From crossbreeding to biotechnology-facilitated improvement of banana and plantain

Biotechnology Advances 32 (2014) 158–169

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

From crossbreeding to biotechnology-facilitated improvement of banana and plantain Rodomiro Ortiz a,⁎, Rony Swennen b a b

Swedish University of Agricultural Sciences (SLU), Department of Plant Breeding, Sundsvagen 14, Box 101, 23053 Alnarp, Sweden Katholieke Universiteit Leuven – International Institute of Tropical Agriculture (IITA) – Bioversity International, Willem de Croylaan 42, Box 2455, 3001 Heverlee, Belgium

a r t i c l e

i n f o

Available online 1 October 2013 Keywords: Diversity Genebanks Genomics DNA marker-aided breeding Musa Ploidy Proteomics Sequencing Transcriptomics Transgenics

a b s t r a c t The annual harvest of banana and plantain (Musa spp.) is approximately 145 million tons worldwide. About 85% of this global production comes from small plots and kitchen or backyard gardens from the developing world, and only 15% goes to the export trade. Musa acuminata and Musa balbisiana are the ancestors of several hundreds of parthenocarpic Musa diploid and polyploid cultivars, which show multiple origins through inter- and intra-specific hybridizations from these two wild diploid species. Generating hybrids combining host plant resistance to pathogens and pests, short growth cycles and height, high fruit yield, parthenocarpy, and desired quality from the cultivars remains a challenge for Musa crossbreeding, which started about one century ago in Trinidad. The success of Musa crossbreeding depends on the production of true hybrid seeds in a crop known for its high levels of female sterility, particularly among polyploid cultivars. All banana export cultivars grown today are, however, selections from somatic mutants of the group Cavendish and have a very narrow genetic base, while smallholders in subSaharan Africa, tropical Asia and Latin America use some bred-hybrids (mostly cooking types). Musa improvement goals need to shift to address emerging threats because of the changing climate. Innovative cell and molecular biology tools have the potential to enhance the pace and efficiency of genetic improvement in Musa. Micropropagation has been successful for high throughput of clean planting materials while in vitro seed germination assists in obtaining seedlings after inter-specific and across ploidy hybridization. Flow cytometry protocols are used for checking ploidy among genebank accessions and breeding materials. DNA markers, the genetic maps based on them, and the recent sequencing of the banana genome offer means for gaining more insights in the genetics of the crops and to identifying genes that could lead to accelerating Musa betterment. Likewise, DNA fingerprinting has been useful to characterize Musa diversity. Genetic engineering provides a complementary tool to Musa breeders who can introduce today transgenes that may confer resistance to bacteria, fungi and nematodes, or enhance pro-vitamin A fruit content. In spite of recent advances, the genetic improvement of Musa depends on a few crossbreeding programs (based in Brazil, Cameroon, Côte d'Ivoire, Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda) or a handful of genetic engineering endeavors (Australia, Belgium, India, Kenya, Malaysia and Uganda). Development investors (namely international aid and philanthropy) should therefore increase their funding to genetically enhance this crop that ranks among the 10-top staple foods of the developing world. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Inter-disciplinary research shed light on the origins of our favorite fruit Genetic resources characterization . . . . . . . . . . . . . . . . . Crossbreeding and (quantitative) genetics . . . . . . . . . . . . . . Host plant resistance through crossbreeding . . . . . . . . . . . . . Fruit quality, human nutrition and health . . . . . . . . . . . . . . Tissue culture: micro-propagation of clean planting materials . . . . . Molecular cytogenetics and cytometry . . . . . . . . . . . . . . . DNA marker-facilitated diversity, origin and relatedness assessments . Genetic maps and marker-aided breeding . . . . . . . . . . . . . . Mutations and “tilling” . . . . . . . . . . . . . . . . . . . . . . The genome sequencing provides new insights and DNA markers . . .

⁎ Corresponding author. E-mail address: [email protected] (R. Ortiz). 0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biotechadv.2013.09.010

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13. Other genetic and omics-based resources . . . . . . . . . . . . . . . . . 14. Genetic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . 15. Outlook: genetic enhancement to meet global demand in a changing climate References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The giant, perennial, herbaceous bananas and plantains (Musa spp.) were grown in 10.6 million ha in 2011 with an average fruit yield of 13.6 t ha−1 (FAO, 2013). The crop includes about 1000 dessert, cooking and beer cultivars derived after intra- or inter-specific hybridization of the wild diploid (2n = 2x = 22 chromosomes) ancestor species Musa acuminata (A genome) and Musa balbisiana (B genome) (Ortiz, 2011). The cultivars (mostly triploids or 2n = 3x = 33) show fruit parthenocarpy and often sterility, which makes challenging the crossbreeding of this crop (Ortiz, 2013). The crop ranks among the 10 most important staples and feeds million of people worldwide, but there are not even 10 active Musa crossbreeding programs, namely in Brazil, Cameroon, Côte d'Ivoire, Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda. The oldest breeding program in operation is that of the Fundación Hondureña de Investigación Agrícola (FHIA, Honduras), whose initial focus was on dessert bananas, and later on included cooking bananas and plantains. After almost a century of Musa crossbreeding, smallholders only grow, however, a few bred-cultivars of banana and plantain in the developing world (especially for local markets and almost nil for export trade), while farmers selected most of today's cultivars and their somatic mutations. Genebanks in the tropical world include Musa wild species and cultivars, but Bioversity International Transit Center (ITC) at the Katholieke Universiteit Leuven (Belgium) holds the world's largest in vitro repository of Musa diversity. There are very vibrant biotechnology-facilitated research undertakings with potential impacts in genetic enhancement through tissue culture, omics and transgenic methods, particularly in Australia, Belgium, Brazil, China, France, India, Kenya, Malaysia, Nigeria, South Africa, Uganda, United Kingdom and USA. Without doubt the releases – after several years of painstaking ploidy manipulations and field trials – of new secondary triploid matooke banana hybrids (Musa, AAA) in Uganda (Lorenzen, 2012), the field-testing of transgenic East African highland banana with host plant resistance to Xanthomonas wilt (Tripathi et al., 2010), and the sequencing of the M. acuminata genome (D'Hont et al., 2012) are among the most significant achievements in recent years. They ensued from long-term research-for-development partnerships involving national and international institutes plus academics, and with the aim of meeting the needs of banana and plantain farming in the tropics. Two recent books (Pillay and Tenkouano, 2011; Pillay et al., 2012) and comprehensive overviews (Ortiz, 2011, 2013) about Musa genetic resources, breeding, genetics, genetic engineering and omics give details of some of their advances, bottlenecks and remaining challenges. There was also, at the end of the last decade, a special journal issue that included archeobotany, genetics, linguistics and phytogeography articles unraveling partially Musa domestication (De Langhe et al., 2009). This review article updates such reports mostly summarizing research progress of the last four years, thereby giving the state of the art on Musa genetic enhancement through crossbreeding and using various biotech-facilitated methods.

