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Research review paper
From crossbreeding to biotechnology-facilitated improvement of bananaand plantain
Rodomiro Ortiz a,⁎, Rony Swennen b
a Swedish University of Agricultural Sciences (SLU), Department of Plant Breeding, Sundsvagen 14, Box 101, 23053 Alnarp, Swedenb Katholieke Universiteit Leuven – International Institute of Tropical Agriculture (IITA) – Bioversity International, Willem de Croylaan 42, Box 2455, 3001 Heverlee, Belgium
The annual harvest of banana and plantain (Musa spp.) is approximately 145 million tonsworldwide. About 85% ofthis global production comes fromsmall plots andkitchenor backyard gardens from thedevelopingworld, andonly15% goes to the export trade.Musa acuminata andMusa balbisiana are the ancestors of several hundreds of parthe-nocarpic Musa diploid and polyploid cultivars, which show multiple origins through inter- and intra-specific hy-bridizations from these two wild diploid species. Generating hybrids combining host plant resistance topathogens and pests, short growth cycles and height, high fruit yield, parthenocarpy, and desired quality fromthe cultivars remains a challenge forMusa crossbreeding, which started about one century ago in Trinidad. The suc-cess of Musa crossbreeding depends on the production of true hybrid seeds in a crop known for its high levels offemale sterility, particularly amongpolyploid cultivars. All banana export cultivars grown today are, however, selec-tions from somaticmutants of the groupCavendish andhave a very narrowgenetic base,while smallholders in sub-Saharan Africa, tropical Asia and Latin America use some bred-hybrids (mostly cooking types).Musa improvementgoals need to shift to address emerging threats because of the changing climate. Innovative cell and molecularbiology tools have the potential to enhance the pace and efficiency of genetic improvement in Musa. Micro-propagation has been successful for high throughput of clean plantingmaterialswhile in vitro seed germination as-sists in obtaining seedlings after inter-specific and across ploidy hybridization. Flow cytometry protocols are usedfor checking ploidy among genebank accessions and breeding materials. DNA markers, the genetic maps basedon them, and the recent sequencing of the banana genome offer means for gaining more insights in the geneticsof the crops and to identifying genes that could lead to acceleratingMusa betterment. Likewise, DNA fingerprintinghas been useful to characterize Musa diversity. Genetic engineering provides a complementary tool to Musabreeders who can introduce today transgenes that may confer resistance to bacteria, fungi and nematodes, or en-hance pro-vitamin A fruit content. In spite of recent advances, the genetic improvement of Musa depends on afew 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, Malaysiaand Uganda). Development investors (namely international aid and philanthropy) should therefore increasetheir funding to genetically enhance this crop that ranks among the 10-top staple foods of the developing world.
The giant, perennial, herbaceous bananas and plantains (Musa spp.)were grown in 10.6 million ha in 2011 with an average fruit yield of13.6 t ha−1 (FAO, 2013). The crop includes about 1000 dessert, cookingand beer cultivars derived after intra- or inter-specific hybridization ofthe wild diploid (2n = 2x = 22 chromosomes) ancestor species Musaacuminata (A genome) and Musa balbisiana (B genome) (Ortiz, 2011).The cultivars (mostly triploids or 2n = 3x = 33) show fruit partheno-carpy and often sterility, whichmakes challenging the crossbreeding ofthis crop (Ortiz, 2013).
The crop ranks among the 10 most important staples and feedsmillion of people worldwide, but there are not even 10 active Musacrossbreeding programs, namely in Brazil, Cameroon, Côte d'Ivoire,Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda. The oldestbreeding program in operation is that of the Fundación Hondureña deInvestigación Agrícola (FHIA, Honduras), whose initial focus was ondessert 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 developingworld (especially for local markets and almost nil for export trade),while farmers selectedmost of today's cultivars and their somaticmuta-tions. Genebanks in the tropical world include Musa wild speciesand cultivars, but Bioversity International Transit Center (ITC) at theKatholieke Universiteit Leuven (Belgium) holds the world's largestin vitro repository ofMusa diversity.
There are very vibrant biotechnology-facilitated research undertak-ings with potential impacts in genetic enhancement through tissue cul-ture, 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 – afterseveral years of painstaking ploidy manipulations and field trials – ofnew secondary triploid matooke banana hybrids (Musa, AAA) inUganda (Lorenzen, 2012), the field-testing of transgenic East Africanhighland banana with host plant resistance to Xanthomonas wilt(Tripathi et al., 2010), and the sequencing of theM. acuminata genome(D'Hont et al., 2012) are among themost significant achievements in re-cent years. They ensued from long-term research-for-developmentpartnerships involving national and international institutes plus aca-demics, and with the aim of meeting the needs of banana and plantainfarming in the tropics.
Two recent books (Pillay and Tenkouano, 2011; Pillay et al., 2012)and comprehensive overviews (Ortiz, 2011, 2013) about Musa geneticresources, breeding, genetics, genetic engineering and omics give de-tails of some of their advances, bottlenecks and remaining challenges.There was also, at the end of the last decade, a special journal issuethat included archeobotany, genetics, linguistics and phytogeographyarticles unraveling partially Musa domestication (De Langhe et al.,2009). This review article updates such reports mostly summarizing re-search progress of the last four years, thereby giving the state of the artonMusa genetic enhancement through crossbreeding and using variousbiotech-facilitated methods.
2. Inter-disciplinary research shed light on the origins of ourfavorite fruit
Perrier et al. (2009) advocate a multi-disciplinary approach toelucidate the process of Musa domestication and deciphering the
diversity of banana and plantain. A further article by Perrier et al.(2011) provides insights onto the complex geo-domestication path-ways that generated the various Musa cultivars. Molecular phyloge-netics (using microsatellites and diversity array technology — DArT)facilitated identifying putative wild ancestors of modern cultivars andthe key domestication steps for each of the most important cultivargroups, while archeology and linguistics shed light on the humanspread of Musa and its farming from Papua New Guinea to WestAfrica during the Holocene. Kennedy (2009) also suggests consideringmiscellaneous uses ofMusa plants (beyond the edible fruits) to accountthe spreading of this species. For example, Iles (2009) found impres-sions of banana pseudostems in an iron slag from Eastern Africa,which could prove its use in iron-producing technology. Likewise, therare Musa seeds can be used in archeology (De Langhe, 2009) or an al-ternative source for DNA fingerprinting of its crop wild relatives (Umaet al., 2011b).
Linguistic evidence can fill gaps of archeo-botanical records, therebyallowing mapping and dating crop dispersal. The names used for ba-nana within island Southeast Asia and Melanesia suggest a westwarddispersal of the crop from New Guinea, mixing with a Philippine culti-var, then westward again to mainland Southeast Asia, and onward toSouth Asia's west (Donohue and Denham, 2009). Linguistic researchalso indicates that plantains were likely introduced toWest and CentralAfrica through an unknown route 2500 to 3000 BP (Blench, 2009;Mbida Mindzie et al. 2001, 2004, 2005), becoming thereafter an impor-tant crop in Central African rainforest and supporting the further Bantuexpansion in the continent (Neumann and Hildebrand, 2009).
