scholarly journals Screening of Cultivars for Tissue Culture Response and Establishment of Genetic Transformation in a High-yielding and Disease-resistant Cultivar of Theobroma cacao

2020 ◽  
Author(s):  
Jesse Jones ◽  
Elaine Zhang ◽  
Dominick Tucker ◽  
Daniel Rietz ◽  
Doug Dahlbeck ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Genetic Transformation ◽  
Theobroma Cacao ◽  
Dna Delivery ◽  
Transformation Frequency ◽  
Resistant Cultivar ◽  
Tissue Culture Response ◽  
Disease Resistant ◽  
Delivery Efficiency

AbstractA highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning the initiation of floral material to the regeneration of plantlets into soil has been tested and improved in cacao. Fourteen cultivars were screened for their tissue culture response and transfer DNA (T-DNA) delivery efficiency via Agrobacterium. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from immature somatic embryos ranging in size between 4-10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in secondary callus growth medium increased the percentage of explants producing greater than ten embryos by 504% and 443%, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant level. Grafting transgenic scions with undeveloped roots to wild-type seedlings with strong, healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The present methods can be applied to improve tissue culture response and transformation frequency in other cacao cultivars.Key messageTissue culture and genetic transformation methods for a high-yielding, disease-resistant cultivar of Theobroma cacao were established while factors affecting T-DNA delivery and somatic embryogenesis were identified.

scholarly journals Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

2021 ◽  
Author(s):  
Jesse Jones ◽  
Elaine Zhang ◽  
Dominick Tucker ◽  
Daniel Rietz ◽  
Douglas Dahlbeck ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Theobroma Cacao ◽  
Dna Delivery ◽  
Transformation Frequency ◽  
Resistant Cultivar ◽  
Cupric Sulfate ◽  
Secondary Somatic Embryogenesis ◽  
Tissue Culture Response ◽  
Disease Resistant

AbstractA highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

scholarly journals Pengaruh Sitokinin, Jenis Eksplan, dan Genotipe terhadap Embriogenesis Somatik Kakao

2016 ◽  
Vol 3 (2) ◽  
pp. 71
Author(s):  
Nur Ajijah ◽  
RR. Sri Hartati
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Somatic Embryo ◽  
Theobroma Cacao ◽  
Somatic Embryos ◽  
Interaction Effects ◽  
Embryo Formation ◽  
Primary Somatic Embryogenesis ◽  
Primary Somatic Embryos

<p><em>Information on the effect of cytokinins on cacao (</em>Theobroma cacao<em> L.) primary somatic embryogenesis and its interaction with explant types and genotypes is not yet known. This study aimed to evaluate the effect of cytokinins and its interaction with explant types and genotypes on cacao somatic embryogenesis. The study was conducted at tissue culture laboratory of IAARD, Bogor from April until December 2012 and October 2014 until February 2016. Three types of cytokinins i.e. kinetin (0.58, 1.16, and 2.32 </em><em>μ</em><em>M), thidiazuron (0.01, 0.02, and 0.04 </em><em>μ</em><em>M) and benzylaminopurine (0.55, 1.11, and 2.22 </em><em>μ</em><em>M) in combination with 9 </em><em>μ</em><em>M 2,4-D were tested for their effectiveness in inducing somatic embryogenesis from petals and staminoid explants of Cimanggu 1 genotype. Furthermore, three levels of kinetin (0.58, 1.16, and 2.32 </em><em>μ</em><em>M</em><em>) also in combination with 9 </em><em>μ</em><em>M 2,4-D were evaluated for their influences on the somatic embryogenesis from petals and staminoid explants of three cacao genotypes i.e. Sulawesi 02, ICCRI 04 and Cimanggu 3. The result demonstrated that 2.32 </em><em>μ</em><em>M kinetin and staminoids explant were more effective to induce cacao somatic embryogenesis of Cimanggu 1 genotype (7%, 0.23 embryos/explant). Additionally, there were interaction effects between the level of kinetin with explant types and genotype on the percentage of explants forming embryo at 12 weeks after culture. The highest percentage of somatic embryo formation was shown by ICCRI 04 genotype with the use of petals explant and a kinetin level of 1.16 </em><em>μ</em><em>M (31.85%), but not significantly different from the level of kinetin 2.23 </em><em>μ</em><em>M (25.55%). The formation of primary somatic embryos of cacao is largely determined by the type and level of cytokinins, type of explant, and genotype.</em></p>

scholarly journals Coffee Yield and Mineral Cycle in Intercropping of Coffea canephora and Some Species of Timber Shade Trees

