Embryogenic Callus Induction Research Articles

Genetic architecture of embryogenic callus induction in maize from the perspective of population genomics

Genetic architecture of embryogenic callus induction in maize from the perspective of population genomics

QTL Mapping of Somatic Regeneration-Related Traits in Maize

The somatic regeneration of maize depends on its genotypes, so improving its variety with modern biotechnology is severely restricted. Locating the quantitative trait loci (QTLs) associated with somatic regeneration is important for breeding elite inbred lines that undergo genetic transformations. Here, by crossing the high-regeneration inbred line H99 and non-regeneration inbred line Fr993, an F2 population and its F2:3 and F2:4 population families were constructed. Immature embryos from the family populations were subjected to tissue culture in two independent seasons to determine their embryogenic callus induction rates (EIRs), green callus rates (GCRs) and plantlet regeneration rates (PRRs). Genetic linkage maps were constructed for the F2 population to locate somatic regeneration QTLs. The results showed that variation in the EIR, GCR and PRR ranged from 0.00–99.33%, and their broad-sense heritabilities were 0.50, 0.52 and 0.53, respectively. The total genetic distance of the linkage maps constructed by the GenoBaits 10 K chip was 2319.50 cM, and twelve QTLs were associated with somatic regeneration traits, accounting for 3.90–14.06% of the phenotypic variation. Expression analysis revealed six candidate genes screened from the QTLs with distinct responses to induction culture in the parental lines, suggesting that they may impact commitment to somatic cell fate. These results provide a basis for the molecular breeding of maize varieties with high-frequency somatic regeneration.

A Temporary Immersion System Improves Regeneration of In Vitro Irradiated Recalcitrant Indica Rice (Oryza sativa L.) Embryogenic Calli.

The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray (60Co) mutagenesis reduces chimeras and allows for a faster selection of desirable traits but requires the optimization of the laboratory procedure. The objectives of the present work were sequencing of matK and rbcL, the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity to gamma radiation, and optimization of the generation procedure. All sequenced genes matched perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction improved using MS medium containing 2 mg L−1 2,4-D, and regeneration was achieved with MS medium with 3 mg L−1 BA and 0.5 mg L−1 NAA. The optimized radiation condition was 60 Gy, (LD20 = 64 Gy) with 83% regeneration. An immersion system (RITA®, Saint-Mathieu-de-Tréviers, France) of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved regeneration in the immersion system. To our knowledge, this is the first study reporting the use of an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica rice materials.

Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants

Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L−1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

Efficient plant regeneration via meristematic nodule culture in Paeonia ostii ‘Feng Dan’

Tree peony (Paeonia sect. Moutan) is an economically important multipurpose woody plant in terms of its medical, ornamental and oil values, but its breeding and industrial development are severely limited due to inefficient traditional propagation methods and existing in vitro regeneration systems. Meristematic nodules (MNs) are an attractive alternative to solve this problem. This study first presented a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ via MN culture with four consecutive steps, including embryogenic callus (EC) formation, MN induction and leaf cluster differentiation, shoot elongation, rooting and acclimatization. The highest EC induction rate (81.25%) was achieved when cotyledons were cultured on modified Murashige and Skoog (mMS) medium with 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 5.37 µM α-naphthylacetic acid (NAA) for 30 days. The optimal MN induction rate (100%) and leaf cluster differentiation rate (45.83%) were obtained when ECs were cultured on modified woody plant medium (mWPM) supplemented with 2.02 µM CPPU + 2.27 µM thidiazuron (TDZ) for a subculture time of 10 days. The combination of 1.29 µM 6-benzyladenine (BA) + 0.58 µM gibberellin (GA3) yielded the best shoot elongation (13.40 shoots per nodule), rooting rate (43.33%) and consequently survival rate (45.83%). The study will be beneficial to the mass propagation, breeding and genetic improvement of tree peony.