Phytoliths are rigid, microscopic plant silica bodies whose shapesand sizes are used in paleo-ecology, archeology and food science. Theygave evidence supporting New Guinea's key role in the domesticationof Eumusa (recently renamed as Musa) and Australimusa bananas(Lentfer, 2009b). Banana seeds also have diagnostic phytoliths that dis-criminate between these two main sections, the giantMusa ingens andEnsete and can be further used for tracing domestication. Vrydaghs et al.(2009) were able to link banana phytolith diversity with M. acuminataphylogeny (including edible diploid and triploid derivatives). They fur-ther indicated that extra sampleswould be necessary for understandingthe extent of variation and identifying discriminating traits. A systemat-ic recording of phytolith data could assist filling gaps of banana past dis-tribution in Asia, particularly China and India — which seem to havecontributed little to early domestication of the crop (Fuller andMadella, 2009). Lentfer (2009a) also shows that well-preserved starchgranules can be used for tracking the domestication and dispersal ofMusa and related species.
Buerkert et al. (2009) found a triploid banana cultivar surviving in alimestone rock niche in Oman. This hyperarid country has a narrow di-versity of banana, which grows only in northern oases in the monsoon-dominated southeastern tip of the Sultanate. Thisfinding gives evidenceof how some locations in the Arabian Peninsula could provide a refugeto banana cultivars brought from humid regions, e.g. coastal EastAfrica and nearby islands or Indonesia.
3. Genetic resources characterization
The endowments of Musa diversity available in genebanks serve assources for banana and plantain crossbreeding. They can also providemeans for exploring other uses ofMusa 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 diver-gence across species and cultivar groups, defining the best germplasmgrouping, assigning accessions to previously agreed diversity clusters,eliminating synonyms in genebanks, identifying useful sources of varia-tion for further genetic enhancement, managing intellectual propertyand marketing (Dheda Djailo et al., 2011; Hasan et al., 2011; Nunes deJesus et al., 2009; Saraswathi et al., 2011; Yan et al. 2011).
Channelière et al. (2011) claim that standardizing protocols and de-scriptors for characterizing Musa diversity will facilitate its use and as-sessment across environments, clustering accessions, and developing ameta-core subset. For example, using planting date data from variousexperiments in the tropics and subtropics, Fortescue et al. (2011)were able to determine the contributions of photoperiod and soilwater balance to time of flowering. This research also gave evidencethat banana has a facultative long-day response to photoperiod.
A standard descriptor list should include reliable morphologicaltraits and DNA markers capable of describing the diversity of the cropand its wild relatives. In this regard, Onyango et al. (2011) indicatedthat male bud, fruit and sucker traits were useful to distinguish withingenome groups and subgroups, and to isolate various subgroups withingenomegroupwhen assessingdiversity of bananas grown in East Africa.Likewise, Karamura et al. (2011) found that East African farmers use sizeand shape, texture, appearance, agronomic and commercial aspects todescribe and name their banana cultivars, thereby highlighting theneed of trait complementarity for assessing banana diversity. Variableplant and inflorescence descriptors can be also used for cultivar registra-tion as noted by Nunes de Jesus et al. (2009).
4. Crossbreeding and (quantitative) genetics
Lorenzen et al. (2012) report advances in introgressing host plant re-sistance to various pathogens and pests affecting the crop, particularly inAfrica. They acknowledge, however, thatmore efforts are needed for pro-ducing high-yielding cultivars showing multiple host plant resistanceand same fruit quality as local cultivars. To succeed in this endeavor,multi-environment trials (across locations and several crop cycles) areneeded to assess the performance of selected germplasm prior to theirrelease as new cultivars. Incidentally, Tenkouano et al. (2012b) wereable to establish the relationship between sample size, repeatabilityand level of confidence for phenotypic discrimination of Musa breedingmaterials. This information will assist the planning of Musa multi-environment trials.
The genetic base of triploid banana and plantain cultivars seems tobe very narrow because they likely ensued from 20 to 25meiosis events(and just 1 or 2 in Africa), whichmakes them very fragile in spite of theavailable diversity inMusawild species and diploid cultivars (MusaNet,2012). In this regard, Nyine and Pillay (2011) demonstrated that bananabreeding schemes relying on crossing wild diploids with geneticallyuniform landraces increased the genetic diversity of East African high-land bananas.
Broadening the genetic base of banana and plantain can be achievedby recurrent selection including cropwild relatives and diploid cultivarsin the source population (C0) and further use as parents of derived ma-terials from subsequent cycles of selection(C1 onwards) in crossbreed-ing schemes. Recurrent selection may consider progeny testing toimprove diploid populations and generate elite diploid hybrids to beused as parents in 3x–2x or 4x–2x crossing schemes. The use of DNAmarkers coupled with field trial data and sound biometrics could assistto estimate the diversity in diploid breeding stocks (Martins Pereiraet al., 2012) or induced mutants (Pestana et al., 2011). This approachalso provides means to selecting parents for the genetic enhancementof banana and plantain.
Diploid breeding remains an important activity in some of the pro-grams engaged in Musa genetic enhancement operating today in theworld (Amorim et al., 2013; Ortiz, 2013; Uma et al., 2011a). Toolssuch as marker-aided breeding, doubled-haploids, genomic selection
(including the use of genotyping-by-sequencing or next generation se-quencing) may further accelerate population improvement at the dip-loid level. Intermediate diploid-breeding sources may enhance geneticgains in the banana and plantain cultigen pools when including themin various 4x–2x reciprocal recurrent selection schemes aiming the re-lease of advanced triploid hybrids. Their impact will be measured interms of diversity of diploid sets of elite parents with required targettraits 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) toproduce triploids following 4x–2x crossings can be another breedingapproach for incorporating diversity in the cultigen breeding pool(Goigoux et al., 2013; Tomepke and Sadon, 2013). Moreover, Jennyet al. (2013) indicated that fertility could be achieved in 4x-CD derivedfrom treating their AB sterile ancestors with colchicine. They furtherused a resynthesized AABB hybrid (named ‘Kunnan’) in crossingschemes with AA and BB parents to produce AAB and ABB secondarytriploids, respectively.
The use of “brute force” can facilitate the crossbreeding of the almostfemale sterile ‘Cavendish’ cultivar – the most important banana for theexport trade today – as noticed before when hand pollinating a fewplants and getting seeds that germinated in Honduras and Nigeria. Forexample, Aguilar Morán (2013) indicated that he got 200 hybrid seedsafter mass-pollinating 20,000 ‘Cavendish’ bunches (consisting of about2 million fruits) with pollen from diploid parents. Only 40 of themhad, however, viable embryos, which produced 20 tetraploid hybrids.These hybrids were further used in 4x–2x crossing schemes for produc-ing two promising secondary triploid hybrids due to their host plant re-sistance to black Sigatoka and Fusariumwilt race 1 and with same fieldperformance as the ‘Cavendish’ “grandmother”.
