Isoforms of Linamarase in Cassava (Manihot esculenta)

Linamarase (EC. 3.2.1.21) was purified from different tissues of cassava (leaf, rind and tuber) to compare the kinetic properties and characteristics of the enzyme in these tissues. Purified enzyme preparation appeared as single band of average molecular size 70 kD in SDS-PAGE gels. The kinetic properties of linamarase with respect to pH and temperature indicated that tuber linamarase possessed a broader pH optimum and higher temperature stability as compared to leaf and rind enzymes. Differences in Km values for linamarin were observed with leaf linamarase having the highest Km value (500 μM) followed by rind (400 μM) and then tuber (250 μM) linamarases. Rind enzyme appeared to be less susceptible to urea denaturation than the leaf enzyme. Comparison of elution profiles from DEAE-Cellulose indicated that the relative amounts of the various ionic forms of the enzyme differed in the case of each tissue. Elution patterns of the enzyme from Con A-Sepharose also differed, suggesting difference in glycosylation among leaf, rind and tuber enzymes. This was confirmed by carbohydrate analysis which showed that the tuber linamarase contained significantly higher amount of protein bound carbohydrate. These results suggest the possible occurrence of different forms of linamarase in cassava.     

Cassava, Manihot esculenta Crantz, genetic resources: origin of the crop, its evolution and relationships with wild relatives

About 98 species of Manihot are known. All of them are native to the New World and are concentrated in four regions in Brazil and Central America. All the Manihot species so far examined have 2n = 36 chromosomes. Interspecific hybrids between cassava and its wild relatives show relatively normal meiosis, and further generations can be obtained. Electrophoresis shows affinity among wild species of different sections, and between some of them and cassava. Both polyploidy and apomixis may have contributed to speciation in this genus. Polyploidy produced genetic variability, while apomixis is responsible for perpetuating new hybrid types adapted to different environments. Cassava may have originated by hybridization between two wild Manihot species, followed by vegetative reproduction of the hybrid.     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

Isozyme Diversity in Cassava Cultivars (Manihot esculenta Crantz)

Isoenzyme electrophoresis was used as a method to determine genetic diversity in various M. esculenta cultivars collected in the Southwestern (SW) and Northwestern (NW) regions of the State of Parana, in the South region of Brazil, and in cultivars produced at the Agronomic Institute of Campinas (IAC), S~ao Paulo State, Southeastern region of Brazil. The cultivars have been maintained by vegetative propagation for 5 years and are useful in production programs. A total of 28 loci in the acid phosphatase (ACP; EC 3.1.3.2), esterases (EST; EC 3.1.1.1), malate dehydrogenase (MDH; EC 1.1.1.37), and shikimate dehydrogenase (SKDH; EC 1.1.1.15) isozymes was analyzed. The proportion of polymorphic loci for NW, SW, and IAC cultivars was 57.14, 50.0, and 53.6%, respectively. Genetic diversity calculated by Nei's genetic identity (I) showed high I values for the three M. esculenta subpopulations. The high degree of polymorphism expressed by cassava cultivars is highly relevant to stimulate breeding programs with M. esculenta species.     

Gene flow between cassava, Manihot esculenta Crantz, and wild relatives

Controlled and natural hybridization between cassava and wild relatives does occur. Barriers within the genus appear to be weak due to recent evolution of the group. All Manihot species examined cytogenetically have a chromosome number of 2n = 36. However, they behave meiotically as diploids. The weak interspecific barriers have led to an extremely heterozygous gene pool that may begin a sequence of hybridization followed by speciation. Introgression from cassava into a number of wild species (M. neusana, M. alutacea, M. reptans and M. anomala) has been detected by both morphological marker genes and molecular techniques. Winged fruit, setaceous bracteoles, and wide leaf sinus were dominant genes that came from cassava and appeared in the hybrids. The characteristic protein bands of cassava were recognized in the hybrid seed protein electrophoresis.     

木薯体细胞胚胎发生及植株再生研究

对木薯体细胞胚胎发生的影响因素进行了优化研究。结果表明,基因型对木薯体细胞胚胎发生影响很大,在供试的六个品种中,“华南 8 号”的体细胞胚胎发生率和产胚量最高,分别为 65% 和19个;侧芽茎尖为最佳外植体,体细胞胚胎发生的最佳培养基为MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D。同时,对木薯体细胞胚再生成完整植株的主要影响因素作了分析,建立了一个高效的植株再生体系。     

Cassava (Manihot esculenta) leaf and tuber concentrate in diets for broiler chickens

Experiment 1 was conducted to determine the nutritive quality of cassava tuber leaf concentrate prepared either by mixing cassava tubers and leaves together before grinding or grinding them separately before mixing. Three mixing proportions of 50:50, 60:40 and 80:20 were used. Samples were subjected to either sundrying or oven drying. Physical examination of samples, proximate analysis and cyanide content of each of the samples were determined. In experiment 2 the sample of highest nutritive value was selected for inclusion in four diets at levels of 0%, 10%, 20% and 30% respectively. A total of 120 day-old broilers were divided into four groups and each group further into three subgroups. The four experimental diets were fed to each of the groups for a period of 9 weeks. Growth parameters, carcass characteristics and haematological parameters were also determined. Results showed that in experiment 1 the grinding together of cassava tubers and leaves in the proportion of 50:50 before sun-drying gave the best texture, highest CP content with low HCN content. Inclusion of 10% cassava concentrate gave good performance in terms of growth and feed conversion, with no detrimental effects on haematological parameters and carcass characteristics.     

Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication

Genetic variation at five microsatellite loci was used to investigate the evolutionary and geographical origins of cassava (Manihot esculenta subsp. esculenta) and the population structure of cassava's wild relatives. Two hundred and twelve individuals were sampled, representing 20 crop accessions, 27 populations of cassava's closest wild relative (M. esculenta subsp. flabellifolia), and six populations of a potentially hybridizing species (M. pruinosa). Seventy-three alleles were observed across all loci and populations. These data indicate the following on cassava's origin: (1) genetic variation in the crop is a subset of that found in the wild M. esculenta subspecies, suggesting that cassava is derived solely from its conspecific wild relative. (2) Phenetic analyses group cassava with wild populations from the southern border of the Amazon basin, indicating this region as the likely site of domestication. (3) Manihot pruinosa, while closely related to M. esculenta (and possibly hybridizing with it where sympatric), is probably not a progenitor of the crop. Genetic differentiation among the wild populations is moderately high (F:(ST) = 0.42, rho(ST) = 0.54). This differentiation has probably arisen primarily through random genetic drift (rather than mutation) following recent population divergence.     

A Rapid and Inexpensive Enzymatic Assay for Total Cyanide in Cassava (Manihot esculenta Crantz) and Cassava Products

The well-known alkaline picrate test for cyanide has been improved by incorporating an enzymatic step to make the assay much more specific and quantitative. The sensitivity or detection limit of this method was found to be 0.16 μg/cm³ while the precision as indicated by the coefficient of variation was 3%. The method was, in addition, found to be rapid, simple, inexpensive and ideally suited for the analysis of large number of cassava tissues and products, such as may be encountered in cassava agronomy and breeding work or in industrial quality control laboratories. A trained operator working alone consistently analyzed at least 700 samples/day using this assay method.     

Antholysis in Cassava (Manihot esculenta Crantz) Possibly Caused by Mycoplasma-Like Organisms

Phyllody and apostasis of cassava plants were frequently observed during recent disease surveys in the Cauca Valley of Colombia. Many valuable cassava clones have been affected rendering them unsuitable for hybridization. Light and electron microscopic observations have revealed the presence of mycoplasma-like organisms in the diseased phloem tissues. The causal agent is sensitive to tetracycline and streptomycin at 1000 ppm a. i., but not to penicillin.     

A High-throughput Platform for the Production and Analysis of Transgenic Cassava (Manihot esculenta) Plants

A platform for high-throughput production and analysis of transgenic cassava (Manihot esculenta) has been developed for the variety 60444 and implemented to generate plants expressing traits for nutritional enhancement, modified metabolism, promoter analysis and disease resistance. Over a three and a half year period this system has been utilized to produce more than 3500 independent transgenic plant lines from 50 different genetic constructs within a single laboratory. Plants recovered through this system have proven robust and efficacious for engineered traits under greenhouse conditions and within the first confined field trials of transgenic cassava carried out in Uganda, Kenya, Nigeria and Puerto Rico. Detailed procedures are described for the operation of this platform, including all steps in tissue culture, genetic transformation, copy number estimation, greenhouse establishment for shoot and storage root formation and systems for centralized quality control, transgenic plant tracking and regulatory compliance. In addition to providing reliable transgenic plant production for proof of concept studies in the variety 60444, the systems implemented and described here form the structure for high throughput production of transgenic farmer-preferred cultivars of cassava.     

Microsatellites in Cassava (Manihot esculenta Crantz): discovery, inheritance and variability

 Fourteen microsatellites containing GA-repeats were isolated and characterized in cassava (Manihot esculenta Crantz, Euphorbiaceae). Microsatellite heterozygosity (h) was estimated in 48 accessions using (³²P)-end-labeled primers and in more than 500 accessions using fluorescence-based genotyping. Heterozygosity values ranged from 0.00 to 0.88 and the number of alleles detected varied from 1 to 15. The reproducibility of allele sizing was also assessed using fluorescence-based genotyping. The average inter-gel size difference was 1.03 nucleotides. Chi-square tests (χ²) were performed to analyse segregation distortion and the linkage between alleles segregating from either or both parents in an F₁ mapping population. Most microsatellite loci segregated in the expected 1 : 1, 1 : 2 : 1 or 1 : 1 : 1 : 1 ratio. Linkage was detected between loci segregating from either parent, and segregation distortion from the male parent was detected for locus GA-131. Approximately 80% of the microsatellites detected one or two alleles per accession, suggesting a low degree of microsatellite locus duplication, an unexpected finding for a putative allopolyploid, highly heterozygous species. The high h values of most microsatellites, their amplification in other Manihot taxa and their suitability for high-throughput, fluorescence-based genotyping, make microsatellites the marker of choice for germplasm characterization and saturation of the cassava map. Received: 4 September 1997 / Accepted 16 March 1998     