Somatic Embryogenesis of Anthurium andraeanum Linden., -ATropical Florists'Plant.

The global floriculture market is expected to reach US$41.1 billion by 2027 at a CAGR of 5% over the analysis period 2020-2027; on the year 2020, the recorded market value in this trade was US$29.2 billion. The florists mainly useAnthurium andraeanum flowers in fashionable bouquets and floral arrangements because of their beautiful, attractive bright colored eye-catching spathe, candle-like spadix, prolonged vase life, etc. The cut flower industry always seeks elite cultivars and new hybrids of A. andraeanum, that in turn depend on the availability of large numbers of clonal planting propagules. In vitro somatic embryogenesis is an important technique for large-scale clonal propagation, development of transgenic plants, creation of new variety by somaclonal variation, mutagenesis on in vitro plants, and germplasm preservation for future use. Here, we describe the protocol of somatic embryogenesis of Anthurium andraeanum cv. Cancan, an important commercial cultivated variety. The protocol has been optimized by using 4 different types of culture mediawhich are used during embryogenic callus induction, multiplication of callus, induction of somatic embryogenesis, and maturation plus conversionof embryosinto plantlets. The protocol outlines the detailed methods from mother plant procurement to hardening of micro plants that is ready to transfer in the field and itcan be used for large-scale commercial propagation.

Somatic Embryogenesis from Immature Olive Zygotic Embryos.

The effects of plant growth regulators (PGRs) and the explant type on the embryogenesis and plant regeneration of olive (Olea europaea L. ssp europaea var. sativa) cv. "Chetoui" were studied using immature zygotic embryos. Embryogenic callus induction was achieved on OMc medium supplemented with different concentrations of BAP and NAA at low levels. Immature zygotic embryos as juvenile tissues are competent for somatic embryogenesis independently of PGRs supplementation. Repetitive somatic embryogenesis was obtained on PGRs-free media or at low concentrations in the dark.

Influence of Plant Growth Regulators on Embryogenic Callus and Somatic Embryos Induction in Date Palm Cv. Zaghloul Uses Leaves in Vitro

Influence of Plant Growth Regulators on Embryogenic Callus and Somatic Embryos Induction in Date Palm Cv. Zaghloul Uses Leaves in Vitro

Optimization of Somatic Embryo Regeneration of <i>Larix olgensis</i> Through Multiple Media

To establish a stable and efficient  Larix olgensis  somatic embryo regeneration system. In this study, immature zygotic embryos of  Larix olgensis  were used as explant materials for embryogenic callus induction, and embryogenic callus with good growth were selected to induce somatic embryo formation by liquid medium A, semi-solid medium B, and solid medium C. The callus induction medium is S medium+2,4-D 1.0 mg/L + 6-BA 0.5 mg/L + KT 0.3 mg/L, sucrose 20 g/L, agar 3 g/L; the subculture medium is S medium+2, 4-D 0.15 mg/L + 6-BA 0.05 mg/L + KT 0.05 mg/L, sucrose 30 g/L, agar 6 g/L. Mature medium A is S medium + PEG4000 100 g/L + ABA 15 mg/L + AgNO 3  10 mg/L, sucrose 45 g/L liquid medium. Mature medium B and C are mature medium A supplemented with 2 g/L and 10 g/L agar, respectively. The maturation time of this method is about 32 days, the synchronization rate is 78.0%, and the proportion of somatic embryos in normal morphology is 83.0%. Somatic embryo formation efficiency in normal morphology can reach 223.0 embryos/g embryogenic callus, somatic embryo germination rate can reach 47.2%. Compared with a single maturation medium, the maturation time is shortened, somatic embryo generation is more synchronized, and high somatic embryo yield and quality are improved. It provides a way for the commercialization of somatic embryo regeneration of  Larix olgensis , brings great convenience to large-scale breeding and reproduction, physiological and biochemical research, and genetic modification, and has great practical application value.

Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis.