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diversity of banana and plantain. A further article by Perrier et al. (2011) provides insights onto the complex geo-domestication pathways that generated the various Musa cultivars. Molecular phylogenetics (using microsatellites and diversity array technology — DArT) facilitated identifying putative wild ancestors of modern cultivars and the key domestication steps for each of the most important cultivar groups, while archeology and linguistics shed light on the human spread of Musa and its farming from Papua New Guinea to West Africa during the Holocene. Kennedy (2009) also suggests considering miscellaneous uses of Musa plants (beyond the edible fruits) to account the spreading of this species. For example, Iles (2009) found impressions of banana pseudostems in an iron slag from Eastern Africa, which could prove its use in iron-producing technology. Likewise, the rare Musa seeds can be used in archeology (De Langhe, 2009) or an alternative source for DNA fingerprinting of its crop wild relatives (Uma et al., 2011b). Linguistic evidence can fill gaps of archeo-botanical records, thereby allowing mapping and dating crop dispersal. The names used for banana within island Southeast Asia and Melanesia suggest a westward dispersal of the crop from New Guinea, mixing with a Philippine cultivar, then westward again to mainland Southeast Asia, and onward to South Asia's west (Donohue and Denham, 2009). Linguistic research also indicates that plantains were likely introduced to West and Central Africa through an unknown route 2500 to 3000 BP (Blench, 2009; Mbida Mindzie et al. 2001, 2004, 2005), becoming thereafter an important crop in Central African rainforest and supporting the further Bantu expansion in the continent (Neumann and Hildebrand, 2009). Phytoliths are rigid, microscopic plant silica bodies whose shapes and sizes are used in paleo-ecology, archeology and food science. They gave evidence supporting New Guinea's key role in the domestication of Eumusa (recently renamed as Musa) and Australimusa bananas (Lentfer, 2009b). Banana seeds also have diagnostic phytoliths that discriminate between these two main sections, the giant Musa ingens and Ensete and can be further used for tracing domestication. Vrydaghs et al. (2009) were able to link banana phytolith diversity with M. acuminata phylogeny (including edible diploid and triploid derivatives). They further indicated that extra samples would be necessary for understanding the extent of variation and identifying discriminating traits. A systematic recording of phytolith data could assist filling gaps of banana past distribution in Asia, particularly China and India — which seem to have contributed little to early domestication of the crop (Fuller and Madella, 2009). Lentfer (2009a) also shows that well-preserved starch granules can be used for tracking the domestication and dispersal of Musa and related species. Buerkert et al. (2009) found a triploid banana cultivar surviving in a limestone rock niche in Oman. This hyperarid country has a narrow diversity of banana, which grows only in northern oases in the monsoondominated southeastern tip of the Sultanate. This finding gives evidence of how some locations in the Arabian Peninsula could provide a refuge to banana cultivars brought from humid regions, e.g. coastal East Africa and nearby islands or Indonesia.

3. Genetic resources characterization 2. Inter-disciplinary research shed light on the origins of our favorite fruit Perrier et al. (2009) advocate a multi-disciplinary approach to elucidate the process of Musa domestication and deciphering the

The endowments of Musa diversity available in genebanks serve as sources for banana and plantain crossbreeding. They can also provide means for exploring other uses of Musa plants, e.g. ornamental bananas (Häkkinen, 2005; Santos-Serejo et al., 2012). Characterizing Musa

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genetic resources will assist to determine the extent of genetic divergence across species and cultivar groups, defining the best germplasm grouping, assigning accessions to previously agreed diversity clusters, eliminating synonyms in genebanks, identifying useful sources of variation for further genetic enhancement, managing intellectual property and marketing (Dheda Djailo et al., 2011; Hasan et al., 2011; Nunes de Jesus et al., 2009; Saraswathi et al., 2011; Yan et al. 2011). Channelière et al. (2011) claim that standardizing protocols and descriptors for characterizing Musa diversity will facilitate its use and assessment across environments, clustering accessions, and developing a meta-core subset. For example, using planting date data from various experiments in the tropics and subtropics, Fortescue et al. (2011) were able to determine the contributions of photoperiod and soil water balance to time of flowering. This research also gave evidence that banana has a facultative long-day response to photoperiod. A standard descriptor list should include reliable morphological traits and DNA markers capable of describing the diversity of the crop and its wild relatives. In this regard, Onyango et al. (2011) indicated that male bud, fruit and sucker traits were useful to distinguish within genome groups and subgroups, and to isolate various subgroups within genome group when assessing diversity of bananas grown in East Africa. Likewise, Karamura et al. (2011) found that East African farmers use size and shape, texture, appearance, agronomic and commercial aspects to describe and name their banana cultivars, thereby highlighting the need of trait complementarity for assessing banana diversity. Variable plant and inflorescence descriptors can be also used for cultivar registration as noted by Nunes de Jesus et al. (2009). 4. Crossbreeding and (quantitative) genetics Lorenzen et al. (2012) report advances in introgressing host plant resistance to various pathogens and pests affecting the crop, particularly in Africa. They acknowledge, however, that more efforts are needed for producing high-yielding cultivars showing multiple host plant resistance and same fruit quality as local cultivars. To succeed in this endeavor, multi-environment trials (across locations and several crop cycles) are needed to assess the performance of selected germplasm prior to their release as new cultivars. Incidentally, Tenkouano et al. (2012b) were able to establish the relationship between sample size, repeatability and level of confidence for phenotypic discrimination of Musa breeding materials. This information will assist the planning of Musa multienvironment trials. The genetic base of triploid banana and plantain cultivars seems to be very narrow because they likely ensued from 20 to 25 meiosis events (and just 1 or 2 in Africa), which makes them very fragile in spite of the available diversity in Musa wild species and diploid cultivars (MusaNet, 2012). In this regard, Nyine and Pillay (2011) demonstrated that banana breeding schemes relying on crossing wild diploids with genetically uniform landraces increased the genetic diversity of East African highland bananas. Broadening the genetic base of banana and plantain can be achieved by recurrent selection including crop wild relatives and diploid cultivars in the source population (C0) and further use as parents of derived materials from subsequent cycles of selection(C1 onwards) in crossbreeding schemes. Recurrent selection may consider progeny testing to improve diploid populations and generate elite diploid hybrids to be used as parents in 3x–2x or 4x–2x crossing schemes. The use of DNA markers coupled with field trial data and sound biometrics could assist to estimate the diversity in diploid breeding stocks (Martins Pereira et al., 2012) or induced mutants (Pestana et al., 2011). This approach also provides means to selecting parents for the genetic enhancement of banana and plantain. Diploid breeding remains an important activity in some of the programs engaged in Musa genetic enhancement operating today in the world (Amorim et al., 2013; Ortiz, 2013; Uma et al., 2011a). Tools such as marker-aided breeding, doubled-haploids, genomic selection

