In vitro multiplication of cassava varieties / Multiplicação in vitro de variedades de mandioca / Multiplicación in vitro de variedades de mandioca

The implantation of a model of sustainable development for agriculture can happen with the contribution of the mandiocultura. But for this, culture needs to be strengthened. In vitro propagation is an instrument for this purpose. Micropropagation can provide growers with large quantities of vigorous and healthy cassava seedlings in a short time. The objective of this study was to evaluate the in vitro establishment of four varieties of cassava cultivated in the municipality of Colorado do Oeste, State of Rondônia, popularly known as Arara, Caturra, Cacau Vermelha and Roxinha. For that, an experiment was carried out in the Laboratory of In Vitro Cultivation at the Pole of Technological Innovation of the University of Cruz Alta (UNICRUZ), with a completely randomized design in a 4 x 2 factorial scheme, with 6 replications. The treatments consisted of explants grown in Murashige and Skoog (MS) medium without the presence of growth regulator and MS medium supplemented with of 6-benzylaminopurine (BAP). The results indicate that the mean contamination percentage of the explants was 47.19%, differing among the varieties. The best growth response in culture media, in the multiple comparison of means (Scott-Knott's test, 5%), was obtained with MS medium without BAP addition, with significant difference between varieties. Under the conditions of this experiment, it was evidenced that micropropagation is a viable tool for obtaining varieties of interest, with desired phytosanitary qualities, with varietal and large-scale authenticity.(AU)

A implantação de um modelo de desenvolvimento sustentável para a agricultura pode acontecer com a contribuição da mandiocultura. Mas para isso, a cultura precisa ser fortalecida. A propagação in vitro é um instrumento para este fim. A micropropagação pode proporcionar aos produtores grande quantidade de mudas de mandioca vigorosas e sadias em um curto espaço de tempo. O objetivo deste trabalho foi avaliar o estabelecimento in vitro de quatro variedades de mandioca cultivadas no município de Colorado do Oeste, Rondônia, popularmente conhecidas como Arara, Caturra, Cacau Vermelha e Roxinha. Para isso, foi realizado um experimento no Laboratório de Cultivo In Vitro no Polo de Inovação Tecnológica da Universidade de Cruz Alta (UNICRUZ), com delineamento inteiramente casualizado em esquema fatorial 4 x 2, com 6 repetições. Os tratamentos consistiram em explantes cultivados em meio Murashige e Skoog (MS) sem a presença de regulador de crescimento e meio MS suplementado com 6-benzilaminopurina (BAP). Os resultados indicam que a porcentagem média de contaminação dos explantes foi de 47,19%, diferindo entre as variedades. A melhor resposta de crescimento em meios de cultura, na comparação múltipla de médias (teste de Scott-Knott, 5%), foi obtida com meio MS sem adição de BAP, com diferença significativa entre as variedades. Nas condições deste experimento, ficou evidenciado que a micropropagação é uma ferramenta viável para obtenção de variedades de interesse, com qualidades fitossanitárias desejadas, com autenticidade varietal e em larga escala.(AU)

La implantación de un modelo de desarrollo sostenible para la agricultura puede suceder con el aporte de la mandiocultura. Pero, para eso, la cultura necesita ser fortalecida. La propagación in vitro es un instrumento para ese fin. La micropropagación puede proporcionar a los cultivadores grandes cantidades de plántulas de mandioca vigorosas y saludables en poco tiempo. El objetivo de esta investigación ha sido evaluar el establecimiento in vitro de cuatro variedades de mandiocas cultivadas en el municipio de Colorado del Oeste, Rondônia, popularmente conocidas como Arara, Caturra, Cacau Vermelha y Roxinha. Para eso, se realizó un experimento en el Laboratorio de cultivo in vitro en el Polo de Innovación Tecnológica de la Universidad de Cruz Alta (UNICRUZ), con delineamiento completamente casualizado en esquema factorial 4 x 2, con 6 repeticiones. Los tratamientos consistieron en explantes cultivados en medio Murashige y Skoog (MS) sin la presencia de regulador de crecimiento y medio MS suplementado con 6-bencilaminopurina (BAP). Los resultados indican que el porcentaje de contaminación promedio de los explantes fue de 47.19%, diferenciándose entre las variedades. La mejor respuesta de crecimiento en medios de cultivo, en la comparación múltiple de medias (prueba de Scott-Knott, 5%), se obtuvo con medio MS sin adición de BAP, con una diferencia significativa entre las variedades. Bajo las condiciones de este experimento, se evidenció que la micropropagación es una herramienta viable para obtener variedades de interés, con cualidades fitosanitarias deseadas, con autenticidad varietal y de gran escala.(AU)

Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate.

