogenase in IbLEA14-overexpressing lines than in control or RNAi lines. These results suggest that IbLEA14 might positively regulate the response to various stresses by enhancing lignification.

Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange

Somatic embryogenesis (SE) is a remarkable process of plant somatic cells developing into an embryo capable of forming a complete plant. MiRNAs play important roles in plant development by regulating expression of their target genes, but its function in SE has rarely been studied. Herein, ten conserved miRNAs with critical functions in plant development are detected by stem-loop qRT-PCR in the SE system of Valencia sweet orange. Sixteen unigenes from citrus are predicted to be targeted by six of the miRNAs. Cleavage sites on 15 of these target mRNAs are mapped by 5'RACE, of which ten are reported in this study. Transcript abundances of the 16 target unigenes are detected by qRT-PCR during SE process. Stage and tissue-specific expressions of miRNAs and their targets suggest their possible modulation on SE of citrus callus: miR156, 168 and 171 exert regulatory function during somatic embryo induction process; miR159, 164, 390 and 397 are related to globular-shaped embryo formation; miR166, 167 and 398 are required for cotyledon-shaped embryo morphogenesis; in addition, target genes of miR164, 166 and 397 are associated with SE disability of nonembryogenic callus. Exploration of miRNA-mediated modulation on SE is expected to provide new insights into plant cell totipotency, as well as how to maintain and enhance the embryogenic capacity of somatic cells.

IN VITRO CULTURE OF Pogostemon cablin BENTH. (NILAM PLANT): THE EFFECT OF NAA AND BAP ON EMBRYOGENIC CALLUS PROLIFERATION AND SUBSEQUENT SOMATIC EMBRYOGENESIS

An experiment to investigate the somatic embryogenesis from shoot-derived callus of Pogostemon cablin (nilam plant) has been conducted at the Plant Biotechnology Laboratory, Agricultural Faculty, University of Jambi from January through to July 2004. Callus proliferation was induced on explants taken from young shoots cultured on solid MS medium supplemented with phytohormones NAA (0.8, 1.1, 1.4, and 1.7 ppm) and BAP (1.1, 1.4, 1.7, and 2.0 ppm) under in vitro conditions. Cultures were maintained at 25 􀂱 1 oC, light intensity 50 􀁭mol m-2 s-1, and 16 hours photoperiod. The results indicated that all cultured explants showed positive responses on callus proliferation on all treatments within two weeks of culture initiation. The effect of phytohormones, however, was unspecific as all callus showed similar properties, from non-embryogenic to embryogenic. The addition of NAA and/or BAP to the culture medium was not significantly affected the number of days to callus proliferation. Callus fresh weight was significantly affected by NAA (P = 0.01) or BAP (P = 0.05), but the interaction of these phytohormones resulted in a non-significant effect on callus fresh weight (P = 0.18). Also, BAP significantly affected callus dry weight (P =0.03). However, neither NAA nor its interaction with BAP significantly affected callus dry weight (P = 0.07 and 0.16, subsequently). Embryogenic and non-embryogenic callus were subcultured separately onto new fresh media with the same composition as for callus induction. Following this subculture, embryogenic callus regenerated somatic embryos within ten days, whereas non-embryogenic callus did not show any symptom of embryogenesis, and lost their proliferative capacity after six weeks of subculture. The regenerated somatic embryos continued to grow to form profuse mass of young plantlets ready for in vivo acclimatization. Keywords: Pogostemon cablin, nilam, patchouli oil, tissue culture, in vitro culture, NAA, BAP

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

Four abiotic stress-induced miRNA families differentially regulated in the embryogenic and non-embryogenic callus tissues of Larix leptolepis

Somatic embryogenesis involves complex molecular signaling pathways. Deregulation of these signaling pathways can transform the embryogenic callus to non-embryogenic callus. To investigate the miRNA regulation underlying this detrimental transformation in Japanese Larch ( Larix leptolepis), we compared miRNA expression profiles between embryogenic and non-embryogenic callus at day 3 and day 14 after sub-culture. Four miRNA families dominated the 165 differentially expressed miRNAs between embryogenic and non-embryogenic callus. Of the four, miR171 was up-regulated, and miR159, miR169, and miR172 were down-regulated in the embryogenic callus. These four families are all abiotic stress-induced miRNAs, and all target transcription factors that regulate a group of genes important for cell differentiation and development, including scarecrow-like (SCL) transcription factor (miR171), apetala2 (miR172), MYB transcription factors (miR159), and NF-YA transcription factor (miR169). Three down-regulated miRNA families in the embryogenic callus are also regulated by ABA, which further shed light into the potential mechanisms underlying the transformation of the embryogenic competence in L. leptolepis. This study represents the first report on the miRNA regulation of the embryogenic and non-embryogenic callus in plant, and thus these four miRNA families provide important clues for further functional investigation.