(including the use of genotyping-by-sequencing or next generation sequencing) may further accelerate population improvement at the diploid level. Intermediate diploid-breeding sources may enhance genetic gains in the banana and plantain cultigen pools when including them in various 4x–2x reciprocal recurrent selection schemes aiming the release of advanced triploid hybrids. Their impact will be measured in terms of diversity of diploid sets of elite parents with required target traits as defined by the end users. Chromosome doubling (CD) of diploid species or selected stocks (e.g. putative ancestral 2x) and the use of their CD-derivative(s) to produce triploids following 4x–2x crossings can be another breeding approach for incorporating diversity in the cultigen breeding pool (Goigoux et al., 2013; Tomepke and Sadon, 2013). Moreover, Jenny et al. (2013) indicated that fertility could be achieved in 4x-CD derived from treating their AB sterile ancestors with colchicine. They further used a resynthesized AABB hybrid (named ‘Kunnan’) in crossing schemes with AA and BB parents to produce AAB and ABB secondary triploids, respectively. The use of “brute force” can facilitate the crossbreeding of the almost female sterile ‘Cavendish’ cultivar – the most important banana for the export trade today – as noticed before when hand pollinating a few plants and getting seeds that germinated in Honduras and Nigeria. For example, Aguilar Morán (2013) indicated that he got 200 hybrid seeds after mass-pollinating 20,000 ‘Cavendish’ bunches (consisting of about 2 million fruits) with pollen from diploid parents. Only 40 of them had, however, viable embryos, which produced 20 tetraploid hybrids. These hybrids were further used in 4x–2x crossing schemes for producing two promising secondary triploid hybrids due to their host plant resistance to black Sigatoka and Fusarium wilt race 1 and with same field performance as the ‘Cavendish’ “grandmother”. Oselebe et al. (2010) noticed that 3x hybrid offspring are predominantly produced in 4x–2x crossings while the reciprocal (2x–4x) yields 2x hybrid offspring, thereby suggesting an unequal contribution of the parents to their offspring in the former scheme and double-reduction in the 4x parent for producing n gametes in the latter. Oselebe and Tenkouano (2009) indicated previously that 2x offspring from 2x–4x crossings had short plants, showed early flowering and produced small bunches. Hence, Oselebe et al. (2010) further redefined models for predicting hybrid performance after interploidy crosses in Musa. Tenkouano et al. (2012a) provide formulae that describe the relationships between parents and their derived offspring for various interploidy crossing schemes. This information can be also used to estimate narrowsense heritability for quantitative traits in Musa. Additive genetic effects are significant on the expression of bunch weight, fruit filling time, fruit length, plant height and leaf number while non-additive effects account for suckering behavior and fruit circumference in secondary 3x hybrids derived from 4x–2x inter-mating (Tenkouano et al., 2012c). Maternal general combining ability (GCA) explains most of the variation in plant height and leaf number, thereby suggesting selection for both traits on the 4x female parent previous to their use in crossing blocks. Paternal GCA was the main contributor to variation in fruit filling time, bunch weight, and fruit length, thereby confirming that these traits could be improved in diploid parents through recurrent selection. Specific combining ability (SCA) effects were significant for many traits, except fruit filling time. Hence, additional genetic gain could be achieved through reciprocal recurrent selection considering tetraploid and diploid parents as independent but complementary population sources for this breeding scheme. The euploid (2x and 4x) hybrid offspring derived by crossing 3x French plantains from West Africa and the 2x M. acuminata spp. burmannicoides ‘Calcutta 4’ segregated for the fruit parthenocarpy P1gene (Ortiz and Vuylsteke, 1995) because of the heterozygous locus of the former cultivar group and the recessive genotype for this locus (i.e., lacking P1) of the wild banana. Recent research by Okoro et al. (2011) found that the 2x banana ‘Borneo’ has P1 but lacks the other two dominant alleles (P2 and P3) needed for fruit parthenocarpy. They

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further hypothesized that P2 and P3, which appear to correlate with rates of genome-by-environment dependent seed development, when non-synchronized with fruit development could lead to dehiscence, death of developing fruit, or non-parthenocarpic fruit. A reliable drought phenotyping should be pursued to breed for this trait in Musa. Very recently Ravi et al. (2013) assessed stomatal conductance, cell membrane stability, leaf emergence rate, rate of leaf senescence, relative water content, and bunch yield under water scarcity because they were thought to be associated with enhanced adaptation to drought. They concluded that recording bunch weight under drought stress should be the target trait for phenotyping. 5. Host plant resistance through crossbreeding Fungi, bacteria, viruses, nematodes and insects are the main pathogens and pests and causing pests of banana and plantain affecting their farming worldwide. Lorenzen et al. (2009) advocate host-plant resistance as the most economic and sustainable means for plant health management. They admit that the gene introgression process, linkage drag, complex trait genetics, lack of adequate screening protocols or host-plant interactions (e.g. various strains and R gene specifity) may confound deploying host plant resistance in banana and plantain farming systems. Furthermore, monitoring and pre-emptive breeding of host plant resistance are mandatory to address any changes of pathosystems because of emerging new strains due to recombination, mutation in, or migration of the pathogen(s). Breeding host plant resistance to black Sigatoka dominates the agenda of most breeding programs worldwide (Abadie et al., 2009; Kobenan et al., 2009; Lorenzen et al., 2009; Menon et al., 2011; Ortiz, 2013 and references therein). Ortiz and Vuylsteke (1994a) found that host resistance to black Sigatoka in euploid plantain–banana hybrids was due to one major recessive gene (bs1) and two minor independent alleles with additive effects (bsr2 and bsr3). Although Vroh-bi et al. (2009) indicated that host plant resistance was “quantitative” in 2x segregating offspring derived by crossing Calcutta 4 and M. balbisiana Montpellier, they also noticed that the results fit into a tri-hybrid segregation ratio, thereby confirming the oligo-genic system. Some of the newly bred hybrids pleased consumers and were released for farming — particularly in Africa (Kobenan et al., 2009; Ortiz, 2013; Tenkouano and Swennen, 2004), while others require improving some fruit quality traits (Garruti et al., 2013) or can be used for further food processing (de Godoy et al., 2013). Fusarium wilt, Moko/bugtok, blood bacterial wilt and Xanthomonas wilt are other biotic factors affecting various banana-based farming systems. Resistant cultivars should be therefore bred and integrated into a plant health management approach to increase their potential success in fighting banana wilts (Daniells, 2011). The diploid banana hybrid M53 seems to be a promising source for developing wilt-resistant cultivars to Fusarium oxysporum f. sp. cubense or Foc (de Matos et al., 2011). The wild-seeded M. acuminata ssp. malaccensis could be another source for breeding host plant resistant to Foc tropical race 4 and it has been used for developing two linkage maps to identify putative resistance markers to this wilt (Kayat et al., 2009). Plant parasitic nematodes can cause a significant yield loss (25–50%) in banana and plantains. Resistant germplasm shows low percentage of dead roots and high percentage of functional roots (Kumar et al. 2009). An index, which combines records of dead root percentage, large lesion number and nematode population density, could facilitate field selection for host plant resistance to burrowing and spiral nematodes (Hartman et al., 2010). There could be, however, variability in reproductive fitness and virulence of nematodes populations (Dochez et al., 2013a), which calls for host plant resistance screening of Musa germplasm across available strains. In this regard, Dochez et al. (2013b) found a burrowing nematode population from Mbarara (Uganda) that broke the host plant resistance of the 2x banana ‘Pisang Jari Buaya’, a source of resistance to this nematode known worldwide. Their results