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava.

Cytogenetic and anatomic behavior of cytochimeras and total polyploids in cassava.

Cassava periclinal cytochimeras, cultivars, and interspecific hybrid and polyploid types were studied in relation to embryonic, cytogenetic, and anatomical behavior. Their apical shoots, pollen grains, male and female buds, roots, stomata, and flowering period were analyzed. Chimeras exhibited increased size of L1 and L2 cells. Polyploidy led to enlargement of stomata in chimeras whereas L2 gave tetraploid chromosome configurations, tetrad irregularity, decrease of pollen viability, and increase in frequency of polyembryo sacs. The chimeric composition of tetraploids L1 and L2 and diploid L3 expressed a notable epigenetic effect seen in a marked enlargement of edible roots compared to total diploid. One of the chimeric types was accompanied by complete flowering inhibition. Pollen viability and diameter appeared to be reliable markers to determine ploidy levels.

Empowering biotechnology in southern Africa: establishment of a robust transformation platform for the production of transgenic industry-preferred cassava.

Knowledge and technology transfer to African laboratories and farmers is an important objective for achieving food security and sustainable crop production on the sub-Saharan African continent. Cassava (Manihot esculenta Crantz) is a vital source of calories for more than a billion people in developing countries, and its potential industrial use for starch and bioethanol in the tropics is increasingly being recognized. However, cassava production remains constrained by the susceptibility of the crop to several biotic and abiotic stresses. For more than a decade, biotechnology has been considered an attractive tool to improve cassava as it substantially circumvents the limitations of traditional breeding, which is particularly time-consuming and tedious because of the high heterozygosity of the crop. A major constraint to the development of biotechnological approaches for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite some success achieved in genetic modification of the model cassava cultivar Tropical Manihot Series (TMS), TMS 60444, in some European and U.S. laboratories, the lack of a reproducible and robust protocol has not allowed the establishment of a routine transformation system in sub-Saharan Africa. In this study, we optimized a robust and efficient protocol developed at ETH Zurich to successfully establish transformation of a commercially cultivated South African landrace, T200, and compared this with the benchmark model cultivar TMS 60444. Results from our study demonstrated high transformation rates for both T200 (23 transgenic lines from 100 friable embryogenic callus (FEC) clusters) compared with TMS 60444 (32 transgenic lines from 100 FEC clusters). The success in transforming landraces or farmer-preferred cultivars has been limited, and the high transformation rate of an industry-preferred landrace in this study is encouraging for a feasible transformation program for cassava improvement in South Africa (SA), which can potentially be extended to other countries in southern Africa. The successful establishment of a robust cassava transformation and regeneration system in SA demonstrates the relevance of technology transfer to sub-Saharan Africa and highlights the importance of developing suitable and reliable techniques before their transfer to laboratories offering less optimal conditions.

Evaluation of cassava plants generated by somatic embryogenesis at different stages of development using molecular markers

Background: Cassava (Manihot esculenta Crantz) is a crop that is high in carbohydrates in the roots and in protein in the leaves, important for both human consumption and animal feed, and also has a significant industrial use for its starches. In this study we evaluated the genetic variability with molecular markers in different stages in micropropagated plants from somatic embryos of Venezuelan native clone 56. Results: Three markers were used: ISTR, AFLP and SSR, finding that ISTR showed the highest polymorphism among individuals tested. With AFLP a high similarity between the evaluated individuals was observed and with SSR total monomorphism was seen. Using cluster analysis it was found that individuals from an embryo labeled as fasciated at the beginning of the somatic embryogenesis process were grouped as independent of the other plants when analyzed at the acclimatization stage. The differences found with the different markers used are discussed. In field trials, micropropagated plants had a yield between 4 and 5 times the average yield of cassava in Venezuela. Conclusion: Despite variability in terms of DNA markers, somatic embryogenesis is suitable for mass propagation of highly performing cassava clones.

Efficient transformation and regeneration of transgenic cassava using the neomycin phosphotransferase gene as aminoglycoside resistance marker gene.