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

Induction of Embryogenic Callus in Cashew (Anacardium occidentale L.) by Cotyledon, Nucellus and Testa with Antioxidants Effect

Aims: This work aims to evaluate the influence of different growth regulators combined with antioxidants on the induction of callus with different explants in cashew.
 Materials and Methods: Nucellar tissues, cotyledons and testa from elite tree nuts are cultured on media that differ in their concentration of 6-Benzylaminopurine (BAP: 0 mg/l; 0.25 mg/l; 0.5 mg/l) and Acid 2,4 dichlorophenoxyacetic (2,4-D: 0 mg/l; 0.8 mg/l). Therefore, for the control of the browning of the explants, the effect of activated charcoal and polyvinylpyrrolidone was tested. The amount of callus formed is evaluated after 28 days and after 90 days by simple observation according to a given scale. 
 Results: Analysis of variance of callus formation 28 days after the culture of explants shows that the interaction between growth regulators and antioxidants significantly influences (p < 0.05) the induction of callus. The combination BAP 0.25mg/l and 2,4-D 0.8mg/l produces on average more callus (0.47 ± 0.00). There is a very significant difference (P < 0.05 to p<0.001) in the effect of growth regulators and antioxidants on obtaining callus after 90 days of cultivation. The 0.25 mg/l combination of BAP and 0.8 mg/l of 2,4-D still appears to be the best combination of growth regulators for callus with 58% of callus formed. Cotyledons in the presence of PVP respond better than activated charcoal. The nucellus are the explants that respond better in the presence of activated charcoal. The test for Least Significant Difference reveals that PVP significantly promotes (p <0.05) the production of a large number of calli (77%) unlike activated charcoal (9%) after 90 days of culture.
 Conclusion: Summary, for obtaining viable and embryogenic callus in cashew, the combination of BAP at 0.25 mg/L and 0.8 mg/L of 2,4-D is the best and this in the presence of PVP with cotyledons.

Embryogenic callus induction, cell suspension culture, and spectrum-effect relationship between antioxidant activity and polyphenols composition of Siraitia grosvenorii cultured cells

Siraitia grosvenorii (S. grosvenorii), a perennial vine of Cucurbitaceae family, has been widely used in medicine and food products. However, its application is hindered due to the limited output of natural and cultivated plants. Thus, to expand plant resource and application of S. grosvenorii, the current research study comprises embryogenic callus induction, development of suspension culture, and analysis on the spectrum-effect relationship between antioxidant activity and polyphenols composition of S. grosvenorii cultured cells. Adding 0.5 g·L−1 ascorbic acid and 1.0 g·L−1 choline could increase the induction rate from 0.75% to 68% and enhances the antioxidant enzyme activities of loose embryogenic calluses. A total of 15 polyphenols (14 flavonols and 1 phenolic acid) in the callus extracts were identified via liquid chromatography-mass spectrometry. Through Plackett-Burman's design and response surface methodology, optimal proportionings of plant growth regulator (PGRs) for cell suspension culture were obtained. With the optimized PGRs, the obtained cell biomass and polyphenols content were 32.18% and 13.86%, respectively, more than PGRs proportioning before optimization. Furthermore, a homogeneous and rapidly grown suspension cell culture was developed with a maximum biomass and polyphenols content of 25.5 g·L−1 DW and 30.75 mg·g−1 DW, respectively. The results of spectrum-effect relationships analysis indicated a strong linear correlation between 15 above-identified polyphenols and the antioxidation activity of the extracts. In addition, the partial least square method result revealed that all 15 polyphenols positively contributed to antioxidant capacity, including 3,4-di-p-coumaroyl, 5-tri-hydroxycoumaroyl quinic acid and kaempferol-3-O-Glc-7-O-Rha with vital roles. Our results indicated that the extracts from S. grosvenorii cell have excellent antioxidant activity, and therefore have potential applications in healthy food and daily cosmetics.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.).