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highlight the importance of taking into account strain pathogenicity when engaging in banana and plantain germplasm enhancement for host plant resistance to nematodes. 6. Fruit quality, human nutrition and health Banana and plantain can provide essential micronutrients such as vitamin A to various populations in the developing world (Englberger et al. 2003). The pulp of their fruits shows significant variation of provitamin A carotenoids (Davey et al., 2011), but very low iron and zinc contents (Davey et al., 2009d). Davey et al. (2009b) indicated, however, that treatments for the extraction and analysis of carotenoids in Musa fruits need to prevent the breakdown and loss of fruit carotenoids, which are often unstable and sensitive to light exposure. Diploid (AA) bananas from Papua New Guinea had the highest levels of β-carotene when assessing this germplasm along with dessert (AAA) and East African highland (AAA) bananas (Fungo and Pillay, 2011). The orange pulp plantains (AAB) exhibit higher provitamin A carotenoids than the dessert (AAA) bananas with white-cream pulp (Davey et al., 2007). Ekesa et al. (2012) indicated that the bio-accessibility of provitamin A carotenoids depends however on the food recipes and other ingredients included in the dish. The consumption of 100 g of steamed African highland bananas or roasted plantains can provide 24 to 35% and 16 to 20% of the vitamin A recommended dietary allowances for pre-school children and women of reproductive age, respectively (Ekesa et al. 2013). These results suggest that the availability of Musa germplasm whose modest and realistic consumption levels may impact positively on populations at risk of vitamin A deficiency. Fungo and Pillay (2011) noticed a positive correlation between pulp color intensity and β-carotene concentration. This correlation was strong when using a yellowness index based on colorimetry vis-à-vis color chart scores (Pereira et al. 2011). Davey et al. (2009c) also proposed using visible and near infrared reflectance spectroscopy for high-throughput screening of fruit pulp samples for vitamin A content. 7. Tissue culture: micro-propagation of clean planting materials Tissue culture can provide “pathogen-free propagules” or “cleanplanting materials” for smallholders growing banana and plantain in the tropics (Lule et al. 2013a,b). They can also facilitate the safe movement of germplasm across borders and accelerate multiplication of desired cultivars. Quality standards and plant health certification for propagules and training for users are needed to ensure sustainable micro-propagation of banana and plantain (Dubois et al., 2013), particularly in the developing world, where most governments are yet to enact policy or provide incentives to facilitate the embracing of this technology by banana smallholders. Crop management may benefit from micro-propagation as observed in Kenya by Njuguna et al. (2011). While working with several hundred banana farmers, they noted that crop yield increased from 10 t ha−1 to 30 t ha−1 when using tissue culture-derived propagules, which were more expensive than traditional suckers. Nonetheless, farmers using this biotechnology increased by 145% their income due to the enhanced crop yield. Fungal endophytes can be further inoculated into banana tissue culture plantlets to extend the benefits of this “clean planting material” technology (Dubois et al., 2006). In this way, the endophytes are included in the planting material sold to banana farmers. 8. Molecular cytogenetics and cytometry Cytogenetic research in Musa during the first half of the 20th century dealt with determining chromosome numbers of wild species and cultivars, and chromosome pairing in hybrids (Pillay et al. 2012). This early work paved the path for today's molecular cytology. Fluorescence in situ hybridization (FISH) has been advocated as a new tool in Musa breeding. It may help validating collinearity between

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parents included in crossing schemes, gene-mapping research, and identifying chromosomal rearrangements between related Musa species and their putative derived cultivars (De Capdeville et al., 2009). For example, FISH with probes for satellite DNA sequences (which had a high sequence conservation within, and a high homology between Musa species), rRNA genes and a single-copy BAC clone 2G17 led to identify distinct chromosome banding patterns in M. acuminata and M. balbisiana (Čížková et al., 2013),thereby determining the genomic constitution in interspecific hybrids. This research also provided means for adding new cytogenetic markers in Musa. Jeridi et al. (2011) used a new protocol for preparing chromosomes of AAB and ABB cultivars at metaphase I that are suitable for genomic in situ hybridization (GISH). Their GISH research shows interspecific recombination between M. acuminata and M. balbisiana chromosomes, thereby confirming an early hypothesis of Ortiz and Vuylsteke (1994b) based on segregation data in 2x arising from crossing plantains (3x) with a wild banana (2x). This homoeologous pairing allow chromosome exchanges between both species. Very recently Nunes de Jesus et al. (2013) indicated that the analyses of 221 Musa accession microsatellites using simple-sequence repeats (SSR), internal transcribed spacer (ITS) polymorphism and flow cytometry further support the occurrence of recombination between A and B genomes. Two-dimensional electrophoresis (2DE) and two-dimensional difference gel electrophoresis (2D DIGE) with de novo tandem mass spectrometry (MS/MS) sequence determination were used to characterize inter- and intra-cultivar protein polymorphism in various Musa cultivars (Carpentier et al., 2011). Together with multi-variate statistics, they were able to classify some cultivars according to their putative genomes because the proteome does not always match the former. 9. DNA marker-facilitated diversity, origin and relatedness assessments DNA markers facilitate taxonomy, help cultivar true-to-type assessment and are useful for both population and quantitative genetics research in Musa (Ortiz, 2011). New microsatellites were widely used in the past years for assessing diversity in bananas, plantains and other related crop wild relatives (Table 1). Some of these new microsatellites were derived from expressed sequenced tags (EST) or from genomic sequence surveys (GSS). Furthermore, a robust approach for genotyping in Musa – based on 19 microsatellite loci that are scored with fluorescently labeled primers and using high-throughput capillary electrophoresis separation with high resolution – became available recently (Hřibová et al., 2013). Garcia et al. (2011) also used a banana transcriptome database containing 42,724 EST (approx. 24 Mb of DNA sequence) to design primers that were able to distinguish 32 variable number of tandem repeat (VNTR) and 119 target region amplified polymorphism (TRAP) alleles in 14 diploid Musa accessions. Their research shows the advantage of engaging EST-derived markers for genetic diversity analysis and gene discovery in Musa. Microsatellites and amplified fragment length polymorphisms (AFLP) were useful for identifying somaclonal variants in Musa (Vroh-Bi et al., 2011). One SSR locus very similar to an arcelin gene revealed a deletion

in a subculture variant, while AFLP analysis attributed most of the in vitro-derived variants to internal 5′-cytosine methylation events. The sequence-related amplified polymorphism (SRAP) technique, based on amplifying open reading frames (ORFs), was used to assess genetic diversity and relationships among Musa accessions. For example, SRAP revealed that most genetic relationships among 29 polyploid banana cultivars were correlated to their region of origin (Wei et al., 2011). The clustering of these cultivars agreed with the putative genomes given to them. SRAP also exhibited more variation than AFLP when used in a sample of 40 Musa accessions (cultivars and wild species) relevant to the genetic enhancement of this crop (Youssef et al., 2011). Likewise, SRAP was able to discriminate among species within Eumusa and between triploid cooking bananas and plantains. Diversity array technology (DArT) has been also successful for high throughput genotyping and diversity analysis in Musa (Risterucci et al., 2009). This DNA hybridization-based molecular marker technique detects simultaneously polymorphism at many genomic loci without needing sequence information. Risterucci et al. (2009) indicated that DArT provided the same genetic relatedness among Musa accessions, as previously noted with other DNA markers, but with a high resolution and speed, plus a low cost. There were recent in-country assessments with DNA markers that provided new insights about Musa diversity not included in previous surveys, e.g. wild Musa in China (Qin et al., 2011), cultivars in Indonesia (Retnoningsih et al., 2011) or landraces in Myanmar (Wan et al., 2005). Similarly, Agoreyo et al. (2008) were able to discriminate plantains from Jamaica and Nigeria using the arbitrary primer polymerase chain reaction (AP-PCR) technique. AP-PCR also revealed the relatedness among the Nigerian plantain cultivars included in their research. Table 2 lists other recent systematic botany research in Musa and related species as well as that for elucidating the origin of today's cultivars using nuclear and plastid DNA markers. Based on chloroplast and mitochondrial genomes, De Langhe et al. (2010) hypothesized that “backcrossing” of an unknown parent could account for the unbalanced genomes as well as inter-genome translocation found in some edible cultivars. Moreover, after analyzing with 22 microsatellites the diversity of 561 Musa accessions, Hippolyte et al. (2012) determined the closest diploid ancestors of the triploid ‘Cavendish’ and ‘Gros Michel’ dessert bananas. They also indicated that significant morphological variation in both did not ensue from recombination but from epigenetic regulations. Likewise, DNA sequence analysis of four genes in a set of 100 cultivars and wild accessions revealed multiple domestications of edible Musa (Volkaert, 2011). The clusters of M. acuminata ssp. banksii/errans and M. acuminata subsp. malaccensis/microcarpa/zebrina/burmannica/ siamea plus M. balbisiana appear to be involved in the domestication of bananas and plantains. Diversity assessment with DNA markers of many diploid accessions from these subspecies may further assist to select distinct parents for new crossbreeding schemes. Very recently, Němcová et al. (2011) concluded that comparative sequence analysis of single-copy genes may resolve the evolutionary history of Musaceae and could complement the analyses ensuing from the use of both ribosomal DNA ITS1-5.8S-ITS2 region and DArT. In this regard, Hřibová et al. (2011) using ITS sequence-derived phylogenetic

