Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB)

Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile.     

Genetic transformation of Cymbidium orchid by particle bombardment

 A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse. Received: 20 July 1998 / Revision received: 2 December 1998 / Accepted: 17 December 1998     

Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain

The most devastating disease currently threatening to destroy the banana industry worldwide is undoubtedly Sigatoka Leaf spot disease caused by Mycosphaerella fijiensis. In this study, we developed a transformation system for banana and expressed the endochitinase gene ThEn-42 from Trichoderma harzianum together with the grape stilbene synthase (StSy) gene in transgenic banana plants under the control of the 35S promoter and the inducible PR-10 promoter, respectively. The superoxide dismutase gene Cu,Zn-SOD from tomato, under control of the ubiquitin promoter, was added to this cassette to improve scavenging of free radicals generated during fungal attack. A 4-year field trial demonstrated several transgenic banana lines with improved tolerance to Sigatoka. As the genes conferring Sigatoka tolerance may have a wide range of anti-fungal activities we also inoculated the regenerated banana plants with Botrytis cinerea. The best transgenic lines exhibiting Sigatoka tolerance were also found to have tolerance to B. cinerea in laboratory assays.     

A combined use of microprojectile bombardment and DNA imbibition enhances transformation frequency of canola (Brassica napus L.)

Efforts to increase the frequency of recovered homozygous transgenic B. napus plants from direct DNA transformation treatments led to the development of a method of combined microprojectile bombardment and desiccation/DNA imbibition. The combined method was compared to individual treatments in two experiments utilizing microspore-derived embryo hyocotyls as targets for the -glucuronidase (GUS) and NPT II genes. Both the transient gene expression of -GUS and the stable transformation by NPT II demonstrated that the combined use of microprojectile bombardment and desiccation/DNA imbibition yielded more transgenic plants (at least three-times more) than either individual transformation protocol. In a histochemical analysis for -GUS activity, an average of 37% of the hypocotyls receiving the combined treatment displayed a positive response, whereas only 8% of the hypocotyls showed a positive response following microprojectile bombardment alone. The hypocotyls obtained by the joint treatment also showed more multisite expression of the -GUS gene per hypocotyl than those treated only with microprojectile bombardment. Southern analysis of NPT II gene integration into subsequently-derived secondary embryos indicated that the transformation efficiency of the combined treatment was 2% in comparison to 0.6% for that of the singular microprojectile bombardment. The number of inserts integrating per transformation event appears to be independent of the transformation methods. Neither of the marker genes was expressed in hypocotyls treated only with desiccation/DNA imbibition. Utilization of hypocotyl regeneration from microspore-derived embryos via a secondary embryogenesis system provided a reliable method for producing transgenic plants. The combined use of microprojectile bombardment and desiccation/DNA imbibition proved to be an efficient approach to obtain homozygous transgenic canola plants.     

Plant regeneration from protoplasts of dessert banana cv. Grande Naine (Musa spp., Cavendish sub-group AAA) via somatic embryogenesis

Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine (Musa spp., Cavendish sub-group AAA) were achieved through somatic embryogenesis. Protoplasts were isolated from cell suspensions at a yield of 3᎒⁷ protoplasts/ml packed cell volume (0.5 g). For the induction of cell divisions, two banana cell suspensions, SF265 (AA) and IRFA903 (AA), were used as feeder layers. SF265 (AA) was found to be more efficient for inducing cell divisions than IRFA903 (AA). The first embryogenic cell suspensions were established from protoplast-derived microcalli. The transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators. Plant regeneration was achieved in the same regeneration medium.     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA)

Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV) was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana.     

Comparative analysis of phenotypic and genotypic diversity among plantain landraces (Musa spp., AAB group)

Genetic diversity amongst 76 plantain landraces has been studied using RAPD analysis at two levels of intensity and compared with groupings based on phenotypic indices and morphotype. There was a good correlation (R²=0.78) between estimates of genetic diversity based on 76 RAPD bands and 164 RAPD bands. However, there was a poor correlation between RAPD-based estimates of genetic diversity and a phenotypic index based on agronomic characters. There was also a poor correlation between RAPD analyses and morphotype group (based on bunch type and stature). These results suggest that the traditional designations of plantain landraces based on morphotype do not provide a true reflection of overall genetic divergence. Similarly, classification systems using phenotypic indices based on agronomic characters may not provide accurate taxonomic differentiation. The level of genetic divergence within morphogroups based on bunch type suggests that True Horn plantains are derived from False Horn plantains which in turn are derived from French plantains. Genetic divergence was found to be generally quite low within the plantain landrace genepool, which is consistent with the proposed evolution of this germplasm through somatic mutation of a relatively small number of introductions. However, putative synonyms/duplicates have been shown to be genetically distinct. In contrast, a group of 12 landraces have been identified that are highly distinct from one another (showing 20–35% dissimilarity). Fertile members of this group may be useful for generating genetically diverse 2x and 4x breeding populations that can be used in breeding secondary triploid hybrid plantain varieties. Received: 8 January 2000 / Accepted: 2 March 2000     