Oselebe et al. (2010) noticed that 3x hybrid offspring are predomi-nantly produced in 4x–2x crossings while the reciprocal (2x–4x) yields2x hybrid offspring, thereby suggesting an unequal contribution of theparents to their offspring in the former scheme and double-reductionin the 4x parent for producing n gametes in the latter. Oselebe andTenkouano (2009) indicated previously that 2x offspring from 2x–4xcrossings had short plants, showed early flowering and producedsmall bunches. Hence, Oselebe et al. (2010) further redefined modelsfor predicting hybrid performance after interploidy crosses in Musa.Tenkouano et al. (2012a) provide formulae that describe the relation-ships betweenparents and their derived offspring for various interploidycrossing schemes. This information can be also used to estimate narrow-sense heritability for quantitative traits inMusa.
Additive genetic effects are significant on the expression of bunchweight, fruit filling time, fruit length, plant height and leaf numberwhile non-additive effects account for suckering behavior and fruit cir-cumference in secondary 3x hybrids derived from 4x–2x inter-mating(Tenkouanoet al., 2012c).Maternal general combining ability (GCA) ex-plains most of the variation in plant height and leaf number, therebysuggesting selection for both traits on the 4x female parent previousto their use in crossing blocks. Paternal GCA was the main contributorto variation in fruit filling time, bunch weight, and fruit length, therebyconfirming that these traits could be improved in diploid parentsthrough recurrent selection. Specific combining ability (SCA) effectswere significant for many traits, except fruit filling time. Hence, addi-tional genetic gain could be achieved through reciprocal recurrent se-lection considering tetraploid and diploid parents as independent butcomplementary population sources for this breeding scheme.
The euploid (2x and 4x) hybrid offspring derived by crossing 3xFrench plantains from West Africa and the 2x M. acuminata spp.burmannicoides ‘Calcutta 4’ segregated for the fruit parthenocarpyP1gene (Ortiz and Vuylsteke, 1995) because of the heterozygous locusof 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 othertwo dominant alleles (P2 and P3) needed for fruit parthenocarpy. They
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further hypothesized that P2 and P3, which appear to correlate withrates of genome-by-environment dependent seed development, whennon-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 thistrait inMusa. Very recently Ravi et al. (2013) assessed stomatal conduc-tance, cell membrane stability, leaf emergence rate, rate of leaf senes-cence, relative water content, and bunch yield under water scarcitybecause they were thought to be associated with enhanced adaptationto drought. They concluded that recording bunchweight under droughtstress should be the target trait for phenotyping.
5. Host plant resistance through crossbreeding
Fungi, bacteria, viruses, nematodes and insects are the main patho-gens and pests and causing pests of banana and plantain affectingtheir farmingworldwide. Lorenzen et al. (2009) advocate host-plant re-sistance as the most economic and sustainable means for plant healthmanagement. They admit that the gene introgression process, linkagedrag, complex trait genetics, lack of adequate screening protocols orhost-plant interactions (e.g. various strains and R gene specifity) mayconfound deploying host plant resistance in banana and plantain farm-ing systems. Furthermore,monitoring andpre-emptive breeding of hostplant resistance are mandatory to address any changes of pathosystemsbecause of emerging new strains due to recombination, mutation in, ormigration of the pathogen(s).
Breeding host plant resistance to black Sigatoka dominates the agen-da of most breeding programsworldwide (Abadie et al., 2009; Kobenanet al., 2009; Lorenzen et al., 2009; Menon et al., 2011; Ortiz, 2013 andreferences therein). Ortiz and Vuylsteke (1994a) found that host resis-tance to black Sigatoka in euploid plantain–banana hybrids was due toone major recessive gene (bs1) and two minor independent alleleswith additive effects (bsr2 and bsr3). Although Vroh-bi et al. (2009) indi-cated that host plant resistance was “quantitative” in 2x segregatingoffspring 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 bredhybrids pleased consumers andwere released for farming— particularlyin 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 (deGodoy et al., 2013).
Fusarium wilt, Moko/bugtok, blood bacterial wilt and Xanthomonaswilt are other biotic factors affecting various banana-based farming sys-tems. Resistant cultivars should be therefore bred and integrated into aplant health management approach to increase their potential successin fighting banana wilts (Daniells, 2011). The diploid banana hybridM53 seems to be a promising source for developingwilt-resistant culti-vars to Fusarium oxysporum f. sp. cubense or Foc (de Matos et al., 2011).The wild-seededM. acuminata ssp.malaccensis could be another sourcefor breeding host plant resistant to Foc tropical race 4 and it has beenused for developing two linkage maps to identify putative resistancemarkers 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 ofdead roots and high percentage of functional roots (Kumar et al. 2009).An index, which combines records of dead root percentage, large lesionnumber and nematode population density, could facilitate field selec-tion for host plant resistance to burrowing and spiral nematodes(Hartman et al., 2010). There could be, however, variability in reproduc-tive fitness and virulence of nematodes populations (Dochez et al.,2013a), which calls for host plant resistance screening of Musa germ-plasm across available strains. In this regard, Dochez et al. (2013b)found a burrowing nematode population from Mbarara (Uganda) thatbroke the host plant resistance of the 2x banana ‘Pisang Jari Buaya’, asource of resistance to this nematode known worldwide. Their results
highlight the importance of taking into account strain pathogenicitywhen engaging in banana and plantain germplasm enhancement forhost plant resistance to nematodes.
6. Fruit quality, human nutrition and health
Banana andplantain can provide essentialmicronutrients such as vi-tamin A to various populations in the developing world (Englbergeret al. 2003). The pulp of their fruits shows significant variation of provi-tamin A carotenoids (Davey et al., 2011), but very low iron and zinc con-tents (Davey et al., 2009d). Davey et al. (2009b) indicated, however,that treatments for the extraction and analysis of carotenoids in Musafruits need to prevent the breakdown and loss of fruit carotenoids,which are often unstable and sensitive to light exposure.
Diploid (AA) bananas fromPapuaNewGuinea had the highest levelsof β-carotenewhen assessing this germplasm alongwith dessert (AAA)and East African highland (AAA) bananas (Fungo and Pillay, 2011). Theorange pulp plantains (AAB) exhibit higher provitamin A carotenoidsthan the dessert (AAA) bananas with white-cream pulp (Davey et al.,2007). Ekesa et al. (2012) indicated that the bio-accessibility of provita-min A carotenoids depends however on the food recipes and other in-gredients included in the dish. The consumption of 100 g of steamedAfrican highland bananas or roasted plantains can provide 24 to 35%and 16 to 20% of the vitamin A recommended dietary allowances forpre-school children and women of reproductive age, respectively(Ekesa et al. 2013). These results suggest that the availability of Musagermplasmwhosemodest and realistic consumption levels may impactpositively on populations at risk of vitamin A deficiency.
Fungo and Pillay (2011) noticed a positive correlation between pulpcolor intensity and β-carotene concentration. This correlation wasstrong when using a yellowness index based on colorimetry vis-à-viscolor chart scores (Pereira et al. 2011). Davey et al. (2009c) also pro-posed using visible and near infrared reflectance spectroscopy forhigh-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 “clean-planting materials” for smallholders growing banana and plantain inthe tropics (Lule et al. 2013a,b). They can also facilitate the safe move-ment of germplasm across borders and accelerate multiplication of de-sired cultivars. Quality standards and plant health certification forpropagules and training for users are needed to ensure sustainablemicro-propagation of banana and plantain (Dubois et al., 2013), partic-ularly in the developing world, where most governments are yet toenact policy or provide incentives to facilitate the embracing of thistechnology by banana smallholders.