Cassava is one of the most important crops in the tropics. Its industrial use for starch and biofuel production is also increasing its importance for agricultural production in tropical countries. In the last decade cassava biotechnology has emerged as a valuable alternative to the breeding constraints of this highly heterozygous crop for improved trait development of cassava germplasm. Cassava transformation remains difficult and time-consuming because of limitations in selecting transgenic tissues and regeneration of transgenic plantlets. We have recently reported an efficient and robust cassava transformation protocol using the hygromycin phosphotransferase II (hptII) gene as selection marker and the aminoglycoside hygromycin at optimal concentrations to maximize the regeneration of transgenic plantlets. In the present work, we expanded the transformation protocol to the use of the neomycin phosphotransferase II (nptII) gene as selection marker. Several aminoglycosides compatible with the use of nptII were tested and optimal concentrations for cassava transformation were determined. Given its efficiency equivalent to hptII as selection marker with the described protocol, the use of nptII opens new possibilities to engineer transgenic cassava lines with multiple T-DNA insertions and to produce transgenic cassava with a resistance marker gene that is already deregulated in several commercial transgenic crops.

Apomixis in different ploidy levels of cassava.

Two polyploid hybrids between cassava (Manihot esculenta) cultivar 307-2 and its wild relatives M. glaziovii and M. anomala, were studied to examine the relationship between ploidy level and the production of seeds without fertilization. A clearing method was applied to assess ovule sizes as an indication of multiembryony. The diploid cultivar 307-2 had regular 18 bivalents at meiotic metaphase 1 while the polyploid types showed chromosome configurations varying from 3 to 4 quadrivalents and 28 to 30 bivalents. A total of 14% of studied ovules of the polyploid hybrid involving M. glaziovii were multiebryonic, while the percentage of multiembryony was as low as 2% in the polyploid hybrid M. anomala×M. esculenta. Diploid hybrid types did not show any multi embryony. Adventitious embryos were found and documented for the first time in polyploid hybrids M. esculenta×M. glaziovii. The association of multiple embryo formation with ovary size and pollination showed that apomictic embryos form independently from fertilization. Simple iodized carmine stain for measuring pollen viability proved as efficient as the sophisticated Alexander method.

Genetic, embryonic and anatomical study of an interspecific cassava hybrid.

A molecular, anatomical and cytogenetic study of an interspecific hybrid between Manihot esculenta (cassava) and the wild species M. oligantha was carried out. Cytogenetics revealed relatively complete chromosome pairing and high viability of the pollen grains. Ovule structure examined by the clearing method showed polyembryony in 2.7% of the ovules. Doubling of the chromosome number resulted in an increase in polyembryony of up to 18% and a reduction in pollen viability. Multivalent formation was also observed. An anatomical study of stems of diploid and tetraploid hybrids showed a larger number of vascular bundles in the tetraploid type.

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

CaCl2 enhanced somatic embryogenesis in Manihot esculenta Crantz.

Primary cassava somatic embryos were induced on a medium without CaCl(2), however, no or only a few secondary somatic embryos were formed from them. With 15 mM CaCl(2) in the medium for induction of cassava primary embryos, more secondary somatic embryos were produced from them, and they were much effective in maintaining their embryogenic capacity than the controls of embryos which were induced without CaCl(2).

Molecular analysis of apomixis in cassava.

Cassava is the main staple for more than 800 million people in the tropics. It is propagated vegetatively by stem cuttings, which maintains superior genotypes but favors disease accumulation and spread. In this report, we present the results of the screening of the progeny and the second generation of the clone UnB 307 for apomixes using microsatellites. A total of 29 plants were screened, representing the maternal plant, its first and second generations that were left to open pollination. About 20% of the offspring were rated as genetically identical plants. This result confirms the facultative apomictic nature of cassava, with high environmental effect.

Indirect somatic embryogenesis in cassava for genetic modification purposes.

In cassava both direct and indirect somatic embryogenesis is described. Direct somatic embryogenesis starts with the culture of leaf explants on Murashige and Skoog (MS) medium supplemented with auxins. Somatic embryos undergo secondary somatic embryogenesis when cultured on the same medium. Indirect somatic embryogenesis is initiated by subculture of directly induced embryogenic tissue on auxin-supplemented medium with Gresshoff and Doy salts and vitamins. A very fine friable embryogenic callus (FEC) is formed after a few rounds of subculture and stringent selection. This FEC is maintained by subculture on auxin supplemented medium. Lowering of the auxin concentration allows the FEC to form mature somatic embryos that develop into plants when transferred to a cytokinin-supplemented medium.

Chromosome doubling induces apomixis in a cassava x Manihot anomala hybrid.

A new species was synthesized artificially by chromosome doubling of an interspecific hybrid between cassava and Manihot anomala. The ensuing polyploid type exhibits an apomictic nature and maintains its morphological characteristics in the progeny. It showed a 29% frequency of multiembryonic sacs in its examined ovules whereas the multiembryonic sacs were absent in the diploid type.