Table 1 Recent Musa diversity assessment using new microsatellites. Microsatellites

Finding

Specific primers for 41 loci from 5 ‘Calcutta 4’bacterial artificial chromosome 20 (out of 33) loci had polymorphism when screened across 21 diploid (BAC) consensi datasets M. acuminata accessions, contrasting in resistance to Sigatoka 23 primers from cultivar ‘Gongjiao’ Polymorphisms assessed on 26 banana cultivars and 11 related species/subspecies 19 microsatellite loci scored using fluorescently labeled primers and Genotyping platform tested and optimized on a set of 70 diploid and 38 high-throughput capillary electrophoresis separation with high resolution triploid banana accessions 52 new primer pairs 34 microsatellites identified in expressed sequenced tags (EST) and BAC clones from ‘Calcutta 4’ were validated in 22 wild and improved diploids 229 primers based on genomic sequence data survey 26 markers amplified in 15 banana accessions

Reference Miller et al. (2010) Wang et al. (2010) Christelová et al. (2011b) Amorim et al. (2012) Ravishankar et al. (2012)

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Table 2 DNA marker-research on Musa relatedness and origin of modern cultivars. DNA marker

Finding

Reference

Chloroplast and mitochondrial primer pairs from polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLP)

Primary centers of origin for both chloroplast and mitochondrial genomes of M. acuminata and M. balbisiana revealed. Most cultivars had a single M. acuminata cytotype (or combinations of chloroplast and mitochondrial gene-pools), while a single M. balbisiana noted in interspecific hybrids Four gene pools among M. acuminata wild types, while M. balbisiana did not show sequence divergence Monophyly of Musaceae family as per phylogenetic analyses, which indicated that Musella and Ensete to be congeneric or closely related. None of the five Musa sections previously defined based on morphology was a monophyletic group but 2 infra-generic clades identified based on basic chromosome numbers: x = 11 (Musa and Rhodochlamys) and x = 10 (Callimusa), 9 (Australimusa) or 7 (Ingentimusa) Non-clear-cut distinction between Eumusa and Rhodochlamys, thereby confirming their genetic relationships. ‘Matti’ (Eumusa) could be a parthenocarpic derivative of M. acuminata ssp. burmannica, while 4 unique wild M. acuminata from northeastern India during recent explorations had high relatedness to Rhodochlamys Close relationship of Australimusa and Callimusa, but Eumusa and Rhodochlamys are not reciprocally monophyletic, thereby supporting merge between these two sections Putative family trees suggesting simple ways for the evolution of the today's hybrid cultivars and some wild genotypes lacking in genebanks

Boonruangrod et al. (2008)

5′ external transcribed spacer (ETS) rDNA sequence information Nuclear ribosomal internal transcribed spacer (ITS) and chloroplast sequences

Microsatellites (coupled with morpho-taxonomy)

DNA sequences from 19 unlinked nuclear genes

Cytoplasmic and nuclear marker systems

reconstruction divided the genus Musa into two distinct clades: Callimusa and Australimusa and Eumusa and Rhodochlamys. They also noticed that many intraspecific banana hybrids have conserved parental ITS sequences, thereby suggesting an incomplete concerted evolution of rDNA loci. Their research also found one type of ITS sequence in some putative interspecific cultivars, which challenges their hybrid origin. 10. Genetic maps and marker-aided breeding Heslop-Harrison (2011) argues very eloquently how knowledge-led breeding based on genomics and crop design will allow Musa “superdomestication”; i.e., inter-disciplinary research to find and evaluate genes for target traits and incorporate them into new cultivars. Certainly such search and use for genes and traits will significantly depend on the available diversity endowment in Musa genebanks, and on combining expertise with the aim of exploiting gene pools as well as on using omics-led science for improving banana and plantain. As noted by Lorenzen et al. (2011) DNA marker-aided breeding (MAB) provides means for accelerating today's slow and land-intensive Musa genetic enhancement. As indicated in a previous section, DNA markers are providing insights into Musa diversity, origin and relatedness and putative ancestors, which will assist selecting parents for crossbreeding. Horry (2011) also indicate that DNA markers are revealing the population structure of Musa, clarifying gamete behavior, and giving insights for manipulating ploidy and inter-specificity. New genetic maps – based mostly on DArT and microsatellite s– became available

Boonruangrod et al. (2009) Li et al. (2010)

Durai et al. (2011)

Christelová et al. (2011a) Boonruangrod et al. (2011)

in the last 3 years (Table 3). They, along with other segregating materials (Rehka et al., 2011) and DNA marker systems such as new microsatellites derived from pyrosequencing-based transcriptome analysis (Cruz et al., 2013), expressed sequence tags–simple sequence repeats or EST–SSR (Mbanjo et al., 2012a) and single nucleotide polymorphism (SNP) markers (Adesoye et al., 2012) may be used to locate target genes, understand genetics of complex traits and be landmarks for MAB, thereby increasing Musa crossbreeding efficiency. The completion of the banana genome sequence and next-generation sequencing technology will further add new DNA markers and increase the precision of MAB. 11. Mutations and “tilling” Targeting induced local lesions in genomes (TILLING) could be a useful tool for reverse genetics in banana and plantain (Wang et al. 2012). For this purpose, Jankowicz-Cieslak et al. (2012) treated shoot apical meristems of banana with ethyl methanesulphonate (EMS) — a chemical mutagen. They found a high density of GC–AT transition mutations and noticed that genotypically heterogeneous stem cells resulting from the mutagenic treatment were rapidly sorted to fix a single genotype in the meristem. Their research further demonstrated the accumulation of potentially deleterious heterozygous alleles, thereby suggesting that mutation induction may reveal recessive traits. The use of TILLING can further extended to assess Musa cultivars, ecotypes, landraces and wild accessions; i.e., eco-TILLING. Till et al.