Cropmanagementmay benefit frommicro-propagation as observedin Kenya by Njuguna et al. (2011).While workingwith several hundredbanana farmers, they noted that crop yield increased from 10 t ha−1 to30 t ha−1 when using tissue culture-derived propagules, which weremore expensive than traditional suckers. Nonetheless, farmers usingthis biotechnology increased by 145% their income due to the enhancedcrop yield. Fungal endophytes can be further inoculated into banana tis-sue culture plantlets to extend the benefits of this “clean plantingmate-rial” technology (Dubois et al., 2006). In this way, the endophytes areincluded in the planting material sold to banana farmers.
8. Molecular cytogenetics and cytometry
Cytogenetic research inMusa during the first half of the 20th centurydealt with determining chromosome numbers of wild species and culti-vars, and chromosome pairing in hybrids (Pillay et al. 2012). This earlywork paved the path for today's molecular cytology.
Fluorescence in situ hybridization (FISH) has been advocated as anew tool inMusa breeding. It may help validating collinearity between
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parents included in crossing schemes, gene-mapping research, andidentifying chromosomal rearrangements between related Musa spe-cies and their putative derived cultivars (De Capdeville et al., 2009).For example, FISH with probes for satellite DNA sequences (which hada high sequence conservation within, and a high homology betweenMusa species), rRNA genes and a single-copy BAC clone 2G17 led toidentify distinct chromosome banding patterns in M. acuminata andM. balbisiana (Čížková et al., 2013),thereby determining the genomicconstitution in interspecific hybrids. This research also providedmeans for adding new cytogenetic markers in Musa.
Jeridi et al. (2011) used a new protocol for preparing chromosomesof AAB and ABB cultivars atmetaphase I that are suitable for genomic insitu hybridization (GISH). Their GISH research shows interspecific re-combination 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 awild banana (2x). This homoeologous pairing allow chromo-some exchanges between both species. Very recently Nunes de Jesuset al. (2013) indicated that the analyses of 221 Musa accessionmicrosatellites using simple-sequence repeats (SSR), internal tran-scribed spacer (ITS) polymorphism and flow cytometry further supportthe occurrence of recombination between A and B genomes.
Two-dimensional electrophoresis (2DE) and two-dimensional dif-ference gel electrophoresis (2D DIGE) with de novo tandemmass spec-trometry (MS/MS) sequence determination were used to characterizeinter- and intra-cultivar protein polymorphism in various Musa culti-vars (Carpentier et al., 2011). Together with multi-variate statistics,they were able to classify some cultivars according to their putative ge-nomes because the proteome does not always match the former.
9. DNA marker-facilitated diversity, origin andrelatedness assessments
DNA markers facilitate taxonomy, help cultivar true-to-type assess-ment and are useful for both population and quantitative genetics re-search in Musa (Ortiz, 2011). New microsatellites were widely used inthe past years for assessing diversity in bananas, plantains and other re-lated crop wild relatives (Table 1). Some of these new microsatelliteswere derived from expressed sequenced tags (EST) or from genomic se-quence surveys (GSS). Furthermore, a robust approach for genotyping inMusa – based on 19microsatellite loci that are scoredwith fluorescentlylabeled primers and using high-throughput capillary electrophoresisseparation with high resolution – became available recently (Hřibováet al., 2013). Garcia et al. (2011) also used a banana transcriptome data-base containing 42,724 EST (approx. 24 Mb of DNA sequence) to designprimers that were able to distinguish 32 variable number of tandem re-peat (VNTR) and 119 target region amplified polymorphism (TRAP) al-leles in 14 diploidMusa accessions. Their research shows the advantageof engaging EST-derivedmarkers for genetic diversity analysis and genediscovery 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
Table 1Recent Musa diversity assessment using new microsatellites.
Microsatellites Finding
Specific primers for 41 loci from 5 ‘Calcutta 4’bacterial artificial chromosome(BAC) consensi datasets
20 (out of 33M. acuminata
23 primers from cultivar ‘Gongjiao’ Polymorphismspecies/subsp
19 microsatellite loci scored using fluorescently labeled primers andhigh-throughput capillary electrophoresis separation with high resolution
Genotyping ptriploid bana
52 new primer pairs 34 microsateclones from ‘
229 primers based on genomic sequence data survey 26 markers a
in a subculture variant, while AFLP analysis attributed most of the invitro-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 ge-netic diversity and relationships among Musa accessions. For example,SRAP revealed that most genetic relationships among 29 polyploid ba-nana cultivars were correlated to their region of origin (Wei et al.,2011). The clustering of these cultivars agreed with the putative ge-nomes given to them. SRAP also exhibited more variation than AFLPwhen used in a sample of 40 Musa accessions (cultivars and wild spe-cies) relevant to the genetic enhancement of this crop (Youssef et al.,2011). Likewise, SRAP was able to discriminate among species withinEumusa and between triploid cooking bananas and plantains.
Diversity array technology (DArT) has been also successful for highthroughput genotyping and diversity analysis in Musa (Risterucciet al., 2009). This DNA hybridization-basedmolecularmarker techniquedetects simultaneously polymorphism at many genomic loci withoutneeding sequence information. Risterucci et al. (2009) indicated thatDArT provided the same genetic relatedness among Musa accessions,as previously noted with other DNAmarkers, but with a high resolutionand speed, plus a low cost.
There were recent in-country assessments with DNA markers thatprovided new insights about Musa diversity not included in previoussurveys, e.g. wild Musa in China (Qin et al., 2011), cultivars inIndonesia (Retnoningsih et al., 2011) or landraces in Myanmar (Wanet al., 2005). Similarly, Agoreyo et al. (2008) were able to discriminateplantains from Jamaica and Nigeria using the arbitrary primer polymer-ase chain reaction (AP-PCR) technique. AP-PCR also revealed the relat-edness among theNigerian plantain cultivars included in their research.
Table 2 lists other recent systematic botany research in Musa andrelated species as well as that for elucidating the origin of today's culti-vars using nuclear and plastid DNA markers. Based on chloroplast andmitochondrial genomes, De Langhe et al. (2010) hypothesized that“backcrossing” of an unknown parent could account for the unbalancedgenomes as well as inter-genome translocation found in some ediblecultivars.Moreover, after analyzingwith 22microsatellites the diversityof 561 Musa accessions, Hippolyte et al. (2012) determined the closestdiploid ancestors of the triploid ‘Cavendish’ and ‘Gros Michel’ dessertbananas. They also indicated that significant morphological variationin both did not ensue from recombination but from epigenetic regula-tions. Likewise, DNA sequence analysis of four genes in a set of 100 cul-tivars and wild accessions revealed multiple domestications of edibleMusa (Volkaert, 2011). The clusters of M. acuminata ssp. banksii/erransand M. acuminata subsp. malaccensis/microcarpa/zebrina/burmannica/siamea plus M. balbisiana appear to be involved in the domesticationof bananas and plantains. Diversity assessment with DNA markers ofmany diploid accessions from these subspeciesmay further assist to se-lect distinct parents for new crossbreeding schemes.
