Efficient Genetic Transformation and Regeneration of a Farmer-Preferred Cassava Cultivar From Ghana

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa.

The Effect of Medium Type and Subculture Frequency on the Formation of Friable Embryogenic Callus for Coconut Cell Suspension Culture

Coconut, a multipurpose palm, is facing increasing demand for its fruit as well as the pressurefrom industries to produce coconut-derived products. [...]

Studying on regeneration of several cassava varieties (Manihot esculenta Crantz) via friable embryogenic callus

Cassava (Manihotesculenta Crantz) is considered as one of the most important food crops which has high economic value in many areas. It is very neccessay to perfect the cassava regeneration protocol for genetic transformation purpose. In this study, cassava regeneration via friable embryogenic callus (FEC) from tip bud, young stem, pieces of leaf had been optimized in five cassava varieties which were planted in Vietnam including KM 140 (S1), NgheAn white cassava (S2), Lang Son red cassava (S3), HoaBinh high – yield cassava (S4) and Huay Bong (S5). The results indicated that on MS medium supplemented with 10 mg/l Picloram, the proportion of callus formation was very high, reached from 90 to 100%. In the case of using tip buds, after three weeks, calli were transferred to MS medium adding 5 mg/l picloram and 0,2 mg/l IBA. The proportion of FEC formation reached 41,1 – 80,4 % after 8 weeks of cultivation in all studied Cassava varieties. The samples were transferred to MS medium adding 0,3 mg/l BAP to elongate shoots in 4 weeks. The highest regeneration rate belonged to S1, and was 61,67%. Three weeks after shoot transferring on MS medium, the complete seedlings were grown in substrate which was composed by TN01 and husk hun with ratio of 6:4 in greenhouse. As a result, the rate of survival plants reached to 95%. The process of regeneration of cassava through embryonic calli could be applied for the improvement of desired cassava varieties by method of genetic engineering.

Genetic Transformation of Recalcitrant Cassava by Embryo Selection and Increased Hormone Levels

Genetic engineering is considered to be an important tool for the improvement of cassava. Cassava is a highly heterozygous crop species for which conventional breeding is a lengthy and tedious process. Robust transformation is based on Agrobacterium-mediated transformation of friable embryogenic callus (FEC). Production of FEC is genotype-dependent and considered to be a major bottleneck for the genetic transformation of cassava. As a consequence, routine genetic transformation has only been established for a handful of cassava cultivars. Therefore, development of procedures enabling efficient production of high-quality cassava FEC is required to allow the translation of research from the model cultivar to farmer-preferred cassava cultivars. Here we study the FEC production capacity of Brazilian cassava cultivars and report the modification of the protocol for the genetic transformation of Verdinha (BRS 222), a recalcitrant cultivar with high potential for protein production that is extensively used by farmers in Brazil.

Gene expression analysis provides insight into the physiology of the important staple food crop cassava

SummaryCassava (Manihot esculenta) feeds approximately 800 million people worldwide. Although this crop displays high productivity under drought and poor soil conditions, it is susceptible to disease, postharvest deterioration and the roots contain low nutritional content.Here, we provide molecular identities for eleven cassava tissue types through RNA-sequencing and develop an open access, web-based interface for further interrogation of the data.Through this dataset, we report novel insight into the physiology of cassava and identify promoters able to drive specified tissue expression profiles. Specifically, we focus on identification of the transcriptional signatures that define the massive, underground storage roots used as a food source and the favored target tissue for transgene integration and genome editing, friable embryogenic callus (FEC).The information gained from this study is of value for both conventional and biotechnological improvement programs.

Perkembangan kalus embriogenik sagu (Metroxylon sagu Rottb.) pada tiga sistem kultur in vitro Development of embryogenic callus of sago (Metroxylon sagu Rottb.) on three systems of in vitro culture

Summary Embryogenic callus of sago (Metroxylon sagu Rottb.) has been grown on three systems of in vitro culture i.e. agar-solidified medium, liquid medium, and temporary immersion system (TIS) medium to observe and compare the development of embryogenic callus over one passage of six weeks. A-half gram of embryogenic callus was cultured on a modified MS medium containing 10 mg/L 2,4-D and 0.1 mg/L kinetin. For histological studies, embryogenic callus was fixed in FAA and embedded in paraplast wax. Serial sections were stained with safranin 1% and observed microscopically. By the end of culture period, the development of embryogenic callus in TIS medium was relatively better than those of the other two media. Fresh weight of callus in liquid medium and TIS increased by 6.5-fold, while on agar-solidified medium increased by 5.4-fold in six weeks. About 40% of callus in liquid medium and TIS and 20% of callus on agar solidified medium have changed into somatic embryos at globular stage. Histology structure of embryogenic callus of the three systems of in vitro culture shows different pattern. On agar-solidified medium, secondary callus and friable embryogenic callus that consist of meristematic cells were formed. In contrast, more embryogenic cells were formed in liquid medium and TIS to support maturation process to somatic embryos. Therefore, temporary immersion system and liquid medium are recommended for maturation of embryogenic callus, whereas agar-solidified medium is for proliferation of embryogenic callus of sago. Ringkasan Kalus embriogenik sagu (Metroxylon sagu Rottb.) telah ditumbuhkan pada tiga sistem kultur in vitro yaitu medium padat, medium cair, dan medium dengan sistem perendaman sesaat (SPS) untuk mempelajari dan mem-bandingkan perkembangan dari kalus embrio-genik selama periode enam minggu. Setengah gram kalus embriogenik dikulturkan pada medium MS modifikasi yang mengandung 2,4-D 10 mg/L dan kinetin 0,1 mg/L. Untuk studi histologi, kalus embriogenik difiksasi dengan FAA dan embedding menggunakan lilin paraplast. Irisan diwarnai dengan safranin 1% dan diamati menggunakan mikroskop. Pada akhir periode kultur, pertumbuhan kalus pada medium dengan SPS lebih baik dibandingkan dengan medium cair dan padat. Bobot basah kalus pada medium cair dan SPS meningkat 6,5 kali sedangkan pada medium padat meningkat 5,4 kali dalam waktu enam minggu. Sebanyak 40% kalus pada medium cair dan SPS serta 20% kalus pada medium padat berubah menjadi embrio somatik fase globuler. Struktur histologi kalus embriogenik pada ketiga jenis sistem kultur in vitro menunjukkan pola yang berbeda. Pada medium padat terjadi pembentukan kalus sekunder dan kalus embriogenik remah yang terdiri atas sel-sel meristematik. Sebaliknya pada medium cair dan SPS pembentukan sel embriogenik lebih banyak yang menunjang proses pendewasaan menjadi embrio somatik. Oleh karena itu, medium cair dan SPS direkomendasikan untuk pendewasaan kalus embriogenik, sedangkan medium padat untuk proliferasi kalus embriogenik sagu.