Table 3 Banana genetic maps ensuing from recent DNA marker-based research. DNA marker system(s) and mapping population(s)

Map details

Reference

Diversity array technology (DArT) markers and microsatellites (SSR) on segregating (‘Borneo’ × ‘Pisang Lilin’) diploid F1

Synthetic map containing 322 DArT and 167 SSRs on 11 linkage groups (LGs). Two complete parental maps noting the structural rearrangements localized on LGs The first maternal map (6142-1, 81 individuals) included 121 DArT, 106 SSR and 4 AS-PCR markers in 15 LGs adding to 670 cM. The second maternal map (6142-1-S, 58 individuals) based on 71 DArTs, 79 SSRs, and 2 AS-PCRs in 16 LGs spanning across 698 cM. The combined paternal map (139 individuals) comprised 196 DArTs, 117 SSRs and 3 AS-PCRsover 15 LGs and 1004 cM as total length Dense genetic maps used with in excess of 1000 DArT and microsatellites for anchoring 70% of the genome assembly to the 11 Musa chromosomes. LGs 1 and 4 had markers deviating from Mendelian ratio, thereby suggesting structural heterozygosity (or translocations)

Hippolyte et al. (2010)

Allele specific-polymerase chain reactions (AS-PCRs), DArT and microsatellites on two half-sib diploid banana breeding populations segregating for host plant resistance to burrowing nematode: (‘TmB2x 6142-1’ × ‘TmB2x 8075-7’) and (‘TmB2x 6142-1-S’ × ‘TmB2x 8075-7’) DArT and microsatellites on segregating offspring from selfing Pahang — the diploid parent of the genome-sequenced doubled haploid

Mbanjo et al. (2012b)

Carreel et al. (2013)

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(2010) found in excess of 800 novel alleles in 80 accessions using ecoTILLING, which they claim can be a robust and accurate platform for the discovery of polymorphisms in homologous and homoeologous gene targets. Their research was able to identify two SNPs that could be deleterious for the function of a gene putatively important for phototropism. Eco-TILLING can facilitate selecting Musa germplasm for further cross- or mutation breeding. 12. The genome sequencing provides new insights and DNA markers The banana genome sequencing was a global multi-partner endeavor that took about one decade to accomplish its task. This deciphering of the banana genome will lead to the generation and sharing of knowledge on its genome organization, as well as in identifying DNA markers for further use in research and crossbreeding. For example, the bacterial artificial chromosomes (BAC)-end sequencing of M. acuminata doubled haploid (DH)‘Pahang’ revealed that most frequent repeated sequences were homolog to ribosomal RNA genes (particularly 18S rRNA) and that Ty3/gypsy type monkey retro-transposon was the most common (Arango et al., 2011). The repetitive part of a genome can be a useful source for developing DNA markers for further use in Musa diversity analysis and MAB (Hřibová and Doležel, 2011). In this regard, Hřibová et al. (2010) characterized thoroughly the repeat component of the genome of the diploid wild accession ‘Calcutta 4’. Their research improved the knowledge about the organization of banana chromosomes, and provided the sequence resources for repeat masking and annotation for the genome sequencing. Comparative sequence analysis of resistance gene analog (RGA) clusters estimated the degree of sequence conservation and mechanisms of divergence at the intraspecies level (Baurens et al., 2010) as well as provided means for identifying Argonaute gene sequences in bananas (Teo et al., 2011). The Argonaute proteins are involved in RNA silencing. After analyzing the RGA08 gene cluster, Baurens et al. (2010) hypothesized its recent and rapid evolution in M. balbisiana. The DH Pahang (highly resistant to F. oxysporum race 4) was used for drafting the 523-megabase draft sequence of banana, which became the first monocotyledon high-continuity whole-genome sequence outside Poales (D'Hont et al., 2012). This sequence also revealed new whole-genome duplications in the Musa lineage among monocots. Likewise, this reference genome sequence advanced Musa genetics. For example, further analysis led to identifying Musa genes coding for 89 nucleotide-binding site (NBS) leucine-rich repeat proteins. Moreover, transcriptomics research provided means to finding 372 differentially expressed banana genes interacting with the fungal pathogen Mycosphaerella fijiensis, which causes black Sigatoka. Likewise, RNA-Seq analysis showed that receptor-like kinase genes were upregulated in a partially resistant interaction with M. fijiensis, and indicated a strong transcriptional reprogramming in mature green fruits after ethylene treatment. Transcription factors were regulating 597 genes, e.g. upregulated genes encoding cell wall's modifying enzymes, three down-regulated starch synthase genes and one upregulated βamylase gene. This sequencing of the banana should be regarded as a milestone in Musa genetic resource enhancement research and it will be surely further use to gain insights for the betterment of this crop. For example, Maldonado-Borges et al. (2013) annotated differentially expressed genes during somatic embryogenesis of M. acuminata ssp. malaccensis ITC 250 in the Musa genome. Knowing these genes may assist in studying deeply the mechanisms involved throughout the different stages of Musa embryogenesis. 13. Other genetic and omics-based resources As noted above microsatellite polymorphism seems to be abundant in Musa and such DNA markers have been already used in biodiversity assessments, systematics and mapping, while their potential use in MAB is yet to be realized. Retroelement-related sequences are also

abundant and can be exploited as anonymous genetic markers in banana, plantain and their wild relatives. Further research led to design primers – from genomic and EST databases – that were useful for characterizing sequences containing nucleotide binding sites (NBS) and leucine-rich repeat (LRR) motifs, which seem to be associated with host plant resistance genes (Azhar and Heslop-Harrison, 2008; Emediato et al., 2009; Lu et al., 2011; Miller et al., 2008), or with genes enhancing adaptation to abiotic factors such as drought, heat and salinity. Candidate gene discovery was undertaken recently for analyzing differential gene expression from infected leaf cDNA after Musa– Mycosphaerella interactions (Miller et al., 2011). Likewise, Sun et al. (2009) isolated and sequenced resistance gene analogs (RGAs) from the genomic DNA of the tetraploid banana hybrid ‘Goldfinger’, which show resistance to F. oxysporum f. sp. cubense causing Fusarium wilt. The identification and cloning of such genes will assist in Musa genetic enhancement. Emediato et al. (2009) characterized RGAs in M. acuminata cultivars with distinct host plant resistance to M. fijiensis causing black Sigatoka, while Sulliman et al. (2012) assessed the diversity of defense gene analogs (DGA) associated with host plant resistance to various Sigatoka in banana. Their interest was to identify specific markers that could be further use in selecting host plant resistance genes controlling these fungi. Their complete gene sequence characterization could be also useful for Musa genetic engineering. Transcriptomics research provided further analysis of the expression of NBS-LRR RGA in resistant wild diploid ‘Calcutta 4’ and susceptible triploid ‘Cavendish’ banana cultivar, when inoculated in vivo or not with conidiospores of Mycosphaerella musicola causing yellow Sigatoka (Emediato et al., 2013). Similarly, the study of time-course expression of defense genes in banana against the root-lesion nematode Pratylenchus coffeae revealed that mRNA levels of the chalcone synthase gene were higher in the roots of resistant ‘Karthobiumtham’ than in those of susceptible ‘Nendran’ (Backiyarani et al., 2011). This kind of research provides new knowledge on host plant resistance mechanisms and may identify candidate genes for further used in MAB or genetic engineering. Very recently Passos et al. (2013) used 454 GS-FLX Titanium technology to determine the sequence of the gene transcripts from both ‘Calcutta 4’ and ‘Cavendish’, thereby increasing the public domain Musa ESTs. This transcriptome will be also useful for gaining knowledge on banana and plantain performance under stress. MNPR1A and MNPR1B –which are two novel full-length nonexpressor of pathogenesis-relatedgenes1 (NPR1) – were isolated from banana by application of the PCR and rapid amplification of cDNA end (RACE) techniques (Endah et al., 2008). They had distinct expression profiles, as determined by quantitative real time (qRT)-PCR, after either elicitor treatment of by interacting with F. oxysporum f. sp. cubense. The tolerant banana cultivar GCTCV-218 expressed greatly and early MNPR1A while MNPR1B was highly responsive to salicylic acid but not to methyl jasmonate in both GCTCV-218 and the susceptible banana cultivar ‘Grand Naine’. MNPR1A expression was also found to be directly related to pathogenesis-related (PR) gene expression that provides host plant resistance to some fungi. As noted by Podevin et al. (2012), the high sensitivity of gene expression analysis by reverse transcriptase real-time or qRT-PCR requires however both normalization using multiple housekeeping or reference genes, and careful selection of these reference genes. Otherwise, results may not be reliable. Their research showed that the accession or cultivar, plant materials, primer set, and gene identity influence the robustness and outcome of RT-qPCR analysis. The reference genes EF1, TUB and ACT can assist for normalization of gene expression data for Musa leaf samples taken in a greenhouse, whereas the best combination of reference genes (L2 and ACT genes) was still suboptimal for Musa leaf samples from in vitro plants. Peraza-Echeverria et al. (2009) isolated and characterized the entire cDNA sequences of RGC2 and RGC5, which are partial non-TIR-NBS sequences, from the root transcriptome of M. acuminata ssp. malaccensis.