Very recently, Němcová et al. (2011) concluded that comparative se-quence analysis of single-copy genes may resolve the evolutionary his-tory ofMusaceae and could complement the analyses ensuing from theuse of both ribosomal DNA ITS1-5.8S-ITS2 region and DArT. In this re-gard, Hřibová et al. (2011) using ITS sequence-derived phylogenetic
Reference
) loci had polymorphism when screened across 21 diploidaccessions, contrasting in resistance to Sigatoka
Miller et al. (2010)
s assessed on 26 banana cultivars and 11 relatedecies
Wang et al. (2010)
latform tested and optimized on a set of 70 diploid and 38na accessions
Christelová et al. (2011b)
llites identified in expressed sequenced tags (EST) and BACCalcutta 4’ were validated in 22 wild and improved diploids
Amorim et al. (2012)
mplified in 15 banana accessions Ravishankar et al. (2012)
Table 2DNA marker-research onMusa relatedness and origin of modern cultivars.
DNA marker Finding Reference
Chloroplast and mitochondrial primer pairs from polymerase chainreaction–restriction fragment length polymorphisms (PCR–RFLP)
Primary centers of origin for both chloroplast and mitochondrial genomes ofM. acuminata andM. balbisiana revealed. Most cultivars had a singleM. acuminata cytotype (or combinations of chloroplast and mitochondrialgene-pools), while a singleM. balbisiana noted in interspecific hybrids
Boonruangrodet al. (2008)
5′ external transcribed spacer (ETS) rDNA sequence information Four gene pools amongM. acuminatawild types, whileM. balbisiana did notshow sequence divergence
Boonruangrodet al. (2009)
Nuclear ribosomal internal transcribed spacer (ITS) and chloroplastsequences
Monophyly ofMusaceae family as per phylogenetic analyses, which indicatedthatMusella and Ensete to be congeneric or closely related. None of the fiveMusa sections previously defined based on morphology was a monophyleticgroup but 2 infra-generic clades identified based on basic chromosomenumbers: x = 11 (Musa andRhodochlamys) and x = 10 (Callimusa), 9 (Australimusa) or 7 (Ingentimusa)
Li et al. (2010)
Microsatellites (coupled with morpho-taxonomy) Non-clear-cut distinction between Eumusa and Rhodochlamys, therebyconfirming their genetic relationships. ‘Matti’ (Eumusa) could be aparthenocarpic derivative ofM. acuminata ssp. burmannica, while 4 uniquewildM. acuminata from northeastern India during recent explorations hadhigh relatedness to Rhodochlamys
Durai et al. (2011)
DNA sequences from 19 unlinked nuclear genes Close relationship of Australimusa and Callimusa, but Eumusa and Rhodochlamysare not reciprocally monophyletic, thereby supporting merge between thesetwo sections
Christelová et al.(2011a)
Cytoplasmic and nuclear marker systems Putative family trees suggesting simple ways for the evolution of the today'shybrid cultivars and some wild genotypes lacking in genebanks
Boonruangrodet al. (2011)
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reconstruction divided the genus Musa into two distinct clades:Callimusa and Australimusa and Eumusa and Rhodochlamys. They alsonoticed thatmany intraspecific bananahybrids have conserved parentalITS sequences, thereby suggesting an incomplete concerted evolution ofrDNA loci. Their research also found one type of ITS sequence in someputative interspecific cultivars, which challenges their hybrid origin.
10. Genetic maps and marker-aided breeding
Heslop-Harrison (2011) argues very eloquently how knowledge-ledbreeding based on genomics and crop design will allow Musa “super-domestication”; i.e., inter-disciplinary research to find and evaluategenes for target traits and incorporate them into new cultivars. Certainlysuch search and use for genes and traits will significantly depend on theavailable diversity endowment in Musa genebanks, and on combiningexpertise with the aim of exploiting gene pools as well as on usingomics-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-intensiveMusa genetic enhancement. As indicated in a previous section, DNAmarkers are providing insights into Musa diversity, origin and related-ness and putative ancestors, whichwill assist selecting parents for cross-breeding. Horry (2011) also indicate that DNAmarkers are revealing thepopulation structure of Musa, clarifying gamete behavior, and givinginsights for manipulating ploidy and inter-specificity. New geneticmaps – based mostly on DArT and microsatellite s– became available
Table 3Banana genetic maps ensuing from recent DNA marker-based research.
DNA marker system(s) and mapping population(s) Map details
Diversity array technology (DArT) markers and microsatellites(SSR) on segregating (‘Borneo’ × ‘Pisang Lilin’) diploid F1
Synthetic map containTwo complete parenton LGs
Allele specific-polymerase chain reactions (AS-PCRs), DArT andmicrosatellites on two half-sib diploid banana breedingpopulations segregating for host plant resistance to burrowingnematode: (‘TmB2x 6142-1’ × ‘TmB2x 8075-7’) and(‘TmB2x 6142-1-S’ × ‘TmB2x 8075-7’)
The first maternal maand 4 AS-PCR marker(6142-1-S, 58 individuspanning across 698 ccomprised 196 DArTstotal length
DArT and microsatellites on segregating offspring from selfingPahang — the diploid parent of the genome-sequenceddoubled haploid
Dense genetic maps ufor anchoring 70% ofLGs 1 and 4 had markstructural heterozygo
in the last 3 years (Table 3). They, along with other segregating mate-rials (Rehka et al., 2011) and DNA marker systems such as newmicrosatellites derived from pyrosequencing-based transcriptome anal-ysis (Cruz et al., 2013), expressed sequence tags–simple sequencerepeats or EST–SSR (Mbanjo et al., 2012a) and single nucleotidepolymorphism (SNP) markers (Adesoye et al., 2012) may be used tolocate target genes, understand genetics of complex traits and be land-marks for MAB, thereby increasing Musa crossbreeding efficiency. Thecompletion of the banana genome sequence and next-generation se-quencing technology will further add new DNA markers and increasethe precision of MAB.
11. Mutations and “tilling”
Targeting induced local lesions in genomes (TILLING) could be a use-ful tool for reverse genetics in banana and plantain (Wang et al. 2012).For this purpose, Jankowicz-Cieslak et al. (2012) treated shoot apicalmeristems of banana with ethyl methanesulphonate (EMS) — a chemi-cal mutagen. They found a high density of GC–AT transition mutationsand noticed that genotypically heterogeneous stem cells resultingfrom the mutagenic treatment were rapidly sorted to fix a single geno-type in themeristem. Their research further demonstrated the accumu-lation of potentially deleterious heterozygous alleles, thereby suggestingthat 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.
