In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens     

Correction to: Field-transcriptome analyses reveal developmental transitions during flowering in cassava (Manihot esculenta Crantz)

Plant Molecular Biology -     

A molecular genetic map of cassava (Manihot esculenta Crantz)

 A genetic linkage map of cassava has been constructed with 132 RFLPs, 30 RAPDs, 3 microsatellites, and 3 isoenzyme markers segregating from the heterozygous female parent of an intraspecific cross. The F₁ cross was made between ‘TMS 30572’ and ‘CM 2177-2’, elite cassava cultivars from Nigeria and Colombia, respectively. The map consists of 20 linkage groups spanning 931.6 cM or an estimated 60% of the cassava genome. Average marker density is 1 per 7.9 cM. Since the mapping population is an F₁ cross between heterozygous parents, with unique alleles segregating from either parent, a second map was constructed from the segregation of 107 RFLPs, 50 RAPDs, 1 microsatellite, and 1 isoenzyme marker from the male parent. Comparison of intervals in the male-and female-derived maps, bounded by markers heterozygous in both parents, revealed significantly less meiotic recombination in the gametes of the female than in the male parent. Six pairs of duplicated loci were detected by low-copy genomic and cDNA sequences used as probes. Efforts are underway to saturate the cassava map with additional markers, to join the male- and female-derived maps, and to elucidate genome organization in cassava. Received: 5 July 1996/Accepted: 22 November 1996     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz)

Summary In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium.Abbreviations BM Basal Medium - MIE medium volume per initial embryo - E/IE number of Embryos per Initial Embryo     

Evidence for spontaneous polyploidization in cassava Manihot esculenta Crantz

Since high levels of genetic diversity may ensure long-term survival of a plant species, it is essential to preserve the genetic diversity of the species. Tipularia japonica and Epipactis papillosa are rare terrestrial orchids in southern Korea with fewer than 50 mature individuals in a population and southern Japan and considered to be threatened (endangered or vulunerable). To obtain knowledge of how the genetic variation of these species is partitioned within and among populations in Korea, I used enzyme electrophoresis to examine the genetic diversity of each eight known populations of the two species from South Korea. Twenty-three (E. papillosa) and 24 putative loci (T. japonica) resolved from 15 enzyme systems revealed no variation either within or among populations of each species (0.0% of the percentage of polymorphic loci, %P). Previous studies, in contrast, showed that their more widely distributed disjunct congeners T. discolor and E. helleborine harbored high allozyme-based genetic diversity within populations in eastern United States (%P = 75%) and in Denmark (%P = 73.6%), respectively. In theory, small population size leads to allelic fixation at many loci over generations within a population, resulting in population genetic divergence or differentiation. In this regard, the complete lack of genetic differences between conspecific populations of T. japonica and E. papillosa cannot be explained by genetic drift. Instead, the present allozyme data suggest that recent origin from the same genetically depauperate ancestral or source population could result in this observation. The current status of T. japonica and E. papillosa (rarity and lack of genetic variation) significantly threatens the long-term survival of the species in Korea.     

Bemnisia tabaci feeding induces pathogenesis-related proteins in cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Cranzts) plants fed upon by whitefly Bemisia tabaci showed increased levels of pathogenesis-related (PR) proteins, such as beta-1, 3-glucanase, peroxidase and chitinase activities, as compared to uninfested plants. The enzymes increased in specific activities from 2 to 7 fold and protein content in leaf extracts decreased in whitefly-infested plants, compared to uninfested plants. Among the three PR proteins, B. tabaci feeding induced significantly higher beta-1, 3-glucanase activities, when compared with other two PR proteins. Study also discussed the possible application of PR proteins in whitefly control program.     

Minor root rot pathogens of cassava (Manihot esculenta Crantz) in Nigeria

Many different species of fungi are often isolated from rotted cassava root tubers and pathogenicity studies have often implicated Botryodiplodia theobromae and Fusarium solani as the major causal pathogens. Consequently, more attention has often been focused on Botryodiplodia theobromae and Fusarium solani with little or no attention on the other minor pathogens. Considering the increasing importance of cassava to the Nigerian economy and the fact that minor root rot pathogens of cassava today could become major tomorrow, the aim of this research is to determine the incidence, pathogenicity and symptoms of the minor root rot pathogens in cassava from cassava fields within the derived savanna and the humid forest of Nigeria. Isolation of associated fungi was done on rotted root samples and the pathogenicity of these isolates were established by inoculating them into healthy cassava tuberous roots and subsequently reisolating them from resulting rotted tissue. The less frequently isolated fungi where Macrophomina sp., Trichoderma sp., Aspergillus niger, Aspergillus flavus, Sclerotium rolfsii and Fungus ‘A’ (a yet to be identified fungus). Repeated experiments confirmed a constant relationship between inoculated fungus and the resulting rotted tissue colour. The root rot tissue colours associated with inoculated pathogens in the laboratory were identical with the pathogens colony colour on potato dextrose agar.     

Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz)

Plant Molecular Biology - External application of ethanol enhances tolerance to high salinity, drought, and heat stress in various plant species. However, the effects of ethanol application on...     

Characterization of cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz)

Linamarase (EC 3.2.1.21) was purified from cassava petiole, stem, and root cortex by ammonium sulfate precipitation, column chromatography on Sepharose 6B, and chromatofocusing. The last step resolved the enzyme from each source into three forms with pI values of 4.3, 3.3, and 2.9. Each form was found to be oligomeric, consisting of one kind of subunit, Mr 63,000. The major isozyme with a pI of 4.3 from petiole showed a Km for linamarin of 0.6 mM and possessed both beta-glucosidase and beta-fucosidase activities. The former was sensitive to inhibition by delta-gluconolactone, isopropyl-beta-D-thioglucoside, and HgCl2, whereas the latter was inhibited by Tris ion.     

Response of cassava (Manihot esculenta Crantz) to water stress and fertilization

Experiments done in Santander de Quilichao (Cauca, Colombia) on two cassava cultivars indicated that cassava had at least three defence mechanisms against water deficit, enabling it to assimilate and store photosynthates in roots, even during prolonged droughts. These mechanisms include partial stomatal closure, ability of leaves to maintain reasonable net photosynthetic rate for long periods of water stress, reduced leaf area, and exploration of water from deep soil layers. While cassava responded positively to fertilization, no significant statistical differences were found between treatments of stress and non-stress, confirming cassava's ability to tolerate soil water deficit. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid     

Response of cassava (Manihot esculenta Crantz) to water stress and fertilization

Experiments done in Santander de Quilichao (Cauca, Colombia) on two cassava cultivars indicated that cassava had at least three defence mechanisms against water deficit, enabling it to assimilate and store photosynthates in roots, even during prolonged droughts. These mechanisms include partial stomatal closure, ability of leaves to maintain reasonable net photosynthetic rate for long periods of water stress, reduced leaf area, and exploration of water from deep soil layers. While cassava responded positively to fertilization, no significant statistical differences were found between treatments of stress and non-stress, confirming cassava's ability to tolerate soil water deficit.     

SSR and EST-SSR-based genetic linkage map of cassava (Manihot esculenta Crantz)

Simple sequence repeat (SSR) markers provide a powerful tool for genetic linkage map construction that can be applied for identification of quantitative trait loci (QTL). In this study, a total of 640 new SSR markers were developed from an enriched genomic DNA library of the cassava variety 'Huay Bong 60' and 1,500 novel expressed sequence tag-simple sequence repeat (EST-SSR) loci were developed from the Genbank database. To construct a genetic linkage map of cassava, a 100 F(1) line mapping population was developed from the cross Huay Bong 60 by 'Hanatee'. Polymorphism screening between the parental lines revealed that 199 SSRs and 168 EST-SSRs were identified as novel polymorphic markers. Combining with previously developed SSRs, we report a linkage map consisted of 510 markers encompassing 1,420.3?cM, distributed on 23 linkage groups with a mean distance between markers of 4.54?cM. Comparison analysis of the SSR order on the cassava linkage map and the cassava genome sequences allowed us to locate 284 scaffolds on the genetic map. Although the number of linkage groups reported here revealed that this F(1) genetic linkage map is not yet a saturated map, it encompassed around 88% of the cassava genome indicating that the map was almost complete. Therefore, sufficient markers now exist to encompass most of the genomes and efficiently map traits in cassava.     

In vitro cultured primary roots derived from stem segments of cassava (Manihot esculenta) can behave like storage organs