2008 ◽  
Vol 24 (1) ◽  
Author(s):  
Adi Prawoto
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Phenolic Compound ◽  
Embryogenic Callus ◽  
Theobroma Cacao ◽  
Coffea Canephora ◽  
Coffee Yield ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
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

scholarly journals Plant regeneration from proroplasts of alfalfa (Medicago sativa) via somatic embryogenesis

2003 ◽  
Vol 60 (4) ◽  
pp. 683-689 ◽  
Author(s):  
Mariza Monteiro ◽  
Beatriz Appezzato-da-Glória ◽  
Maria José Valarini ◽  
Carlos Alberto de Oliveira ◽  
Maria Lucia Carneiro Vieira
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Plant Regeneration ◽  
Genetic Transformation ◽  
Medicago Sativa ◽  
Cell Suspension ◽  
High Efficiency ◽  
Point Of View ◽  
Ability To Regenerate

Alfalfa is one of the most frequently studied species from the production of tissue culture-derived embryos point of view. In this study, five alfalfa cultivars were analyzed with reference to their ability to regenerate plants from protoplast cultures via somatic embryogenesis. Plant regeneration from leaf-derived protoplasts isolated from the cultivar Rangelander was achieved using a protocol defined for alfalfa cell suspension-derived embryogenesis. Because of its high efficiency, this procedure is recommended for protoplast electroporation-mediated genetic transformation of alfalfa.

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

2015 ◽  
Vol 31 (1) ◽  
pp. 14-20
Author(s):  
Sulistyani Pancaningtyas
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Phenolic Compounds ◽  
Somatic Embryo ◽  
Phenolic Compound ◽  
Embryogenic Callus ◽  
Theobroma Cacao ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
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

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

2009 ◽  
Vol 25 (3) ◽  
Author(s):  
Ketut Anom Wijaya ◽  
Adi Prawoto ◽  
Syrril Ihromi
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Phenolic Compound ◽  
Embryogenic Callus ◽  
Theobroma Cacao ◽  
Dichlorophenoxyacetic Acid ◽  
Pod Borer ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
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

scholarly journals Resterilization in cocoa (Theobroma cacao L.) somatic embryogenesispropagation to save contaminated embryos

2011 ◽  
Vol 27 (1) ◽  
Author(s):  
Sulistyani Pancaningtyas ◽  
Cahya Ismayadi
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Key Words ◽  
Theobroma Cacao ◽  
Contamination Level ◽  
Contaminant Level ◽  
Sodium Hypochloride ◽  
Different Response ◽  
Theobroma Cacao L

Somatic embryogenesis is a technique to produce primary embryos using tissue culture. Contamination in tissue culture can be caused by internal and external contaminant. Resterilization can be performed to save contaminated embryos. The aim of this research is to obtain resterilization method in cocoa micropropagation by tissue culture so that free bacterial explants can be obtained and embryogenic. This experiments used five clones of cocoa, namely Sulawesi 1, KW 514, ICCRI 05, ICCRI 03 and ICCRI 04. Embryogenic clusters in multiplication medium were used as explant. Sodium hypochloride was used as sterilant. Several factors were evaluated using randomized block complete design, i.e. contaminant level, concentration of sterilant and period of sterilant application. Results of resterilization methods showed no significant effect among several factors tested. Among those factors, low contamination level, 10% concentra tion of sterilant and no soaking showed the highest percentage of saving of contaminated embryos. There was different response among five cocoa clones in producing embryogenic explants when using combination of resterilization methods. Key words : Theobroma cacao, somatic embryogenesis, contamination, resterilization.

scholarly journals Carbon stock in different ages and plantation system of cocoa: allometric approach

2009 ◽  
Vol 26 (3) ◽  
Author(s):  
Fitria Yuliasmara ◽  
Aris Wibawa ◽  
Adi Prawoto
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Phenolic Compound ◽  
Embryogenic Callus ◽  
Theobroma Cacao ◽  
Dichlorophenoxyacetic Acid ◽  
Flower Tissue ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
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

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