Reference
ing 322 DArT and 167 SSRs on 11 linkage groups (LGs).al maps noting the structural rearrangements localized
Hippolyte et al. (2010)
p (6142-1, 81 individuals) included 121 DArT, 106 SSRs in 15 LGs adding to 670 cM. The second maternal mapals) based on 71 DArTs, 79 SSRs, and 2 AS-PCRs in 16 LGsM. The combined paternal map (139 individuals), 117 SSRs and 3 AS-PCRsover 15 LGs and 1004 cM as
Mbanjo et al. (2012b)
sed with in excess of 1000 DArT and microsatellitesthe genome assembly to the 11Musa chromosomes.ers deviating fromMendelian ratio, thereby suggestingsity (or translocations)
Carreel et al. (2013)
164 R. Ortiz, R. Swennen / Biotechnology Advances 32 (2014) 158–169
(2010) found in excess of 800 novel alleles in 80 accessions using eco-TILLING, which they claim can be a robust and accurate platform forthe discovery of polymorphisms in homologous and homoeologousgene targets. Their research was able to identify two SNPs that couldbe deleterious for the function of a gene putatively important for pho-totropism. Eco-TILLING can facilitate selectingMusa germplasm for fur-ther cross- or mutation breeding.
12. The genome sequencing provides new insights andDNAmarkers
The banana genome sequencingwas a global multi-partner endeav-or that took about one decade to accomplish its task. This deciphering ofthe banana genome will lead to the generation and sharing of knowl-edge on its genome organization, as well as in identifying DNAmarkersfor further use in research and crossbreeding. For example, the bacterialartificial chromosomes (BAC)-end sequencing ofM. acuminata doubledhaploid (DH)‘Pahang’ revealed that most frequent repeated sequenceswere homolog to ribosomal RNA genes (particularly 18S rRNA) andthat Ty3/gypsy type monkey retro-transposon was the most common(Arango et al., 2011). The repetitive part of a genome can be a usefulsource for developing DNA markers for further use in Musa diversityanalysis and MAB (Hřibová and Doležel, 2011). In this regard, Hřibováet al. (2010) characterized thoroughly the repeat component of the ge-nome of the diploidwild accession ‘Calcutta 4’. Their research improvedthe knowledge about the organization of banana chromosomes, andprovided the sequence resources for repeat masking and annotationfor the genome sequencing.
Comparative sequence analysis of resistance gene analog (RGA)clusters estimated the degree of sequence conservation and mecha-nisms of divergence at the intraspecies level (Baurens et al., 2010) aswell as providedmeans for identifying Argonaute gene sequences in ba-nanas (Teo et al., 2011). The Argonaute proteins are involved in RNA si-lencing. 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 usedfor drafting the 523-megabase draft sequence of banana, which be-came the first monocotyledon high-continuity whole-genome se-quence outside Poales (D'Hont et al., 2012). This sequence alsorevealed new whole-genome duplications in the Musa lineage amongmonocots. Likewise, this reference genome sequence advanced Musagenetics. For example, further analysis led to identifying Musa genescoding for 89 nucleotide-binding site (NBS) leucine-rich repeat pro-teins. Moreover, transcriptomics research provided means to finding372 differentially expressed banana genes interacting with the fungalpathogen Mycosphaerella fijiensis, which causes black Sigatoka. Like-wise, RNA-Seq analysis showed that receptor-like kinase genes wereupregulated in a partially resistant interactionwithM. fijiensis, and indi-cated a strong transcriptional reprogramming in mature green fruitsafter ethylene treatment. Transcription factors were regulating 597genes, e.g. upregulated genes encoding cell wall's modifying enzymes,three down-regulated starch synthase genes and one upregulated β-amylase gene. This sequencing of the banana should be regarded as amilestone in Musa genetic resource enhancement research and it willbe surely further use to gain insights for the betterment of this crop.For example, Maldonado-Borges et al. (2013) annotated differentiallyexpressed genes during somatic embryogenesis of M. acuminata ssp.malaccensis ITC 250 in theMusa genome. Knowing these genes may as-sist in studying deeply themechanisms involved throughout the differ-ent stages of Musa embryogenesis.
13. Other genetic and omics-based resources
As noted above microsatellite polymorphism seems to be abundantin Musa and such DNA markers have been already used in biodiversityassessments, systematics and mapping, while their potential use inMAB is yet to be realized. Retroelement-related sequences are also
abundant and can be exploited as anonymous genetic markers in ba-nana, plantain and their wild relatives. Further research led to designprimers – from genomic andEST databases – thatwere useful for charac-terizing sequences containing nucleotide binding sites (NBS) andleucine-rich repeat (LRR) motifs, which seem to be associated withhost plant resistance genes (Azhar andHeslop-Harrison, 2008; Emediatoet al., 2009; Lu et al., 2011; Miller et al., 2008), or with genes enhancingadaptation to abiotic factors such as drought, heat and salinity.
Candidate gene discovery was undertaken recently for analyzingdifferential 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) fromthe genomic DNA of the tetraploid banana hybrid ‘Goldfinger’, whichshow resistance to F. oxysporum f. sp. cubense causing Fusarium wilt.The identification and cloning of such genes will assist in Musa geneticenhancement.
Emediato et al. (2009) characterized RGAs inM. acuminata cultivarswith distinct host plant resistance to M. fijiensis causing black Sigatoka,while Sulliman et al. (2012) assessed the diversity of defense gene ana-logs (DGA) associated with host plant resistance to various Sigatoka inbanana. Their interestwas to identify specificmarkers that could be fur-ther use in selecting host plant resistance genes controlling these fungi.Their complete gene sequence characterization could be also useful forMusa genetic engineering.
Transcriptomics research provided further analysis of the expressionof NBS-LRR RGA in resistant wild diploid ‘Calcutta 4’ and susceptibletriploid ‘Cavendish’ banana cultivar, when inoculated in vivo or notwith conidiospores of Mycosphaerella musicola causing yellow Sigatoka(Emediato et al., 2013). Similarly, the study of time-course expression ofdefense genes in banana against the root-lesion nematode Pratylenchuscoffeae revealed that mRNA levels of the chalcone synthase gene werehigher in the roots of resistant ‘Karthobiumtham’ than in those of sus-ceptible ‘Nendran’ (Backiyarani et al., 2011). This kind of research pro-vides new knowledge on host plant resistance mechanisms and mayidentify candidate genes for further used inMAB or genetic engineering.
Very recently Passos et al. (2013) used 454 GS-FLX Titanium tech-nology to determine the sequence of the gene transcripts from both‘Calcutta 4’ and ‘Cavendish’, thereby increasing the public domainMusa ESTs. This transcriptomewill be also useful for gaining knowledgeon banana and plantain performance under stress.
MNPR1A and MNPR1B –which are two novel full-length non-expressor of pathogenesis-relatedgenes1 (NPR1) –were isolated from ba-nana by application of the PCR and rapid amplification of cDNA end(RACE) techniques (Endah et al., 2008). They had distinct expressionprofiles, as determined by quantitative real time (qRT)-PCR, after eitherelicitor treatment of by interacting with F. oxysporum f. sp. cubense. Thetolerant banana cultivar GCTCV-218 expressed greatly and earlyMNPR1A while MNPR1B was highly responsive to salicylic acid but notto methyl jasmonate in both GCTCV-218 and the susceptible bananacultivar ‘Grand Naine’.MNPR1A expressionwas also found to be directlyrelated to pathogenesis-related (PR) gene expression that provides hostplant resistance to some fungi.