Variations in vinblastine production at different stages of somatic embryogenesis, embryo, and field-grown plantlets of Catharanthus roseus L. (G) Don, as revealed by HPLC

Catharanthus roseus L. (G) Don. is an important dicotyledonous medicinal plant that produces anticancer compounds, which are used for the treatment of a wide variety of cancers. We have quantified vinblastine (a major dimeric anticancer compound) in various in vitro raised tissues; embryogenic and nonembryogenic calli, three different embryogenic stages (proliferated, matured, and germinating embryo), somatic embryo derived plantlets and in ex vitro grown plantlets by using high performance liquid chromatography. Of the various obtained callus lines and embryogenesis stages, maximum vinblastine content was found in leaf callus and in germinating embryos. The leaves of somatic embryo-derived plantlets contained more vinblastine than did Catharanthus leaves developed ex vitro. The yield of vinblastine was monitored for 30 wk. The production of vinblastine appeared to be age dependent and tissue specific; the finding of our analyses is discussed in detail.

Large impact of the apoplast on somatic embryogenesis in Cyclamen persicum offers possibilities for improved developmental control in vitro.

BackgroundClonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop Cyclamen persicum by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.ResultsThe analysis was based on a cDNA microarray representing 1,216 transcripts and was exemplarily validated by realtime PCR. For this purpose relative transcript abundances of homologues of a putative receptor kinase, two different glutathione S-transferases (GST), a xyloglucan endotransglycosylase (XET) and a peroxidase (POX) were quantitatively measured by realtime PCR for three different comparisons. In total, 417 genes were found to be differentially expressed. Gene Ontology annotation revealed that transcripts coding for enzymes that are active in the extracellular compartment (apoplast) were significantly overrepresented in several comparisons. The expression profiling results are underpinned by thorough histological analyses of somatic and zygotic embryos.ConclusionsThe putative underlying physiological processes are discussed and hypotheses on improvement of the protocol for in vitro somatic embryogenesis in Cyclamen persicum are deduced. A set of physiological markers is proposed for efficient molecular control of the process of somatic embryogenesis in C. persicum. The general suitability of expression profiling for the development and improvement of micropropagation methods is discussed.

Morphological aspects of coconut anther culture derived structures

Anthers excised from male flowers of an adult coconut palm of Sri Lanka Tall cultivar were used to produce plants via androgenesis. Morphological aspects of the anther-derived structures and their plant regeneration pathways were studied. Under the culture conditions employed, both calli and embryo-mediated plant regeneration were observed. Two types of embryos with different germination patterns were identified-one with a germination point and the other type with a blunt haustorium without a prominent germination point which converted into germinated embryos. Three types of calli (embryogenic compact calli, embryogenic friable calli and non-embryogenic fast-growing calli) were also produced and the most commonly identified type was embryogenic compact calli that give rise to somatic embryos. Secondary somatic embryos were produced in both direct embryo and callus mediated embryogenesis. Normal plants with a single shoot were observed in low frequencies while weak plantlets with multiple shoot were observed in abundance. Under similar culture conditions, some plantlets showed vigorous growth whereas the majority had a slow growth rate. Keywords: Androgenesis, anther culture, callus, coconut ( Cocos nucifera L.), embryo, morphology doi :10.4038/jnsfsr.v38i1.1727 J.Natn.Sci.Foundation Sri Lanka 2010 38 (1): 69-74

Nitrogen compounds in embryogenic and non-embryogenic calluses of Medicago arborea L.

Nitrogen (N) metabolism during embryogenesis may be fundamental in the embryogenic response. We used different explants of Medicago arborea L. subsp. arborea seedlings: cotyledons, petioles and leaves, which form calluses with different embryogenic responses. The endogenous contents of total nitrogen, nitrate, nitrite and ammonia and nitrate reductase activity were determined in embryogenic and non-embryogenic calluses induced from the different explants. The endogenous total N content decreased in the calluses as the culture time progressed, this decrease being more pronounced in the more embryogenic calluses obtained from petioles with the H8 and F0 media. Inorganic N decreased during embryogenesis, coinciding with an increase in organic N. Thus, N metabolism somehow seems to be essential in embryogenesis. The N detected in calluses, at the start of culture, was mainly metabolised to nitrite. This metabolism was very pronounced; especially in embryogenic calluses obtained from cotyledons and petioles. That is, the metabolism of N seemed to be more marked in the calluses in which embryogenesis was greater. The nitrite content decreased in all the calluses, the contents being lower, especially in the last months of culture, in the more embryogenic calluses obtained from petioles. In many calluses, ammonia levels did not follow any general pattern. Neither was it possible to detect changes in ammonia levels between the embryogenic and non-embryogenic calluses. Regarding nitrate reductase activity, no clear differences between embryogenic and non-embryogenic calluses were found.

