Jelili T. Opabode

Primary Somatic Embryos from Axillary Meristems and Immature Leaf Lobes of Selected African Cassava Varieties

The study evaluated high-value African cassava varieties for primary somatic embryogenesis usingaxillarymeristems (AM) and immature leaf lobes (LL) on picloram based medium. The study was conducted at the Central Biotech Lab, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria between 2006 and 2009. Completely randomized design with four replicates was used for the study. Using LL explants, there were significan t (P=.05) differences in percent responding leaf lobes, percent explant with pre -embryogenic structure, PSEF and PSEE among cassava varieties. The PSEF of the only three varieties that produced mature somatic embryo were 93.6, 88.5 and 85.7% for TME 12, Ki

Influence of Type and Age of Primary Somatic Embryo on Secondary and Cyclic Somatic Embryogenesis of Cassava ( Manihot esculenta Crantz)

This study investigated the influence of age of the cotyledons, cut from primary somatic embryos (PSE) developed from shoot meristems (SM) or immature leaf lobes (LL), on secondary somatic (SSE) and cyclic (CSE) embryogenesis of two cassava cultivars at th e Central Biotech Laboratory, International Institute of Tropical Agriculture (IITA), Ibadan, between 2006 and 2010. A completely randomized design with three replicates was used for the study. Only PSE at the age of 4 weeks recorded significant (P<0.05) d ifferences in SSE frequency and efficiency between the SM and LL sources. CSE production was highest using 0 to 4 weeks old SSE cotyledons, and significant (P<0.05) differences were only recorded between the SM and LL sources when the age of the SSE cotyle dons was older than 6 weeks. The CSE frequencies from the SM source were significantly greater than that from the LL source when 8 and 10 week -old SSE cotyledons were used. The OriginalResearch Article

In vitro Propagation of Solanecio biafrae and Determination of Genetic Stability of Plantlets Using RAPD and ISSR Markers

Abstract An efficient and reproducible micropropagation protocol of Solanecio biafrae (Oliv. & Hiern) C. Jeffrey has been developed from nodal stem segments. Shoot development was obtained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) alone and in combination with zeatin and 1-naphthaleneacetic acid (NAA). Elongated shoots were rooted in the presence of zeatin or 3-indole-butyric acid (IBA) alone or in combinations. The highest number of explants forming shoots (100%) as well as the highest number of shoots per explant (3.4) and the longest shoots (22 mm) were recorded on medium containing 4.0 mg·dm−3 BAP, 2.0 mg·dm−3 NAA, and 1.0 mg·dm−3 zeatin. About 76% of shoots formed roots on half-strength MS medium free of plant growth regulators. The best root formation (approximately 88%) was recorded on the medium containing 1.0-1.5 mg·dm−3 IBA. The micropropagated shoots with well-developed roots were efficiently acclimatized under greenhouse conditions. The random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) amplification products were monomorphic in micropropagated plants and similar to those of mother plant showing their genetic uniformity. This is the first report of micropropagation of S. biafrae, which will facilitate in vitro mass propagation, conservation, and germplasm exchange of this endangered African vegetable.

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

ABSTRACT Successful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.

In vitro Propagation of Crassocephalum crepidioides—An Endangered African Traditional Leaf Vegetable and Molecular Analysis of Micropropagated Plants

ABSTRACT Crassocephalum crepidioides (Benth) S. Moore is an important African traditional leaf vegetable, but its production is very low and its existence threatened. An efficient and reproducible micropropagation protocol of C. crepidioides was developed from nodal stem segments of pot-grown plants. Shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with benzineamino purine (BAP) alone and in combination with zeatin and naphthalene acetic acid (NAA). Elongated shoots successfully rooted on zeatin alone and in combination with indole butyric acid (IBA). Molecular stability of micropropagated plants was evaluated using random amplified polymorphic DNA (RAPD) markers. Survival of nodal segments in micropropagation medium was greater than 70% in all treatments. The presence of plant growth regulators (PGRs) in MS medium influenced shoot formation, rooting, and number of shoots and roots per explant. The best shoot formation frequency (88.7%), number of shoots per explant (3.7), and shoot length (30 mm) were from a medium containing MS supplemented with 10 mg·L−1 BAP, 15 mg·L−1 NAA, and 1.5 mg·L−1 zeatin. Frequency of root formation and survival of regenerated plants were greater on medium containing half-strength MS salt than medium fortified with full-strength MS salt. The best frequency of root formation (average 87.4%) and survival of regenerated plants (average 95.4%) were from media containing 6.0 mg·L−1 zeatin and 1.0–1.5 mg·L−1 IBA and half-strength MS salt. Micropropagated shoots with well-developed roots were acclimatized under greenhouse conditions in pots containing rich sandy loam soil (pH = 7.2). The RAPD amplification products were monomorphic in micropropagated plants and similar to those of the mother plant. No polymorphism was detected, indicating genetic uniformity of micropropagated plants. This is the first report of micropropagation of C. crepidioides, which will facilitate in vitro mass propagation, conservation, and germplasm exchange of this endangered African vegetable.