Molecular analysis of apomixis in cassava

Cassava is the main staple for more than 800 million people in the tropics. It is propagated vegetatively by stem cuttings, which maintains superior genotypes but favors disease accumulation and spread. In this report, we present the results of the screening of the progeny and the second generation of the clone UnB 307 for apomixes using microsatellites. A total of 29 plants were screened, representing the maternal plant, its first and second generations that were left to open pollination. About 20% of the offspring were rated as genetically identical plants. This result confirms the facultative apomictic nature of cassava, with high environmental effect.

Regeneration of transgenic cassava from transformed embryogenic tissues.

Production of transgenic plants is gradually becoming routine in cassava biotechnology. Green cotyledons of maturing somatic embryos (somatic cotyledons for short) and friable embryogenic suspensions (FES) are the target tissues for transformation by Agrobacterium or biolistics. Putative transgenic shoots develop from transformed somatic cotyledons via shoot organogenesis or from FES via somatic embryogenesis under selection. Maturation of transgenic somatic embryos is induced by transfer to maturation medium with reduced concentrations of selective agents. Mature somatic embryos can also develop directly from FES cells under selection. Transgenic plants are regenerated by the elongation of transgenic shootlets from organogenesis experiments and by the germination of or shoot development from transgenic mature embryos cultured without selection. beta-Glucuronidase (GUS) assays and rooting tests can be used to screen for escapes from selection, which improves the regeneration rate of truly transgenic plants.

Breeding cassava for apomixis.

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.

Breeding cassava for apomixis

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.

Cryopreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation.

A simplified technique which simultaneously induces and cryoprotects embryogenic calli using sucrose followed by dehydration was developed for the cryopreservation of cassava genetic resources. An initial experiment to optimise the sucrose concentration needed for both embryo production and cryoprotection showed that higher concentrations of sucrose-between 0.4 M and 0.5 M-significantly reduced the viability as well as the number of embryos produced by the embryogenic clumps in the absence of freezing. Post-thaw viability as well as embryogenic competence of clumps depended on the percentage moisture lost, duration of exposure to higher sucrose concentrations and the duration of induction of embryogenic clumps. Extending the period of cryoprotection to 21 days coupled with increased moisture loss (greater than 75%) significantly increased both post-thaw viability and the embryogenic competence of cryopreserved clumps to 95%, while reducing the duration decreased post-thaw viability. Cryopreserved callus clumps developed secondary and cyclic embryos similar to those of the non-cryopreserved controls. The optimised protocol was successfully applied to SM1-2075-1 Line 1 somatic embryos. The rate of plant recovery from cryopreserved embryos of both TME 9 and SM1-2075-1 Line 1 was comparable to that of the non-cryopreserved embryos. Successful cryopreservation of embryogenic clumps of cassava can be used to establish in vitro genebanks for long-term conservation of cassava genetic resources to complement field genebanks and other in vitro methods already being used.

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz).

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.

Apomixis and cassava

Apomixis means seed formation without fertilization. In cassava (Manihot esculenta) it is an alternative to reproduction by cuttings, which normally transmits pathogens and leads to an accumulation of viral and bacterial diseases. Apomixis also assures preservation of heterosis and avoids genetic segregation. It occurs in wild relatives of cassava and has been transferred successfully from Manihot glaziovii and M. neusana. It is facultative, and occurs at a low frequency, ranging from 1-2, and apparently is genetically different from apomixis in other crops. With selection, the frequency can reach 13. Apomixis in cassava is frequently associated with aneuploidy but it does occur in some diploid types. It is due to the formation of aposporic sacs, which can easily be detected by clearing tissue preparations. Apomixis appears to have played an important role in speciation during the evolution of Manihot, since it leads to the maintenance and perpetuation of sterile interspecific hybridization. The use of apomixis in cassava breeding could lead to a boom in line improvement and commercial production. In addition to preserving superior genotypes, avoiding contamination of new plants, it would enable international programs to export their germplasm to destination countries. This would allow the use of superior genotypes even if apomixis occurs at a low frequency. A scheme to maximize benefits is to use diploid apomictic clones as maternal parents, which can be crossed with pollinators of polyploid interspecific hybrids, followed by selection among the progeny of new apomictic types that combine the heteroses of both interspecific hybridization and polyploidy. In addition, they acquire favored genes that have been transferred from the wild to the commercial crop