Effect of grape genotype and tissue type on callus growth and production of resveratrols and their piceids after UV-C irradiation

Grape calli systems were used to study the relationships between stilbene production and: (1) four grape genotypes, (2) leaf, berry exocarp and seed tissues and (3) UV-C irradiation. All explants could be de-differentiated. However, subsequent callus growth depended mainly on genotype and tissue type. Non-embryogenic callus accumulated more resveratrols and piceids and had a higher growth index than pro-embryogenic and embryogenic calli. UV-C irradiation for 20 min was most efficient in promoting both the accumulation of resveratrols and piceids and callus growth index. There was dynamic production of resveratrols and piceids in UV-C-irradiated leaf-derived calli over a 72 h period, with optimum harvest time for the highest total stilbene content at 48 h. Accumulation of stilbenes in UV-C-irradiated calli depended upon genetic background and tissue type, with higher stilbene contents in two interspecific root stocks and leaf or exocarp explants.

SOMACLONAL VARIATION AMONG SOMATIC-EMBRYO DERIVED PLANTS OF OLEA EUROPAEA L “CV. KRONEIKI”

Frequency of somaclonal variation in embryogenic calli and plants of Olea europaea L cv.Kroneiki derived from somatic embryogenesis were examined by randomly amplified polymorphic DNA (RAPD) analysis. Radicle and cotyledon (proximal part) of mature zygotic embryo were cultured in OMc medium supplemented with IBA (5 mgL-1) and 2-ip (0.5 mgL-1). Somatic embryogenesis was induced in calli of radicles (55%) and proximal part of cotyledons (8%) on OM medium without any hormones. After culturing the embryogenic calli on OM medium in the presence of BAP (2 mgL-1), regenerated plants were obtained. DNA sample from the leave of the mother plants, seedlings (germinated from zygotic embryo), plantlets (regenerated from somatic embryo), embryogenic calli, non-embryogenic calli and zygotic embryos were subjected to RAPD analysis. 20 arbitrary decamer primers produced polymorphic amplification products. These primers gave a total of 315 reproducible fragments from which 221 (71%) were useful as polymorphic bands with an average of twelve RAPD marks obtained for each primer. A dandogram, based on the unweighted pair group mean average (UPGMA) method of cluster analysis, were constructed using a similarity matrix derived from the RAPD amplification generated by all primers. The estimation of genetic similarity coefficient based on RAPD band sharing data indicated that regenerated plants were less than 75% similar to mother plants.

Isolation and characterization of four somatic embryogenesis receptor-like kinase (RhSERK) genes from miniature potted rose (Rosa hybrida cv. Linda)

The isolation and expression analysis of four partial gene sequences from rose (Rosa hybrida cv. Linda) belonging to the receptor-like kinase gene superfamily are reported. These genes have been designated RhSERK1 to RhSERK4 (Accession No. EF631967 to EF631970) as they exhibit high sequence identities with genes from the somatic embryogenesis receptor-like kinase (SERK) family in other plant species. The RhSERK genes are differentially expressed in non-embryogenic callus, embryogenic callus, mature somatic embryos and a range of tissues from intact plants, indicating a broad role in plant growth and development. However, the expressions of RhSERK3 and RhSERK4 were approximately fivefold higher in embryogenic callus than in non-embryogenic callus, and they are even higher when compared to tissues from intact plants. In addition, RhSERK4 expression was approximately eightfold higher in somatic embryos than in embryogenic callus. These results suggest that the expression pattern of RhSERK3 and RhSERK4 may be used as a marker of somatic embryogenesis.

Regulation of Somatic Embryogenesis in Higher Plants

Somatic embryogenesis is the developmental process by which somatic cells undergo restructuring to generate embryogenic cells. These cells then go through a series of morphological and biochemical changes that result in the formation of a somatic or non-zygotic embryo capable of regenerating plants. Somatic embryogenesis represents a unique developmental pathway that includes a number of characteristic events: dedifferentiation of cells, activation of cell division, and reprogramming of their physiology, metabolism, and gene expression patterns. Valuable studies have elucidated the developmental processes pertaining to the control parameters of somatic embryogenesis. These studies have emphasized the transitional state from somatic to embryogenic cells, identified differentially expressed genes in nonembryogenic and embryogenic calli, isolated varieties of genes that are likely involved in the embryogenic pathway, compared specific gene products that accumulate during different stages of somatic embryogenesis, and discovered molecular or protein markers for somatic embryogenesis. An understanding of embryogenic pathway initiation, and origin of somatic embryos (unicellular or multicellular) is critical to scientific and biotechnological applications. Cell tracking has been successfully applied to determine the fate of embryogenic cells. Identification of hormone-inducible genes has yielded clues to the control of gene expression during embryogenic development. Characterization of genes taking part in signal transduction pathways, such as somatic embryogenesis receptor-like kinases, has generated great interest in the switching of several signal cascades during somatic embryogenesis, and has identified how genes encoding transcription factors such as BBM, LEC1, and LEC2 could be used to regulate somatic embryo development. Protein markers are useful probes for defining embryogenic potential and for marking different phases in plant development. This review provides a systematic and comprehensive analysis of current advances in the development of somatic embryogenesis, as well as the cellular and molecular mechanism of somatic embryogenesis in higher plants. The developmental pathway, differential gene expression, and extracellular protein markers during somatic embryogenesis are especially emphasized.

Induction of Cocoa Natural Resistancy to Cocoa Pod Borer by Silica Application

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