Life tables of Neoseiulus cucumeris exclusively fed with seven different pollens

The juvenile development and survival, and demographic parameters of the predatory mite Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae) fed on pollen of castor bean, tulip, apple, Christmas cactus, horse-chestnut, maize, and birch were assessed under laboratory conditions. Deprivation of food and pollen of castor bean plants resulted in 100 % juvenile mite mortality. Feeding mites with tulip and horse-chestnut pollen resulted in the shortest development and the highest total fecundity. Adult mites fed on birch, tulip, maize, and apple pollen lived significantly longer compared with those fed on pollen of horse-chestnut and Christmas cactus. The intrinsic rate of natural increase ranged between 0.1013 ♀♀/♀/day for maize and 0.1806 ♀♀/♀/day for horse-chestnut pollen as food. Net reproductive rate was the lowest when fed with maize pollen and highest when fed with horse-chestnut pollen. Population doubling time was highest on maize pollen and shortest on horse-chestnut pollen. Our study revealed that birch, tulip, horse-chestnut, apple, and maize pollen can be used by N. cucumeris from early spring to late summer as a suitable alternative food in periods when prey in the field are scarce or absent.     

Heterologous expression of Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) enhanced chilling tolerance in transgenic eggplant (Solanum melongena L.)

Key message

Our study shows that the expression of AtCBF3 and AtCOR15A improved the chilling tolerance in transgenic eggplant.

Abstract

In an attempt to improve chilling tolerance of eggplant (Solanum melongena L) plants, Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) genes both driven by an Arabidopsis RESPONSIVE TO DESSICATION 29A promoter (AtRD29A) were transferred into the plants of eggplant cultivar Sanyueqie. Two independent homozygous transgenic lines were tested for their cold tolerance. The leaves of the transgenic plants in both lines withered much slower and slighter than the wild-type plants after exposure to cold stress treatment at 2 ± 1 °C. The gene expression of AtCBF3 and AtCOR15A was significantly increased as well as the proline content and the levels of catalase and peroxidase activities, while the relative electrical conductivity and the malondialdehyde content were remarkably decreased in the transgenic plants compared with the wild type at 4 ± 0.5 °C. The results showed that the expression of the exogenous AtCBF3 and AtCOR15A could promote the cold adaptation process to protect eggplant plants from chilling stress.     

Identification of small secreted peptides (SSPs) in maize and expression analysis of partial SSP genes in reproductive tissues

Main conclusion

Maize 1,491 small secreted peptides were identified, which were classified according to the character of peptide sequences. Partial SSP gene expressions in reproductive tissues were determined by qRT-PCR. Small secreted peptides (SSPs) are important cell–cell communication messengers in plants. Most information on plant SSPs come from Arabidopsis thaliana and Oryza sativa, while little is known about the SSPs of other grass species such as maize (Zea mays). In this study, we identified 1,491 SSP genes from maize genomic sequences. These putative SSP genes were distributed throughout the ten maize chromosomes. Among them, 611 SSPs were classified into 198 superfamilies according to their conserved domains, and 725 SSPs with four or more cysteines at their C-termini shared similar cysteine arrangements with their counterparts in other plant species. Moreover, the SSPs requiring post-translational modification, as well as defensin-like (DEFL) proteins, were identified. Further, the expression levels of 110 SSP genes were analyzed in reproductive tissues, including male flower, pollen, silk, and ovary. Most of the genes encoding basal-layer antifungal peptide-like, small coat proteins-like, thioredoxin-like proteins, γ-thionins-like, and DEFL proteins showed high expression levels in the ovary and male flower compared with their levels in silk and mature pollen. The rapid alkalinization factor-like genes were highly expressed only in the mature ovary and mature pollen, and pollen Ole e 1-like genes showed low expression in silk. The results of this study provide basic information for further analysis of SSP functions in the reproductive process of maize.     

Development of a complete set of monosomic alien addition lines between Brassica napus and Isatis indigotica (Chinese woad)

Key message

A complete set of monosomic alien addition lines of Brassica napus with one of the seven chromosomes of Isatis indigotica and the recombinant mitochondria was developed and characterized.