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

Improved method for regeneration and Agrobacterium-mediated transformation of Indian short-day onion (Allium cepa L.)

A high-auxin medium, usually used for callus induction, was not effective for Indian short-day onion cv. Bhima super. In this study, we found that the onion seedling radicle was a better explant than shoot tip for embryogenic callus induction, and induction efficiency up to 85.33% along with high embryogenic calli weight was obtained in routinely used medium containing 1.0 mg/L 2,4-D, but specifically supplemented with 0.5 mg/L kinetin. MS medium supplemented with 1.5 mg/L kinetin and 0.125 mg/L ABA showed 73.15% shoot regeneration efficiency from the calli induced from seedling radicle. Geneticin and hygromycin B at 50 mg/L showed optimal selection pressure for 8-week-old onion calli. Agrobacterium-mediated transformation of 8-week-old friable embryogenic calli induced from seedling radicle resulted in phenotypically normal transgenic plants with 1% transformation efficiency. In this study, regeneration and transformation protocols were developed for a widely used Indian short-day onion cultivar, which is instrumental for the development of stable transgenics in this crop.

Embryogenic Calli Induction and Salt Stress Response Revealed by RNA-Seq in Diploid Wild Species Gossypium sturtianum and Gossypium raimondii.

Wild cotton species can contribute to a valuable gene pool for genetic improvement, such as genes related to salt tolerance. However, reproductive isolation of different species poses an obstacle to produce hybrids through conventional breeding. Protoplast fusion technology for somatic cell hybridization provides an opportunity for genetic manipulation and targeting of agronomic traits. Transcriptome sequencing analysis of callus under salt stress is conducive to study salt tolerance genes. In this study, calli were induced to provide materials for extracting protoplasts and also for screening salt tolerance genes. Calli were successfully induced from leaves of Gossypium sturtianum (C1 genome) and hypocotyls of G. raimondii (D5 genome), and embryogenic calli of G. sturtianum and G. raimondii were induced on a differentiation medium with different concentrations of 2, 4-D, KT, and IBA, respectively. In addition, embryogenic calli were also induced successfully from G. raimondii through suspension cultivation. Transcriptome sequencing analysis was performed on the calli of G. raimondii and G. sturtianum, which were treated with 200 mM NaCl at 0, 6, 12, 24, and 48 h, and a total of 12,524 genes were detected with different expression patterns under salt stress. Functional analysis showed that 3,482 genes, which were differentially expressed in calli of G. raimondii and G. sturtianum, were associated with biological processes of nucleic acid binding, plant hormone (such as ABA) biosynthesis, and signal transduction. We demonstrated that DEGs or TFs which related to ABA metabolism were involved in the response to salt stress, including xanthoxin dehydrogenase genes (ABA2), sucrose non-fermenting 1-related protein kinases (SnRK2), NAM, ATAT1/2, and CUC2 transcription factors (NAC), and WRKY class of zinc-finger proteins (WRKY). This research has successfully induced calli from two diploid cotton species and revealed new genes responding to salt stress in callus tissue, which will lay the foundation for protoplast fusion for further understanding of salt stress responses in cotton.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Genotypic variation in the response to embryogenic callus induction and regeneration inSaccharum spontaneum

AbstractEmbryogenic callus induction and regeneration are useful in many aspects of plant biotechnology, especially in the functional characterization of economically important genes. However, in sugarcane, callus induction and regeneration vary across genotypes.Saccharum spontaneumis an important wild germplasm that confers disease resistance and stress tolerance to modern sugarcane cultivars, and its genome has been completely sequenced. The aim of this study was to investigate the effect of genetic variations on embryogenic callus induction and regeneration inS. spontaneumand to screen genotypes having high tissue culture susceptibility. The study was performed using nine genotypes ofS. spontaneumand the following five parameters were assessed to determine the response of genotypes to embryogenic callus induction and regeneration: callus induction, embryogenic callus ratio, embryogenic callus induction, embryonic callus regeneration and regeneration capacity. All the genotypes varied significantly (P< 0.01) in all the parameters, except for embryonic callus regeneration, which was high (>80%) for all the genotypes. High broad-sense heritability (86.1–96.8%) indicated that genetic differences are the major source of genotypic variations. Callus induction was found to be strongly positively correlated with embryogenic callus induction (r= 0.890,P< 0.01) and regeneration capacity (r= 0.881,P< 0.01). Among the nine tested genotypes, VN2 was found to be the most responsive to tissue culture and could therefore be used to characterize functional genes inS. spontaneum. We also suggested an approach with potential applications in facilitating the rapid identification of sugarcane genotypes susceptible to tissue culture.