Scopoletin Involvement in Post-Harvest Physiological Deterioration of Cassava Root (Manihot esculenta Crantz)

A study of the mechanism of the rapid deterioration of cassavaroots has shown that this requires the presence of oxygen andscopoletin, the latter acting, apparently, in some autocatalyticfashion. Roots from plants whose tops were pruned off some daysprior to harvest were less liable to spontaneous deterioration,but responded vigorously to the applied scopoletin, whereasroots stored for some time in the absence of O₂, so-called ‘cured’roots were no longer susceptible to exogenously-applied scopoletin.Clearly, metabolically, the two methods for prevention of deteriorationdiffer pruning may be effective due to internally-reduced scopoletinsupply or absence of some factor involved in the primary oxidation;‘curing’ may involve loss of a scopoletin precursoror inactivation of an enzyme system. Key words: Scopoletin, Manihot esculenta, Cassava, Post-harvest deterioration     

Observations on the Effect of Inoculating Cassava (Manihot esculenta) Plantlets with Fluorescent Pseudomonads

Some 136 isolates of fluorescent pseudomonads were isolated from the rhizosphere of plants growing in 5 different ecosystems. Thirty-four percent of these isolates inhibited the causal agent of cassava stem rot, Erwinia carotovora pv. carotovora, in vitro. One month old plantlets, produced by rooting the shoots of 4 cultivars in distilled water, were inoculated with a suspension (1.1 × 10⁹ cells/ml) of each pseudomonad. Some isolates increased root weight by 95% over uninoculated controls two months after planting when inoculated at planting, and 15, and 30 days afterwards. Inoculated plants were free from symptoms of root pathogens and roots filled earlier than controls. Taxonomic studies showed that these bacterial isolates, were either Pseudomonas putida (90%) or P. fluorescens (10%).     

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta)

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.     

Mining EST-Derived SSR Markers to Assess Genetic Diversity in Cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is a very important staple and industrial crop in tropical and subtropical regions of the world. The paucity of markers is a serious limitation in marker-assisted breeding. A total of 35,992 expressed sequence tags (ESTs) from cassava, which were clustered in 13,173 unigenes, were used in this study. A total of 1,889 microsatellites were identified, with an average density of one simple sequence repeats (SSRs) every 4.40 kb. Of the 1,058 designed EST-SSRs from cultivars SC06, TMS60444, and W14, 431 were polymorphic. Then, 31 randomly selected EST-SSRs from the 431 polymorphic EST-SSRs were used to evaluate the genetic diversity of 76 cassava accessions. A total of 93 alleles were identified, and the number detected for each EST-SSR ranged from one to four. Based on the 93 alleles, the 76 cassava accessions could be classified into six groups, and the genetic similarity coefficient ranged from 0.55 to 0.94. This study demonstrated the potential of EST-derived SSRs in cassava. The resources developed in this study enriched the available molecular markers for cassava.     

A Comparison of Two Approaches for Modelling Cassava (Manihot esculenta Crantz.) Crop Growth

Two approaches for modelling the growth and development of cassava,Manihot esculenta Crantz, are described and evaluated. The twomodels differ only in the hypotheses accounting for storageroot growth. In model 1, assimilate allocation to storage rootsis governed by the combined Chanter's (1976: Mathematical modelsin mushroom research and production. PhD Thesis, Universityof Sussex, UK) growth equation; and in model 2 the spill-overhypothesis for assimilate allocation to storage root governsstorage root growth. In both models, canopy photosynthesis generatesthe carbon substrate required for all growth processes. Thegrowth rates of leaves, stems and storage roots are definedby growth equations subject to substrate saturation kinetics.A key feature of both models is that the growth demands of thestem, fibrous roots and storage roots are related to leaf demandrates. Allocation to stems and branches was modelled by meansof a modified logistic growth equation which includes all theparameters and variables (number of nodes, internode lengths,stem density, stem modulus of elasticity and branch tensilestrength) that define the limits of the load bearing capacityof the shoot's supportive structures. The correlation coefficientsfor determination of yield prediction for the models werer =0.898,P =0.0385 (model 1) and r =0.954, P =0.0117 (model 2). For agrowth season of 290 d (after which leaf area index equals zeroand crop growth ceases), both models simulate the sigmoidaltransition from the lag to exponential phase of crop growth.Both models are equally well corroborated by observed data;however, model 1 has greater explanatory power. Copyright 2000Annals of Botany Company Allocation, simulation, model, crop growth, cassava, Manihot esculenta Crantz.