Tissue- and Organ-Specific Promoters for Expression of Heterologous Genes inTransgenic Cassava (Manihot Esculenta Crantz) Plants

Promoters are regions of DNA that initiates transcription of genes. A number of promoters have been identified that confer high level of expression of heterologous genes in transgenic plants. Some promoters have constitutive expression as they are active in all circumstances in the cell, while others are regulated, becoming active in certain cell or in response to specific stimuli. Despite the availability of tissue- and organ-specific promoters, most transgene expressions in Cassava are driven by constitutive cauliflower mosaic virus promoter. This paper examines the availability of promoters for transgene expression in plants, assesses the use of promoters for transgene expression in Cassava and establishes the need for tissue- and organ-specific promoters for expression of heterologous genes in Cassava.

Responses of cassava (Manihot esculenta Crantz) varieties to in vitro mannitol-induced drought stress

ABSTRACT Rapid selection of drought-tolerant cassava varieties by in vitro screening is essential for efficient breeding for drought tolerance. The objectives of this study were to describe the morphological response of 12 cassava cultivars to in vitro mannitol-induced drought stress and identify tolerant ones. Plantlets were raised from nodal segments on Murashige and Skoog (MS) medium containing either 0 g/L or 20 g/L mannitol. Mannitol increased the number of shoots of plantlets per explant (NSS) of all cultivars by an average of 18.2%. However, mannitol decreased the dry shoot weight (DSW) of all cultivars by half. Mannitol decreased total number of leaves (TNL) of plantlets by more than half in all cultivars, except three, in which mannitol had no effect on the TNL. The average root length (ARL) of each one of the following four varieties: TMS 30572, TMS 50395, MM 96/1751, and TMS 94/0026, was not sensitive to mannitol treatment. However, mannitol increased the ARL of MM 98/3437, TMS 94/0039, TMS 92B/00061, TMS 91934 and TMS 84/00353 but decreased the ARL of TMS 05/1654, TME 419, and TMS 92/0326. Mannitol had no effect on dry root weight (DRW) of plantlets of TMS 91934, TMS 94/0026, TME 419, TMS 30572, TMS 50395, and MM 98/3437. The DRW of each of the following four varieties: TMS 05/1654, TMS 92/0326, TMS 92B/00061, and TMS 94/0039, was decreased by mannitol, whereas the DRW of each of TMS 84/00353 and MM 96/1751 was increased by an average of 42.4%. Stress-tolerance index showed that TMS 84/00353, MM 96/1751, MM 98/3437, and TMS 94/0026 were tolerant of mannitol-induced drought stress and, therefore, recommended for planting in drought-prone regions by farmers.

Exogenously applied gibberellic acid affects shoot regeneration, growth, physiological parameters, and proximate and mineral contents of pot-grown Solanecio biafrae