As noted by Podevin et al. (2012), the high sensitivity of gene ex-pression analysis by reverse transcriptase real-time or qRT-PCR requireshowever both normalization using multiple housekeeping or referencegenes, and careful selection of these reference genes. Otherwise, resultsmay not be reliable. Their research showed that the accession or culti-var, plant materials, primer set, and gene identity influence the robust-ness and outcomeof RT-qPCR analysis. The reference genes EF1, TUB andACT can assist for normalization of gene expression data for Musa leafsamples taken in a greenhouse, whereas the best combination of refer-ence genes (L2 and ACT genes) was still suboptimal for Musa leaf sam-ples from in vitro plants.
Peraza-Echeverria et al. (2009) isolated and characterized the entirecDNA sequences of RGC2 and RGC5, which are partial non-TIR-NBS se-quences, from the root transcriptome of M. acuminata ssp. malaccensis.
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Both partial sequences are in the same cluster, within the ancient non-TIR clade N1.1, with the known but phylogenetically distant FusariumR genes I2 and Fom-2. Searchingwithin this clade may assist identifyingother R genes in Musa.
Knowledge on the expression pattern of ripening genes and mecha-nisms regulating them at ripening may facilitate the post-harvestcontrol of fruit maturity, thereby reducing the fruit spoilage duringtransport and storage. A MADS-box transcription factor gene, which be-longs to the AGAMOUS subfamily and designated asMuMADS1, was iso-lated from banana fruits (Liu et al., 2009).MuMADS1may be induced byethylene biosynthesis associated with postharvest banana ripening, andby exogenous ethylene. Elitzur et al. (2010) cloned and further charac-terized six full-length MADS-box genes from Grand Naine. Half ofthem (MaMADS1, 2, and 3) were highly expressed in fruit only, whilethe other three were expressed in fruit and other organs. Two indepen-dent ripening programs were employed in the pulp and peel and in-volved the activation of mainly MaMADS2, 4, and 5 and later on alsoMaMADS1 in the pulp, and mainly MaMADS1, and 3 in peel. The cDNAofMA-ACS1 – amajor ripening regulating gene in banana – its transcriptandprotein accumulation patterns of this genewere investigated by RoyChoudhury et al. (2011). Their research provided new insights about theexpression pattern and transcriptional regulation of one ofMA-ACS1. Jinet al. (2011) were also able to identify more genes from banana at fruitripening using suppression-subtractive hybridization with cDNAmicro-array. This research relates these genes to early stages of post-harvestbanana ripening, though these authors acknowledge that they need tolink them to their corresponding translated proteins and defining theirroles.
Water scarcity and unusual annual rainfall patterns, which may bebrought by climate change, will affect banana and plantain productivity.Vanhove et al. (2012) developed a drought-screening test for in vitroplants based on amild osmotic stress. The cooking banana (ABB)‘Cachaco’showed the smallest stress-induced growth reduction among the fivetriploid cultivars (dessert bananas, East African highland bananas andplantains) included in this test. The leaf proteome analysis of this cultivarrevealed that the respiration, metabolism of reactive oxygen species(ROS), and several dehydrogenases involved in nicotinamide adenine di-nucleotide (NAD)/NADH homeostasis participate actively in allowingplants under stress to reach a new balance. In this regard, Carpentieret al. (2008a) noted that proteomic research in a non-model plantsuch asMusa species dealt with various issues such as sample prepara-tion, analysis and interpretation of complex data sets, protein identifica-tion via mass spectrometry, plus data management and integration.Vertommen et al. (2011a) also provides an overview on alternativetechniques for membrane proteomics in Musa. They indicated thatgel-based protein separation should not be used in this research, and in-stead an approach that includes peptide separation should be pursuedto increase resolution. Further research by Vertommen et al. (2011b)led to establishing a workflow for peptide-based proteomics of thebanana plasma membrane. Proteomics was also used by Swennenet al. (2011) to study the response of meristem cultures from variousbanana cultivars to osmotic stress related to cryopreservation. Theywere able to construct a proteomemap using two-dimensional gel elec-trophoresis. It includes 637 proteins and some of those differentiallyexpressed under osmotic stresswere further characterized using genet-ic engineering.
Davey et al. (2009a) used cross-hybridization to Affymetrix oligo-nucleotide GeneChip® microarrays for profiling Cachaco's leaftranscriptome after drought stress. They found 2910Musa gene homo-logues when hybridizing to the Affymetrix Rice Genome Array. Thedrought-responsive transcripts included various functional classesassociated with plant stress responses, and a range of regulatorygenes involved in coordinating abiotic stress responses. There were 52drought-sensitive Musa transcripts homologous to genes underlyingQTL for enhanced adaptation to drought and cold in rice, of which twowere within a single gene. More recently, suppression subtractive
hybridization was used for identifying differentially expressed genesin leaves of the M. balbisiana accession ‘Bee Hee Kela’ during waterstress (Ravishankar et al., 2011). Further BLAST search of 50 non-redundant sequences established their similarity with lipoxygenase,rubisco activase, glycine dehydrogenase, catalase and ethylene respon-sive factor, which seem to be involved in cellmembrane integrity, signaltransduction and metabolism in response to dehydration.
The structure and regulation of the Asr gene family in banana andplantain was investigated by Henry et al. (2011) because abscisic acid(ABA), stress, ripening (ASR) proteins are a family of plant-specificsmall hydrophilic proteinswith an unknown function that enhances ad-aptation to drought. This Musa Asr gene family had at least four mem-bers exhibiting two exons and one intron structure and the ABA/waterdeficit stress (WDS) domain — both of which are characteristic of Asrgenes. The genes mAsr1 and mAsr3 were induced by osmotic stressand wounding, while mAsr3 and mAsr4 were induced by exposure toABA in meristem culture. The mAsr3 protein displayed the highest var-iation for amino acid sequence and expression pattern, which makes itthe most interesting candidate for further functional research.
Transcriptomics and proteomics research are complementary be-cause each technique focuses on a subset of genes and proteins.Carpentier et al. (2008b), afterworkingwith banana, indicated that pro-teomics yields a better characterization for poorly characterized species,but transcriptomics can be usedwhen researching low-abundant or hy-drophobic proteins.
14. Genetic engineering
The non-conventional Musa breeding also includes genetic engi-neering, protoplast culture and somatic hybridization (Chen et al.,2011), but the first has shown the most promising results since itsfirst use about 20 years ago (May et al., 1995; Sági et al., 1995). Trans-genics can facilitate the introduction of non-Musa genes into thecultigen pool, and should be incorporated into banana and plantainbreeding programs when lacking natural variation for such trait(s) orfor the genetic amelioration of sterile cultivars. Despite the presence offew research teams on Musa genetic engineering (Remy et al., 2013),noteworthy advances were made both in somatic embryogenesis andgenetic engineering of banana and plantain in recent years (Table 4).