In vitro plant regeneration of some recalcitrant indica rice (Oryza sativa L.) varieties

Rice is the staple diet for the greater part of the world’s population and there is a regular expanding need of rice production. The plant development and productivity are decreased by numerous factors. Utilizing biotechnological instruments is the most useful alternative to create rice varieties with incredible grain quality, improved productivity and protection from different stresses. Nonetheless, the deficiency of a simple, speedy and competent protocol for embryogenic callus induction and plant recovery is a significant draw back and there is substantial genotype-dependence. The current study explores the impact of plant growth regulators on the callus induction, proliferation and recovery from developed seeds in three rice varieties—SR 1, Jehlum and K 332. Greatest callus induction (82.33 ± 2.51% in SR1, 99.66 ± 0.57% in Jehlum and 93.33 ± 2.88% in K332) was seen in type 2 media containing 3% maltose, 500 mg/l casein hydrolysate, 500 mg/l l-proline, 5 mg/l l-glutamine, 40 mg/l cysteine, 5 mg/l l-asparagine, 100 mg/l ascorbic acid, 4 mg/l AgNO3, 600 mg/l MgCl2 enhanced with 4 g/l gelrite, 0.2 mg/l BAP and 2.5 mg/l 2, 4-D. Most noteworthy recurrence of shoot recovery (75.0 ± 3.0% in SR1, 86.66 ± 2.88% in Jehlum and 88.33 ± 2.88% in K332) was seen in the MS B5 medium containing 3 g/l phytagel enhanced with 4% sucrose, 2.5 mg/l BAP, 1 mg/l zeatin, 0.2 mg/l NAA and 0.5 mg/l TDZ. The current protocol gives a quick callus induction and recovery system, which could be utilized in effective genetic transformation and for creating genetically altered plants.

English

Some improved genotypes of cocoa tree (Theobroma cacao L.), have shown in standard study conditions of absence or a very low response to the induction of somatic embryos. This is the case of the recalcitrant genotypes C8 and C15, partially recalcitrant genotypes C14 and C16. This study aims to improve the production of somatic embryos of recalcitrant genotypes. Staminodes and petals excised from the immature buds of the five genotypes C1, C8, C14, C15 and C16 were used as plant material. These floral explants were cultured on different media containing two type of mineral salts such as potassium sulphate (K2SO4) at concentrations 18, 27, 36 and 45 mM and magnesium sulphate (MgSO4) whose concentrations used are 5, 10, 15 and 20 mM. Calli induction was obtained in the five (05) genotypes at percentages ranging from 50 to 100% with staminodes and from 61 to 100% with petals on all media. The transfer of the callogenic explants on the developmental medium allowed the induction of embryogenic calli and somatic embryos after 84 days only with petal explants. The most important PEC and NSE were obtained with two mineral salts concentrations, 27 mM K2SO4 and 15 mM MgSO4 for all genotypes and varied to 20 to 40%. Potassium sulphate (K2SO4) at 27 and magnesium sulphate (MgSO4) at 15 mM are the concentrations of the most suitable mineral salts to overcome recalcitrance of cocoa genotypes.   Key words: Recalcitrant, Theobrama cacao, improved genotypes, mineral salts, somatic embryogenesis.