大田威廉斯香蕉叶片光合特性研究

用Li-6400型便携式光合作用测定系统研究大田威廉斯香蕉叶片光合日变化、光响应曲线和CO₂响应曲线,运用非直角双曲线模型并结合SPSS软件的非线性回归估算其光合作用参数。结果表明,香蕉叶片光合日进程呈单峰曲线,没有明显的午休现象,最大净光合速率出现在10:20左右;光饱和点与光补偿点分别为778.19和61.09μmol/m²·s,表观量子效率为0.0414;CO₂饱和点与补偿点分别为608.97和72.13μmol/mol,羧化效率为0.094。此外,对影响香蕉光合速率的主要环境因子分析表明,在土壤供水充分的条件下,限制光合速率的因子是光合有效辐射而非气孔导度。     

Embryogenic cell suspensions from the male flower of Musa AAA cv. Grand nain

There are very few reports on the establishment of long-term embryogenic cell cultures of banana, especially of triploid cultivars of commercial interest. Embryogenic cell suspensions were prepared using the cultivar Grand nain, the most widely grown dessert banana in the world. After culture for 5 or 6 months of immature male flowerbuds adjacent to the floral apex, yellow, compact calluses and white, friable embryogenic tissues were induced. Suspension cultures were initiated from embryogenic tissues placed in liquid medium. The packed cell volume (PCV) of the suspensions increased 2- to 5- fold with each monthly culture cycle. Plating of the embryogenic suspensions resulted in approximately 370×10³ embryos per ml of PCV. Depending on the size of embryos, 3 to 20% germination was observed. A histological survey of cell suspensions and embryo development was carried out. Cellular aggregates with cells displaying typical embryogenic features were formed. Most of the somatic embryos were probably of unicellular origin.     

Effects of microprojectile bombardment on embryogenic suspension cell cultures of maize (Zea mays L.) used for genetic transformation

We have investigated the interaction between tungsten and gold microprojectiles with suspension-culture cells of maize used for genetic transformation. Particle size measurements were evaluated before and after DNA precipitation to determine mean particle size and the effect of DNA precipitation on particle aggregation. Following particle bombardment, metal foils were examined by scanning electron microscopy to visualize dispersion of individual particles and aggregates. Particle penetration into suspension-culture cell clusters was examined in paraffin-embedded bombarded cells serially sectioned and viewed with light microscopy and by energy dispersive X-ray microanalysis. Acridine-orange-stained bombarded cells were examined to observe cellular response to particle penetration. Transient expression of reporter genes C1 and B and GUS, (-glucuronidase) were used to assess effects of particle bombardment on embryogenic cell types. Autoradiographic analysis of the transformable suspension cell culture SC82 (see Gordon-Kamm et al. 1990, Plant Cell 2, 603–618) was conducted to evaluate the S-phase and mitotic indices in embryogenic and nonembryogenic cells throughout a subculture passage and in response to DNA/particle delivery. The results of these investigations are discussed relative to cytodifferentiation of suspension cell clusters and recovery of transformed clonal sectors.Abbreviations GUS -glucuronidase - FAA formaldehyde-acetic acid-alcohol - SEM scanning electron microscopy     

Genetic transformation of Leymus chinensis with the PAT gene through microprojectile bombardment to improve resistance to the herbicide Basta

Chinese leymus [Leymus chinensis (Trin.) Tzvel.] is a perennial grass (tribe Gramineae) that is widely distributed throughout northern China and Mongolia where it is produced as a forage product. Severe production losses due to weed growth have serious economic consequences, and as non-selective herbicides not only kill the weeds but are also harmful to this forage grass, the introduction of a foreign gene for resistance to the herbicide Basta is necessary since this species lacks herbicide resistance. We have investigated the transformation of a gene for phosphinothricin acetyltransferase (PAT) through microprojectile bombardment in Chinese leymus. Calli from immature inflorescences cultured on N6 medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/l of glutamine were bombarded. The bombarded calli survived on selection medium with 1.0 mg/l of phosphinothricin (PPT). Twenty-three plantlets regenerated from resistant calli on differentiation medium supplemented with 1.0 mg/l 6-benzylaminopurine, 1.0 mg/l kinetin, and 1.0 mg/l PPT, and five of these regenerated plantlets survived on rooting medium with 1.0 mg/l of PPT. PCR and Southern blotting analyses indicated that the PAT gene had been integrated into the genomes of two Chinese leymus plantlets and that the gene was stably transferred to its clonal offsprings. There were no other phenotypic effects associated with transgene expression during vegetative growth except tolerance to the herbicide Basta.The Biotechnology of Pasture Plant Program is funded by the Key Project of the Chinese Academy of Sciences (KSCX1-08)     

Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed

A highly efficient and reproducible transformation system for rice (Oryza sativa L. cv. Taipei 309) was developed using microprojectile bombardment of highly regenerative, green tissues. These tissues were induced from mature seeds on NB-based medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high concentrations of cupric sulfate under dim light conditions; germinating shoots and roots were completely removed. Highly regenerative, green tissues were proliferated on the same medium and used as transformation targets. From 431 explants bombarded with transgenes [i.e. a hygromycin phosphotransferase (hpt) gene plus one of a wheat thioredoxin h (wtrxh), a barley NADP-thioredoxin reductase (bntr), a maize Mutator transposable element (mudrB) or -glucuronidase (uidA; gus) gene], 28 independent transgenic events were obtained after an 8- to 12-week selection period, giving a 6.5% transformation frequency. Of the 28 independent events, 17 (61%) were regenerable. Co-transformation of the second introduced transgene was detected in 81% of the transgenic lines tested. Stable integration and expression of the foreign genes in T₀ plants and T₁ progeny were confirmed by DNA hybridization, western blot analyses and germination tests.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-Benzylaminopurine - BNTR Barley NADP-thioredoxin reductase - DTNB 2,5-Dithiobis-(2-nitrobenzoic acid) - HPT Hygromycin phosphotransferase - IE Immature embryos - MS Murashige and Skoog - PCR Polymerase chain reaction - uidA -Glucuronidase gene - WTRX h Wheat thioredoxin h Communicated by I.S. Chung     

Assessment of a species-specific element (Brep 1) in banana

 The nuclear genome of wild-type banana accessions was investigated for repetitive elements. We report here the occurrence, in the banana genome, of a sequence family of species-specific repetitive elements: Brep 1. This sequence family is distributed throughout the Musaceae with various copy numbers. The two species Musa acuminata and M. schizocarpa carry the highest copy numbers in contrast to M. balbisiana and tested representatives of different other sections. PCR primers were defined in the core consensus sequence for specific amplifications, which allow representatives of this sequence family to be easily detected in wild and cultivated banana clones. Sequence data were analysed and hypotheses on the evolution of banana cultivars from the wild-type banana clones are discussed. Received: 17 January 1997 / Accepted : 7 March 1997     

Morphohistological study of the different constituents of a banana (Musa AAA, cv. Grande naine) embryogenic cell suspension

Five types of cellular aggregates have been characterised in embryogenic cell suspensions of banana (Musa AAA Grande naine cv.). Type I corresponded to isolated cells or to small cell aggregates. Type II were composed of embryogenic cells. Type III can be distinguished from type II due to the presence of peripheral proliferation zones with embryonic cells. Type IV were composed of protodermic masses histologically comparable to proembryos. Type V were nodules composed of a central zone of meristematic cells and of an external zone of starchy cells. Each culture flask of a cell line contained a majority of one of the above-mentioned aggregate types. Histological studies of somatic embryo developement on semi-solid regeneration medium showed that there were close similarities between the initial steps of ontogenesis of the embryos and the different cell aggregates in liquid multiplication medium. It appeared that aggregates II–IV of the suspension belong to the same development continuum which reproduces the initial phases of somatic embryo ontogenesis on semi-solid medium. Type V resulted from the development of type IV, for which ontogenesis is hindered by direct contact with 2,4-dichlorophenoxyacetic acid and the shaken liquid multiplication medium. Type I aggregates probably do not belong to the development continuum but rather correspond to the degeneration of the other types of aggregates in the suspension. The presence of intermediate types in the liquid medium reinforces the hypothesis of a relationship between the aggregates. The aggregates tended to develop through time from a majority of type II or III at the beginning of their culture to types IV–V for older suspensions. Received: 10 August 1999 / Revision received: 8 November 1999 / Accepted: 9 November 1999     

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta^TM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression. Received: 25 September 1998 / Accepted: 27 October 1998     

Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells

Transgenic forage-type Italian ryegrass (Lolium multiflorum Lam.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by riceActl 5 regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric-glucuronidase (gusA) gene driven by the maizeUbi1 promoter. Stably transformed clones were recovered with a selection scheme using hygromycin in liquid medium followed by a plate selection. Plants were regenerated from 33% of the hygromycin-resistant calli. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult Italian ryegrass plants was confirmed by northern analysis and a hygromycin phosphotransferase enzyme assay.Abbreviations 2,4-D 2,4 Dichlorophenoxyacetic acid - GUS Glucuronidase - Hm Hygromycin - HPH Hygromycin phosphotransferase - MS medium Murashige and Skoog medium - PCR Polymerase chain reaction - X-Gluc 5-Bromo-4-chloro--indolyl--D-glucuronic acid     

Mycorrhizal dependency of banana (Musa acuminata,AAA group) cultivar

Seven banana cultivars (Musa acuminata, AAA group) were inoculated with two species of vesicular arbuscular mycorrhizal (VAM) fungi (Glomus mosseae and Glomus macrocarpum) in a greenhouse experiment. Inoculated plants had generally greater shoot dry weight and shoot phosphorus concentrations compared to the noninoculated plants. A great variation in dependency on mycorrhizal colonization was observed among the banana cultivars. Cv. Williams showed the highest relative mycorrhizal dependency (RMD) and cv. Poyo the lowest. For all the cultivars studied, inoculation with G. macrocarpum resulted in the highest RMD values. Both root dry weight and root hair length or density of the noninoculated plants were inverserly correlated with the RMD values of cultivars.