Micronutrient deficiency, particularly of vitamin A, iron and zinc, re-mains amajor challenge because affect several hundredmillions of peo-ple, particularly in the developing world. Dale et al. (2013) have usedgeneric engineering to increased micronutrient content in banana tofight malnutrition in Uganda, where its low-nutrient fruits are usedfor the country'smain dish:matooke. Thefirst harvest of their transgen-ic banana with enhanced vitamin A content was in the South Johnstonearea south of Cairns (Australia). These transgenic bananas over-expressing phytoene synthase (PSY) with either constitutive or fruit-preferred promoters had 15-fold pro-vitamin A levels than the “wild-type”. One of the two phytoene synthase genes was from a naturallyhigh pro-vitamin A banana while the other was a maize gene used fordeveloping ‘Golden Rice 2’ (Paine et al., 2005). The Australian trial wasa proof-of-concept for one of the combinations of transgenes increasingpro-vitamin A. The most promising transgenic bananas with elevatedpro-vitamin A are now undergoing field-testing in Uganda. Further re-search by Mlalazi et al. (2012) led to the isolation and characterizationof PSY genes from the orange-pulp cultivar Auspina, which may beused for breeding new cisgenic or intragenic banana cultivars with en-hanced pro-vitamin A content. This genetic engineering of bananawith sequences originating from its own genomemay increase its pub-lic acceptability.
Transgenic Musa breeding was able to achieve enhanced resistanceto Xanthomonas campestris pv.musacearum causing the devastating ba-nana Xanthomonas wilt in the Great Lakes Region of Africa (Tripathiet al., 2010). Its spread threatens the food security and income of mil-lions of East and Central Africans who depend on this crop for both.
Table 4Advances inMusa somatic embryogenesis and genetic engineering.
Germplasm Achievement Reference
Musa acuminata hybrid CNPMF2803-01 and‘Nanicão’ (Cavendish)
Agrobacterium-mediated transient expression system in immature fruits of banana,which may allow studying gene function and validating those expressed in banana fruit
Matsumoto et al. (2009)
Cavendish (AAA): ‘Grand Naine’ and ‘Williams’ Embryogenic response depends on both genotype and developmental stage of the budsfrom which explants are excised. Selected embryogenic calluses that are successfullyestablished on proliferation medium led to embryogenic cell suspensions (ECS) withgood regeneration capacity
Youssef et al. (2010)
‘Virupakshi’ (AAB banana) Plantlets were regenerated from somatic embryos derived from embryogenic cells ofcalli from immature male flower explants
S. Elayabalan (TNAU, India)et al. (unp.)
‘Nanjangud Rasbale’ (syn. ‘Rasthali’, AAB banana) Highly regenerative ECS were established from floral meristems and thereafter transformedthe antimicrobial peptide (AMP) gene cloned from onion seeds
Mohandas et al. (2011b)
‘Nanjangud Rasbale’ Transgenic plants with AMP and constructs from pCAMBIA 2301 had a small lesion areaafter inoculating with Fusarium oxysporum f. sp. cubense race 1
Mohandas et al. (2011a)
‘Nanjangud Rasbale’ Agrobacterium rhizogenes sonication-aided transformation (≤2.4%) of shoot bud explants Venkatachalam et al. (2011)‘Lady Finger’ (AAB banana) Apoptosis-inhibition-related genes confer resistance to Fusarium wilt race 1 Paul et al. (2011)‘Nangka*’ (AAB) Rice thaumatin-like protein gene enhances resistance to Fusarium wilt race 4 in transgenic
plantsMahdavi et al. (2012)
‘Dwarf Brazilian’ (AAB banana) ECS, which was initiated from immature flowers, transformed using Agrobacteriumtumefaciens containing one construct derived from the replicase-associated protein (Rep)gene of the Hawaiian isolate of Banana bunchy top virus (BBTV)
Borth et al. (2011)
‘Karibale Monthan’ (ABB group) Transgenic banana plants with normal phenotypes constitutively overexpressing novelbanana SK3-type dehydrin geneMusaDHN-1 had enhanced adaptation to drought- andsalt-stress after in vitro and ex vitro assays
Shekhawat et al. (2011)
‘Mpologoma’ (AAA East African highland banana)and ‘Sukali Ndiizi’ (AAB banana)
ECS-derived transgenic plants expressing the Hrap gene from Capsicum pepper under theregulation of the constitutive CaMV35S promoter did not show any infection symptomsafter artificial inoculation of potted plants with banana Xanthomonaswilt in the screenhouse
Tripathi et al. (2010)
‘Sukali Ndiizi’ and ‘Nakinyika’ (AAA East Africanhighland banana)
Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene from sweet pepper(Capsicum annuum) under the regulation of the constitutive CaMV35S promoter showed highresistance to Xanthomonas campestris pv.Musacearum (no disease symptoms after artificialinoculation of in vitro plants in laboratory or in potted plants in the screenhouse)
Namukwaya et al. (2012)
‘Gonja manjaya’(AAB plantain) Development of an embryogenic cell suspension (ECS), regeneration, and transformation afterestablishing ECS using highly proliferative multiple buds
Tripathi et al. (2012)
‘Gonja’ Transgenic host plant resistance to burrowing nematode with maize cystatin that inhibitsnematode digestive cysteine proteinases and a synthetic peptide that disrupts nematodechemoreception
Roderick et al. (2012)
‘Grand Naine’ Transgenic banana plants without marker gene used for selection using the Cre-loxsite-specific recombination system
‘Gros Michel’ (AAA) The potential of rice chitinase genes to enhance host plant resistance to black Sigatokawas shown along with the usefulness of the leaf disk bioassay for early screening intransgenic banana
Kovács et al. (2013)
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The best 65 transgenic plants expressing the hypersensitivity response-assisting protein (Hrap) gene from sweet pepper and not showing anyinfection symptoms after artificial inoculation of potted plants with ba-nana Xanthomonas wilt in the screenhouse were included in confinedfield-testing near Kampala in Uganda. After testing them as motherplants and first ratoon plants, 12 transgenic lines were rated as havingabsolute resistance (Tripathi, 2012). Their plant phenotype and thebunch weight and size were similar to non-transgenic counterparts.These lines will undergo multi-location trials in Uganda and will be fur-ther assessed for environmental and food safety according to Uganda'sbiosafety regulations, risk assessment and management, plus proce-dures for seed registration and release. They may be shared withfarmers in 2017.
15. Outlook: genetic enhancement to meet global demand in achanging climate
Banana and plantain breeding depends on sustaining genetic gains.There have been significant advances in Musa crossbreeding, omics-led genetic enhancement and genetic engineering in recent years assummarized in above sections. Such effort should translate into new ba-nana and plantain cultivars that can address themain issue humankindfaces in this 21st century: meeting the increased demand for food by agrowingpopulationwhowill be improving both their health andwealthwhile adapting to a changing climate. The genetic betterment of Musashould therefore emphasize improvements in crop productivity, hostplant resistance and enhanced use-efficiency of inputs such as water
and fertilizers to ensure it contributes to the sustainable intensificationof banana and plantain farming.
Omics research will continue providing new useful insights for Musagenetic enhancement and may generate new tools for improving furtherthe efficiency of banana andplantain breeding, e.g. bydevelopingmarkersas selection aids or new genomic-led methods that can accelerate the re-lease of cultivars adapted to farming systems where the Musa cropthrives. In this regard, as noted by Roux et al. (2011) it will be a key tobridge the gap between omics and crossbreeding, particularly prioritizingthe following areas to facilitate cooperative research undertakings:collecting and characterizing germplasm, reliable and high throughputphenotyping 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 ba-nana and plantain cultivars, reducing farming costs and enhancingMusafarming systems through eco-efficiency.
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