Perkembangan kalus embriogenik sagu (Metroxylon sagu Rottb.) pada tiga sistem kultur in vitro Development of embryogenic callus of sago (Metroxylon sagu Rottb.) on three systems of in vitro culture

Summary Embryogenic callus of sago (Metroxylon sagu Rottb.) has been grown on three systems of in vitro culture i.e. agar-solidified medium, liquid medium, and temporary immersion system (TIS) medium to observe and compare the development of embryogenic callus over one passage of six weeks. A-half gram of embryogenic callus was cultured on a modified MS medium containing 10 mg/L 2,4-D and 0.1 mg/L kinetin. For histological studies, embryogenic callus was fixed in FAA and embedded in paraplast wax. Serial sections were stained with safranin 1% and observed microscopically. By the end of culture period, the development of embryogenic callus in TIS medium was relatively better than those of the other two media. Fresh weight of callus in liquid medium and TIS increased by 6.5-fold, while on agar-solidified medium increased by 5.4-fold in six weeks. About 40% of callus in liquid medium and TIS and 20% of callus on agar solidified medium have changed into somatic embryos at globular stage. Histology structure of embryogenic callus of the three systems of in vitro culture shows different pattern. On agar-solidified medium, secondary callus and friable embryogenic callus that consist of meristematic cells were formed. In contrast, more embryogenic cells were formed in liquid medium and TIS to support maturation process to somatic embryos. Therefore, temporary immersion system and liquid medium are recommended for maturation of embryogenic callus, whereas agar-solidified medium is for proliferation of embryogenic callus of sago. Ringkasan Kalus embriogenik sagu (Metroxylon sagu Rottb.) telah ditumbuhkan pada tiga sistem kultur in vitro yaitu medium padat, medium cair, dan medium dengan sistem perendaman sesaat (SPS) untuk mempelajari dan mem-bandingkan perkembangan dari kalus embrio-genik selama periode enam minggu. Setengah gram kalus embriogenik dikulturkan pada medium MS modifikasi yang mengandung 2,4-D 10 mg/L dan kinetin 0,1 mg/L. Untuk studi histologi, kalus embriogenik difiksasi dengan FAA dan embedding menggunakan lilin paraplast. Irisan diwarnai dengan safranin 1% dan diamati menggunakan mikroskop. Pada akhir periode kultur, pertumbuhan kalus pada medium dengan SPS lebih baik dibandingkan dengan medium cair dan padat. Bobot basah kalus pada medium cair dan SPS meningkat 6,5 kali sedangkan pada medium padat meningkat 5,4 kali dalam waktu enam minggu. Sebanyak 40% kalus pada medium cair dan SPS serta 20% kalus pada medium padat berubah menjadi embrio somatik fase globuler. Struktur histologi kalus embriogenik pada ketiga jenis sistem kultur in vitro menunjukkan pola yang berbeda. Pada medium padat terjadi pembentukan kalus sekunder dan kalus embriogenik remah yang terdiri atas sel-sel meristematik. Sebaliknya pada medium cair dan SPS pembentukan sel embriogenik lebih banyak yang menunjang proses pendewasaan menjadi embrio somatik. Oleh karena itu, medium cair dan SPS direkomendasikan untuk pendewasaan kalus embriogenik, sedangkan medium padat untuk proliferasi kalus embriogenik sagu.

Somatic Embryogenesis in Four Tree Legumes

Somatic embryogenesis was achieved in four leguminous tree species, that is, Acacia catechu, Acacia arabica, Hardwickia binata, and Dalbergia sissoo using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.25–1.0 mg/l Kn (kinetin) and 2.0–3.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (1-napthaleneacetic acid) and 3% sucrose. MS medium containing 2.0 mg/l 2,4-D and 1.0–1.5 mg/l Kn was noted to be most effective in inducing friable embryogenic callus (FEC); the number of somatic embryos per culture varied in MS medium supplemented with 1.0–2.0 mg/l 2,4-D or NAA and 0.25–1.5 mg/l kinetin. The maximum number of somatic embryos was obtained in MS medium containing 1.5–2.0 mg/l 2,4-D or NAA and 1.0–1.5 mg/l kinetin; proliferation of embryogenic calli was enhanced in cultures having 1.0–2.0 mg/l 2,4-D, 1.0–1.5 mg/l kinetin, and 400–600 mg/l L-Proline. The somatic embryos in various shapes and sizes after the first subculture on MS medium supplemented with 0.1 mg/l IAA and 0.25 mg/l BA; developed shoots and rooted in strength MS medium supplemented with 0.1 mg/l IBA or IAA. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate-controlled hardening chamber.