Abstract

Monosomic alien addition lines (MAALs) are valuable for elucidating the genome structure and transferring the useful genes and traits in plant breeding. Isatis indigotica (Chinese woad, 2n = 14, II) in Isatideae tribe of Brassicaceae family has been widely cultivated as a medicinal and dye plant in China. Herein, the intertribal somatic hybrid (2n = 52, AACCII) between B. napus cv. Huashuang 3 (2n = 38, AACC) and I. indigotica produced previously was backcrossed recurrently to parental B. napus, and 32 MAAL plants were isolated. Based on their phenotype, 5S and 45S rDNA loci and chromosome-specific SSR markers, these MAALs were classified into seven groups corresponding to potential seven types of MAALs carrying one of the seven I. indigotica chromosomes. One of the MAALs could be distinguishable by expressing the brown anthers of I. indigotica, other two hosted the chromosome with 5S or 45S rDNA locus, but the remaining four were identifiable by SSR markers. The simultaneous detection of the same SSR maker and gene locus in different MAALs revealed the paralogs on the chromosomes involved. The recombinant mitochondrial genome in MAALs was likely related with their male sterility with carpellody stamens, while the MAAL with normal brown anthers probably carried the restoring gene for the male sterility. The complete set of MAALs should be useful for exploiting the I. indigotica genome and for promoting the introgression of valuable genes to B. napus.     

Development of somatic hybrids Solanum × michoacanum Bitter. (Rydb.) (+) S. tuberosum L. and autofused 4x S. × michoacanum plants as potential sources of late blight resistance for potato breeding

Key message

Phytophthora infestans resistant somatic hybrids of S. × michoacanum (+) S. tuberosum and autofused 4 x S. × michoacanum were obtained. Our material is promising to introgress resistance from S. × michoacanum into cultivated potato background.

Abstract

Solanum × michoacanum (Bitter.) Rydb. (mch) is a wild diploid (2n = 2x = 24) potato species derived from spontaneous cross of S. bulbocastanum and S. pinnatisectum. This hybrid is a 1 EBN (endosperm balance number) species and can cross effectively only with other 1 EBN species. Plants of mch are resistant to Phytophthora infestans (Mont) de Bary. To introgress late blight resistance genes from mch into S. tuberosum (tbr), genepool somatic hybridization between mch and susceptible diploid potato clones (2n = 2x = 24) or potato cultivar Rywal (2n = 4x = 48) was performed. In total 18,775 calli were obtained from postfusion products from which 1,482 formed shoots. The Simple Sequence Repeat (SSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Random Amplified Polymorphic DNA (RAPD) analyses confirmed hybrid nature of 228 plants and 116 autofused 4x mch. After evaluation of morphological features, flowering, pollen stainability, tuberization and ploidy level, 118 somatic hybrids and 116 autofused 4x mch were tested for late blight resistance using the detached leaf assay. After two seasons of testing three somatic hybrids and 109 4x mch were resistant. Resistant forms have adequate pollen stainability for use in crossing programme and are a promising material useful for introgression resistance from mch into the cultivated potato background.     

The relationship between PMI (manA) gene expression and optimal selection pressure in Indica rice transformation

Key message

An efficient mannose selection system was established for transformation of Indica cultivar IR58025B . Different selection pressures were required to achieve optimum transformation frequency for different PMI selectable marker cassettes.