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Both partial sequences are in the same cluster, within the ancient nonTIR clade N1.1, with the known but phylogenetically distant Fusarium R genes I2 and Fom-2. Searching within this clade may assist identifying other R genes in Musa. Knowledge on the expression pattern of ripening genes and mechanisms regulating them at ripening may facilitate the post-harvest control of fruit maturity, thereby reducing the fruit spoilage during transport and storage. A MADS-box transcription factor gene, which belongs to the AGAMOUS subfamily and designated as MuMADS1, was isolated from banana fruits (Liu et al., 2009). MuMADS1 may be induced by ethylene biosynthesis associated with postharvest banana ripening, and by exogenous ethylene. Elitzur et al. (2010) cloned and further characterized six full-length MADS-box genes from Grand Naine. Half of them (MaMADS1, 2, and 3) were highly expressed in fruit only, while the other three were expressed in fruit and other organs. Two independent ripening programs were employed in the pulp and peel and involved the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in the pulp, and mainly MaMADS1, and 3 in peel. The cDNA of MA-ACS1 – a major ripening regulating gene in banana – its transcript and protein accumulation patterns of this gene were investigated by Roy Choudhury et al. (2011). Their research provided new insights about the expression pattern and transcriptional regulation of one of MA-ACS1. Jin et al. (2011) were also able to identify more genes from banana at fruit ripening using suppression-subtractive hybridization with cDNA microarray. This research relates these genes to early stages of post-harvest banana ripening, though these authors acknowledge that they need to link them to their corresponding translated proteins and defining their roles. Water scarcity and unusual annual rainfall patterns, which may be brought by climate change, will affect banana and plantain productivity. Vanhove et al. (2012) developed a drought-screening test for in vitro plants based on a mild osmotic stress. The cooking banana (ABB)‘Cachaco’ showed the smallest stress-induced growth reduction among the five triploid cultivars (dessert bananas, East African highland bananas and plantains) included in this test. The leaf proteome analysis of this cultivar revealed that the respiration, metabolism of reactive oxygen species (ROS), and several dehydrogenases involved in nicotinamide adenine dinucleotide (NAD)/NADH homeostasis participate actively in allowing plants under stress to reach a new balance. In this regard, Carpentier et al. (2008a) noted that proteomic research in a non-model plant such as Musa species dealt with various issues such as sample preparation, analysis and interpretation of complex data sets, protein identification via mass spectrometry, plus data management and integration. Vertommen et al. (2011a) also provides an overview on alternative techniques for membrane proteomics in Musa. They indicated that gel-based protein separation should not be used in this research, and instead an approach that includes peptide separation should be pursued to increase resolution. Further research by Vertommen et al. (2011b) led to establishing a workflow for peptide-based proteomics of the banana plasma membrane. Proteomics was also used by Swennen et al. (2011) to study the response of meristem cultures from various banana cultivars to osmotic stress related to cryopreservation. They were able to construct a proteome map using two-dimensional gel electrophoresis. It includes 637 proteins and some of those differentially expressed under osmotic stress were further characterized using genetic engineering. Davey et al. (2009a) used cross-hybridization to Affymetrix oligonucleotide GeneChip® microarrays for profiling Cachaco's leaf transcriptome after drought stress. They found 2910 Musa gene homologues when hybridizing to the Affymetrix Rice Genome Array. The drought-responsive transcripts included various functional classes associated with plant stress responses, and a range of regulatory genes involved in coordinating abiotic stress responses. There were 52 drought-sensitive Musa transcripts homologous to genes underlying QTL for enhanced adaptation to drought and cold in rice, of which two were within a single gene. More recently, suppression subtractive

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hybridization was used for identifying differentially expressed genes in leaves of the M. balbisiana accession ‘Bee Hee Kela’ during water stress (Ravishankar et al., 2011). Further BLAST search of 50 nonredundant sequences established their similarity with lipoxygenase, rubisco activase, glycine dehydrogenase, catalase and ethylene responsive factor, which seem to be involved in cell membrane integrity, signal transduction and metabolism in response to dehydration. The structure and regulation of the Asr gene family in banana and plantain was investigated by Henry et al. (2011) because abscisic acid (ABA), stress, ripening (ASR) proteins are a family of plant-specific small hydrophilic proteins with an unknown function that enhances adaptation to drought. This Musa Asr gene family had at least four members exhibiting two exons and one intron structure and the ABA/water deficit stress (WDS) domain — both of which are characteristic of Asr genes. The genes mAsr1 and mAsr3 were induced by osmotic stress and wounding, while mAsr3 and mAsr4 were induced by exposure to ABA in meristem culture. The mAsr3 protein displayed the highest variation for amino acid sequence and expression pattern, which makes it the most interesting candidate for further functional research. Transcriptomics and proteomics research are complementary because each technique focuses on a subset of genes and proteins. Carpentier et al. (2008b), after working with banana, indicated that proteomics yields a better characterization for poorly characterized species, but transcriptomics can be used when researching low-abundant or hydrophobic proteins. 14. Genetic engineering The non-conventional Musa breeding also includes genetic engineering, protoplast culture and somatic hybridization (Chen et al., 2011), but the first has shown the most promising results since its first use about 20 years ago (May et al., 1995; Sági et al., 1995). Transgenics can facilitate the introduction of non-Musa genes into the cultigen pool, and should be incorporated into banana and plantain breeding programs when lacking natural variation for such trait(s) or for the genetic amelioration of sterile cultivars. Despite the presence of few research teams on Musa genetic engineering (Remy et al., 2013), noteworthy advances were made both in somatic embryogenesis and genetic engineering of banana and plantain in recent years (Table 4). Micronutrient deficiency, particularly of vitamin A, iron and zinc, remains a major challenge because affect several hundred millions of people, particularly in the developing world. Dale et al. (2013) have used generic engineering to increased micronutrient content in banana to fight malnutrition in Uganda, where its low-nutrient fruits are used for the country's main dish: matooke. The first harvest of their transgenic banana with enhanced vitamin A content was in the South Johnstone area south of Cairns (Australia). These transgenic bananas overexpressing phytoene synthase (PSY) with either constitutive or fruitpreferred promoters had 15-fold pro-vitamin A levels than the “wildtype”. One of the two phytoene synthase genes was from a naturally high pro-vitamin A banana while the other was a maize gene used for developing ‘Golden Rice 2’ (Paine et al., 2005). The Australian trial was a proof-of-concept for one of the combinations of transgenes increasing pro-vitamin A. The most promising transgenic bananas with elevated pro-vitamin A are now undergoing field-testing in Uganda. Further research by Mlalazi et al. (2012) led to the isolation and characterization of PSY genes from the orange-pulp cultivar Auspina, which may be used for breeding new cisgenic or intragenic banana cultivars with enhanced pro-vitamin A content. This genetic engineering of banana with sequences originating from its own genome may increase its public acceptability. Transgenic Musa breeding was able to achieve enhanced resistance to Xanthomonas campestris pv. musacearum causing the devastating banana Xanthomonas wilt in the Great Lakes Region of Africa (Tripathi et al., 2010). Its spread threatens the food security and income of millions of East and Central Africans who depend on this crop for both.