BACKGROUND AND AIMS: Cassava (Manihot esculenta) has three adventitious root types: primary and secondary fibrous roots, and storage roots. Different adventitious root types can also regenerate from in vitro cultured segments. The aim of this study was to investigate aspects of in vitro production of storage roots. METHODS: Morphological and anatomical analyses were performed to identify and differentiate each root type. Twenty-nine clones were assayed to determine the effect of genotype on the capacity to form storage roots in vitro. The effects of cytokinins and auxins on the formation of storage roots in vitro were also examined. KEY RESULTS: Primary roots formed in vitro and in vivo had similar tissue kinds; however, storage roots formed in vitro exhibited physiological specialization for storing starch. The only consistent diagnostic feature between secondary fibrous and storage roots was their functional differentiation. Anatomical analysis of the storage roots formed in vitro showed that radial expansion as a consequence of massive proliferation and enlargement of parenchymatous cells occurred in the middle cortex, but not from cambial activity as in roots formed in vivo. Cortical expansion could be related to dilatation growth favoured by hormone treatments. Starch deposition of storage roots formed in vitro was confined to cortical tissue and occurred earlier than in storage roots formed in vivo. Auxin and cytokinin supplementation were absolutely required for in vitro storage root regeneration; these roots were not able to develop secondary growth, but formed a tissue competent for starch storing. MS medium with 5 % sucrose plus 0.54 microM 1-naphthaleneacetic acid and 0.44 microM 6-benzylaminopurine was one of the most effective in stimulating the storage root formation. Genotypes differed significantly in their capacity to produce storage roots in vitro. Storage root formation was considerably affected by the segment's primary position and strongly influenced by hormone treatments. CONCLUSIONS: The storage root formation system reported here is a first approach to develop a tuberization model, and additional efforts are required to improve it. Although it was not possible to achieve root secondary growth, after this work it will be feasible to advance in some aspects of in vitro cassava tuberization.     

Inheritance of random amplified polymorphic DNA markers in cassava (Manihot esculenta Crantz)

The informativeness and inheritance of randomly amplified polymorphic DNA (RAPD) markers were investigated in an intraspecific F1 progeny derived from two heterozygous parents. The analysis confirmed the utility of RAPD markers for comparing candidate parents for the development of a molecular genetic map, and provided numerous markers for linkage analysis in a crop with a very limited history of classical or molecular genetic studies. Six potential parental lines (themselves F1 hybrid clones) showed between 1.82 and 0.62 segregating bands per primer in three hybrid families. Forty-three percent (309) of 722 primers produced polymorphic products in the most informative of these three crosses, revealing 328 single-dose (SD) markers segregating 1:1 for presence/absence in a progeny of 90 individuals. A second class of informative markers were those present in both parents but segregating in the progeny. Fifty-seven or 67% of the monomorphic but segregating markers exhibited the 3:1 ratio expected for SD dominant markers in a cross between heterozygotes. Linkage groups were constructed from the segregation of SD RAPD markers originating in the female (TMS 30572) and the male (CM2177-2) parent. Key words : RAPDs, molecular markers, genetic segregation, Manihot, single-dose markers.     

In vitro flowering of orchids

Abstract

Flowering is the most elusive and fascinating of all plant developmental processes. The ability to induce flowering in vitro in orchids would reduce the relatively long juvenile phase and provide deeper insight into the physiological, genetic and molecular aspects of flowering. This review synthesizes all available studies that have been conducted on in vitro flowering of orchids with the objective of providing valuable clues as to the mechanism(s) that is possibly taking place.     

Screening of cassava (Manihot esculenta Crantz) accessions against Phytophthora palmivora induced tuber rot of cassava

Cassava (Manihot esculenta Crantz), is an important tropical tuber crop with global importance and plays a significant role in the food, nutritional and livelihood security of around 500 million people. In India, the low productivity of cassava attributes to the soil borne disease, particularly tuber rot caused by Phytophthora palmivora (Butl.) which is destructive and the attack is spreading in alarming rate in all the cassava growing regions causing heavy yield loss of more than 50%. Introduction of disease resistant varieties may alleviate the problem to a certain extent. This paper describes the screening procedures and findings on the disease resistant variety of cassava accession against tuber rot. Variety Sree Padmanabha imparted high resistance against tuber rot, while Sree Sahya was moderately resistant and all other accessions studied were found to be susceptible in in vitro and in field trials. In screening studies, a reproducible positive correlation was obtained between attached tubers in live plant with detached tubers which showed that detached tuber part can be used for the prediction of resistance in attached live plants of cassava for cultivar resistance. The procedure described here could be used as a simple, rapid and efficient method for screening of cassava accessions against tuber rot of cassava.     

C3-C4 intermediate photosynthetic characteristics of cassava (Manihot esculenta Crantz)

Cassava, bean and maize leaves were fed with¹⁴CO₂ in light and the primary products of photosynthesis identified 5 and 10 seconds after assimilation. In maize, approximately three quarters of the labelled carbon was incorporated in C₄ acids, in beans about two thirds in PGA, and in cassava approximately 40–60% in C₄ acids with 30–50% in PGA. These data indicate that cassava possesses the C₄ photosynthetic cycle, however due to the lack of typical Kranz anatomy appreciable carbon assimilation takes place directly through the Calvin-Benson-Bassham cycle.