Abstract

This study was conducted to establish an efficient transformation system for Indica rice, cultivar IR58025B. Four combinations of two promoters, rice Actin 1 and maize Ubiquitin 1, and two manA genes, native gene from E. coli (PMI-01) and synthetic maize codon-optimized gene (PMI-09) were compared under various concentrations of mannose. Different selection pressures were required for different gene cassettes to achieve corresponding optimum transformation frequency (TF). Higher TFs as 54 and 53 % were obtained when 5 g/L mannose was used for selection of prActin-PMI-01 cassette and 7.5 g/L mannose used for selection of prActin-PMI-09, respectively. TFs as 67 and 56 % were obtained when 7.5 and 15 g/L mannose were used for selection of prUbi-PMI-01 and prUbi-PMI-09, respectively. We conclude that higher TFs can be achieved for different gene cassettes when an optimum selection pressure is applied. By investigating the PMI expression level in transgenic calli and leaves, we found there was a significant positive correlation between the protein expression level and the optimal selection pressure. Higher optimal selection pressure is required for those constructs which confer higher expression of PMI protein. The single copy rate of those transgenic events for prActin-PMI-01 cassette is lower than that for other three cassettes. We speculate some of low copy events with low protein expression levels might not have been able to survive in the mannose selection.     

Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation

In this study, the d-serine ammonia lyase (dsdA) gene from Escherichia coli was evaluated as a selectable marker for maize transformation. Plants are incapable of utilizing the D-form of most amino acids, and d-serine has recently been demonstrated to be phytoinhibitory to plant growth. d-Serine ammonia lyase detoxifies d-serine via a substrate-specific reaction to pyruvate, ammonia, and water. d-Serine inhibits germination of isolated maize immature embryos and growth of embryogenic callus from wild-type plants at concentrations about approx. 2?C15 mM. Transgenic plants were recovered in the presence of d-serine in tissue culture media with dsdA as the selection marker at efficiencies comparable to using a mutated acetohydroxy acid synthase selection marker gene and selection in the presence of imidazolinone herbicides. Immature embryos infected with an Agrobacterium strain containing an acetohydroxy acid synthase gene construct without dsdA did not yield any transgenic events on the selection medium with 10 mM d-serine, indicating that d-serine provided selection tight enough to prevent escapes. Molecular analysis confirmed the integration of the dsdA gene into the genome of the transgenic plants. No adverse phenotypes were observed in the greenhouse, and expression of the dsdA marker had no affect on agronomic characteristics or grain yield in multi-location field trials. Seed compositional analysis demonstrated no significant differences in the contents of seed protein, starch, fatty acids, fiber, phytic acid, and free amino acids between transgenic and non-transgenic control plants. These data indicate that the dsdA gene is properly expressed in maize and the d-serine ammonia lyase (DSDA) enzyme functions appropriately to metabolize d-serine during in vitro selection. Preliminary safety assessments indicated that no adverse affects would be expected if humans were exposed to the DSDA protein in the diet from an allergenicity or toxicity perspective. The dsdA gene in combination with phytoinhibitory levels of d-serine represents a new and effective selectable marker system for maize transformation.     

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

Key message

We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics.

Abstract

Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12–16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1–2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.     

4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA): a versatile building block for labeling of peptides, proteins and oligonucleotides with fluorine-18 via Cu(I)-mediated click chemistry

Cu(I)-mediated [3+2]cycloaddition between azides and alkynes has evolved into a valuable bioconjugation tool in radiopharmaceutical chemistry. We have developed a simple, convenient and reliable radiosynthesis of 4-[¹⁸F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([ ¹⁸ F]F-SA) as a novel aromatic sulfonamide-based click chemistry building block. [ ¹⁸ F]F-SA could be prepared in a remotely controlled synthesis unit in 32 ± 5 % decay-corrected radiochemical yield in a total synthesis time of 80 min. The determined lipophilicity of [ ¹⁸ F]F-SA (logP = 1.7) allows handling of the radiotracer in aqueous solutions. The versatility of [ ¹⁸ F]F-SA as click chemistry building block was demonstrated by the labeling of a model peptide (phosphopeptide), protein (HSA), and oligonucleotide (L-RNA). The obtained radiochemical yields were 77 % (phosphopeptide), 55–60 % (HSA), and 25 % (L-RNA), respectively. Despite the recent emergence of a multitude of highly innovative novel bioconjugation methods for ¹⁸F labeling of biopolymers, Cu(I)-mediated click chemistry with [ ¹⁸ F]F-SA represents a reliable, robust and efficient radiolabeling technique for peptides, proteins, and oligonucleotides with the short-lived positron emitter ¹⁸F.     