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Table 4 Advances in Musa somatic embryogenesis and genetic engineering. Germplasm

Achievement

Reference

Musa acuminata hybrid CNPMF2803-01 and ‘Nanicão’ (Cavendish) Cavendish (AAA): ‘Grand Naine’ and ‘Williams’

Agrobacterium-mediated transient expression system in immature fruits of banana, which may allow studying gene function and validating those expressed in banana fruit Embryogenic response depends on both genotype and developmental stage of the buds from which explants are excised. Selected embryogenic calluses that are successfully established on proliferation medium led to embryogenic cell suspensions (ECS) with good regeneration capacity Plantlets were regenerated from somatic embryos derived from embryogenic cells of calli from immature male flower explants Highly regenerative ECS were established from floral meristems and thereafter transformed the antimicrobial peptide (AMP) gene cloned from onion seeds Transgenic plants with AMP and constructs from pCAMBIA 2301 had a small lesion area after inoculating with Fusarium oxysporum f. sp. cubense race 1 Agrobacterium rhizogenes sonication-aided transformation (≤2.4%) of shoot bud explants Apoptosis-inhibition-related genes confer resistance to Fusarium wilt race 1 Rice thaumatin-like protein gene enhances resistance to Fusarium wilt race 4 in transgenic plants ECS, which was initiated from immature flowers, transformed using Agrobacterium tumefaciens containing one construct derived from the replicase-associated protein (Rep) gene of the Hawaiian isolate of Banana bunchy top virus (BBTV) Transgenic banana plants with normal phenotypes constitutively overexpressing novel banana SK3-type dehydrin geneMusaDHN-1 had enhanced adaptation to drought- and salt-stress after in vitro and ex vitro assays ECS-derived transgenic plants expressing the Hrap gene from Capsicum pepper under the regulation of the constitutive CaMV35S promoter did not show any infection symptoms after artificial inoculation of potted plants with banana Xanthomonas wilt in the screenhouse Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene from sweet pepper (Capsicum annuum) under the regulation of the constitutive CaMV35S promoter showed high resistance to Xanthomonas campestris pv. Musacearum (no disease symptoms after artificial inoculation of in vitro plants in laboratory or in potted plants in the screenhouse) Development of an embryogenic cell suspension (ECS), regeneration, and transformation after establishing ECS using highly proliferative multiple buds Transgenic host plant resistance to burrowing nematode with maize cystatin that inhibits nematode digestive cysteine proteinases and a synthetic peptide that disrupts nematode chemoreception Transgenic banana plants without marker gene used for selection using the Cre-lox site-specific recombination system Somatic embryogenesis (using immature male flower explants), plant regeneration and Agrobacterium tumefaciens-mediated transformation The potential of rice chitinase genes to enhance host plant resistance to black Sigatoka was shown along with the usefulness of the leaf disk bioassay for early screening in transgenic banana

Matsumoto et al. (2009)

‘Virupakshi’ (AAB banana) ‘Nanjangud Rasbale’ (syn. ‘Rasthali’, AAB banana) ‘Nanjangud Rasbale’ ‘Nanjangud Rasbale’ ‘Lady Finger’ (AAB banana) ‘Nangka*’ (AAB) ‘Dwarf Brazilian’ (AAB banana)

‘Karibale Monthan’ (ABB group)

‘Mpologoma’ (AAA East African highland banana) and ‘Sukali Ndiizi’ (AAB banana) ‘Sukali Ndiizi’ and ‘Nakinyika’ (AAA East African highland banana)

‘Gonja manjaya’(AAB plantain) ‘Gonja’

‘Grand Naine’ ‘Dwarf Cavendish’ (AAA) ‘Gros Michel’ (AAA)

The best 65 transgenic plants expressing the hypersensitivity responseassisting protein (Hrap) gene from sweet pepper and not showing any infection symptoms after artificial inoculation of potted plants with banana Xanthomonas wilt in the screenhouse were included in confined field-testing near Kampala in Uganda. After testing them as mother plants and first ratoon plants, 12 transgenic lines were rated as having absolute resistance (Tripathi, 2012). Their plant phenotype and the bunch weight and size were similar to non-transgenic counterparts. These lines will undergo multi-location trials in Uganda and will be further assessed for environmental and food safety according to Uganda's biosafety regulations, risk assessment and management, plus procedures for seed registration and release. They may be shared with farmers in 2017. 15. Outlook: genetic enhancement to meet global demand in a changing climate Banana and plantain breeding depends on sustaining genetic gains. There have been significant advances in Musa crossbreeding, omicsled genetic enhancement and genetic engineering in recent years as summarized in above sections. Such effort should translate into new banana and plantain cultivars that can address the main issue humankind faces in this 21st century: meeting the increased demand for food by a growing population who will be improving both their health and wealth while adapting to a changing climate. The genetic betterment of Musa should therefore emphasize improvements in crop productivity, host plant resistance and enhanced use-efficiency of inputs such as water

Youssef et al. (2010)

S. Elayabalan (TNAU, India) et al. (unp.) Mohandas et al. (2011b) Mohandas et al. (2011a) Venkatachalam et al. (2011) Paul et al. (2011) Mahdavi et al. (2012) Borth et al. (2011)

Shekhawat et al. (2011)

Tripathi et al. (2010)

Namukwaya et al. (2012)

Tripathi et al. (2012) Roderick et al. (2012)

Chong-Pérez et al. (2012a) Chong-Pérez et al. (2012b) Kovács et al. (2013)

and fertilizers to ensure it contributes to the sustainable intensification of banana and plantain farming. Omics research will continue providing new useful insights for Musa genetic enhancement and may generate new tools for improving further the efficiency of banana and plantain breeding, e.g. by developing markers as selection aids or new genomic-led methods that can accelerate the release of cultivars adapted to farming systems where the Musa crop thrives. In this regard, as noted by Roux et al. (2011) it will be a key to bridge the gap between omics and crossbreeding, particularly prioritizing the following areas to facilitate cooperative research undertakings: collecting and characterizing germplasm, reliable and high throughput phenotyping of breeding materials, DNA markers for studying diversity, gene discovery and MAB, and identifying parents for crossbreeding. Their long-term benefits will relate to improving crop productivity, which will be measured by unit of time and space in the newly bred banana and plantain cultivars, reducing farming costs and enhancing Musa farming systems through eco-efficiency.

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