ABSTRACT Solanecio biafrae (Oliv.& Hiеrn) C. Jеffrеу iѕ a trаditiоnаl leafy vеgеtаblе in Afriса, but its vegetative growth is slow, making production fall short of market demands. Gibberellic acid (GA3) is a hormone produced by plants that stimulates growth and enhances absorption of minerals. However, GA3 synthesis is limited in shoots of S. biafrae due to inadequate external stimuli. To increase yield of S. biafrae and its proximate and mineral contents, external application of GA3 is desirable to supplement internally produced GA3. This study examined the influence of exogenously applied GA3 on shoot emergence, growth, fresh shoot weight, physiological parameters, proximate, and leaf mineral element contents of the green-stemmed morphotype of S. biafrae. Stem-cuttings were treated at 1, 3, аnd 6 dауѕ after planting with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg∙L−1 of GA3 in plastic pots. Exogenously applied GA3 influenced most parameters measured except leaf K. Total number of leaves, total leaf area, fresh shoot weight, dry weight, carotenoids, phenolics, total soluble sugars, ash, carbohydrates, leaf Na, P, Mg, Ca, Fe, and Zn increased as GA3 concentration increased up to 80 mg∙L−1. Number of days to shoot emergence and crude fat content were lower in treated plants than controls. Chlorophyll a was highest at 60 mg∙L−1. The study concluded that the optimum GA3 concentration was 80 mg∙L−1 as it promoted the highest total number of leaves, total leaf area, shoot height, dry and fresh shoot weights, and produced the highest synthesis of chlorophyll a, phenolics, total soluble sugars and proteins, crude fiber, protein, ash, carbohydrates, leaf Na, P, Mg, Ca, Fe, and Zn.

Evaluation of Genomic DNA-Extraction Methods for Molecular Analysis of Solanecio biafrae

ABSTRACT Solanecio biafrae (Oliv. & Hiern) C. Jеffrеу, an indigenous vegetable of the humid tropics, is rich in minerals and easy to cultivate, but its vegetative growth is slow, making production fall short of demand. An efficient and effective genomic DNA-extraction method is necessary for a comprehensive molecular analysis to increase leaf yield and further improve proximate and mineral contents of the vegetable. Cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and Qiagen kit (QIAGEN) methods were evaluated for extraction of genomic DNA from young leaves suitable for PCR amplification for molecular analyses. Quantity and quality of the DNA were examined spectrophotometerically and with gel electrophoresis. Amplification of the DNA by PCR for molecular analysis was assessed with five inter-simple sequence repeat (ISSR) primers. The mean DNA yield using QIAGEN, CTAB, and SDS methods were 71.0, 35.4, and 38.8 µg∙µL−1∙g−1 of tissue, respectively. The mean 260/280 nm ratios of QIAGEN, CTAB, and SDS were 1.84, 1.83, and 1.79, respectively. Gel electrophoresis indicated presence of DNA free of contaminants in all samples using three methods. A total of 270 distinct bands were produced by five ISSR primers with similar patterns among three DNA-extraction methods, ranging from 200–2900 base pairs. Three methods are capable of extracting adequate genomic DNA suitable for PCR amplification. The QIAGEN method produced the highest DNA yield, which was of the same quality with DNA obtained using CTAB and SDS methods.

Sustainable Mass Production, Improvement, and Conservation of African Indigenous Vegetables: The Role of Plant Tissue Culture, a Review

ABSTRACT African indigenous vegetables (AIVs) are plants grown and utilized traditionally by communities in Africa because they possess food, nutritive, or medicinal values. Seed dormancy, loss of biodiversity, high perishability of produce, low yield, and inadequacy of planting propagules, among other factors, are constraints against production and distribution of AIVs. This work examines the application of plant tissue culture (PTC) techniques for mass production of propagules, genetic improvement, and biodiversity conservation of AIVs because less than 5% have been tested for responses to PTC techniques. Applicable PTC techniques for sustainable production of AIVs include micropropagation, meristem culture, embryo rescue, shoot–tip culture, callus culture, somatic embryogenesis, protoplast fusion, in vitro flowering, and in vitro selection and mutagenesis. Shoot meristems, hypocotyls, petioles, immature leaves, cotyledons, axillary buds, seed, and epicotyls are suitable explants for PTC initiation. Specific benefits of these techniques include mass and accelerated production of disease-free propagules available in all seasons, an end to genetic erosion using biodiversity conservation and genetic improvement to overcome biotic and abiotic production constraints, and generation of hybrids of AIVs. Uniformity of plants and the ease with which AIVs germplasm are exchanged among African countries make application of the techniques desirable. Challenges facing application of these techniques in Africa are deficit of skilled human resources, underdeveloped laboratories and facilities, irregular power supply and unreliable consumable sources, high cost of PTC research, inadequate research funding, lack of research priority on AIVs, and confusion and controversy regarding PTC and genetic transformation.