The Arabidopsis kinesin gene AtKin-1 plays a role in the nuclear division process during megagametogenesis

Key message

Atkin - 1 , the only Kinesin-1 member of Arabidopsis thaliana , plays a role during female gametogenesis through regulation of nuclear division cycles.

Abstract

Kinesins are microtubule-dependent motor proteins found in eukaryotic organisms. They constitute a superfamily that can be further classified into at least 14 families. In the Kinesin-1 family, members from animal and fungi play roles in long-distance transport of organelles and vesicles. Although Kinesin-1-like sequences have been identified in higher plants, little is known about their function in plant cells, other than in a recently identified Kinesin-1-like protein in a rice pollen semi-sterile mutant. In this study, the gene encoding the only Kinesin-1 member in Arabidopsis, AtKin-1 was found to be specifically expressed in ovules and anthers. AtKin-1 loss-of-function mutants showed substantially aborted ovules in siliques, and this finding was supported by complementation testing. Reciprocal crossing between mutant and wild-type plants indicated that a defect in AtKin-1 results in partially aborted megagametophytes, with no observable effects on pollen fertility. Further observation of ovule development in the mutant pistils indicated that the enlargement of the megaspore was blocked and nuclear division arrested at the one-nucleate stage during embryo sac formation. Our data suggest that AtKin-1 plays a role in the nuclear division cycles during megagametogenesis.     

Purple-leaved Ficus lyrata plants produced by overexpressing a grapevine VvMybA1 gene

Key message

This study established an efficient method of regenerating plants of Ficus lyrata and producing purple-leaved F. lyrata plants through genetic transformation using a VvMybA1 gene of grapevine.

Abstract

Ficus lyrata, a species with unique violin- or guitar-shaped leaves, was regenerated from leaf-derived calli cultured on Murashige and Skoog (MS) basal medium supplemented with 4.5 μM N-phenyl-N’-1, 2, 3-thiadiazol-5-yl urea (TDZ) and 0.5 μM α-naphthalene acetic acid (NAA). Leaf discs were inoculated with Agrobacterium tumefaciens strain EHA 105 harboring a binary vector DEAT that contains the VvMybA1 gene and neomycin phosphotransferase (npt II) gene and subsequently cultured on the established regeneration medium supplemented with 100 mg l^?1 kanamycin. Results showed that 87.5 % of the leaf discs produced kanamycin-resistant callus, and 68.8 % of them produced adventitious shoots. Transgenic plants with three leaf colors including green, green-purple, and purple were produced. Regular and quantitative real-time PCR analyses confirmed the integration of transgenes into the host genome. Semi-quantitative RT-PCR analysis indicated that the VvMybA1 gene was responsible for the purple-colored phenotype. Purple-leaved plants with strong color stability grew vigorously in a greenhouse. This study illustrated the feasibility of using a genetically engineered VvMybA1 gene for drastic modification of leaf color of an important woody ornamental plant.     

Oxidation and reduction of sulfite contribute to susceptibility and detoxification of SO2 in Populus × canescens leaves

Key Message

The critical level for SO ₂ susceptibility of Populus × canescens is approximately 1.2 μL L ^?1 SO ₂ . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO ₂ detoxification.

Abstract

In the present study, uptake, susceptibility and metabolism of SO₂ were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO₂ detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO₂ (0.65, 0.8, 1.0, 1.2 μL L^?1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO₂ assimilation, SO₂ uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO₂ that confers injury to the leaves was 1.2 μL L^?1 SO₂. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO₂ uptake of the plant shoot, whereas SO₂ uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO₂ revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.     

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry

Key message

The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

Abstract

Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6–10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic ‘Aurora’ and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.     

A revision of Erythrochiton sensu lato (Cuspariinae,Rutaceae)

In the rutaceous subtribe Cuspariinae, species with relatively large, valvate, colored calyces have been assigned to Erythrochiton, but differences in arrangement of leaves, type of inflorescence, union of petals, of filaments, and of carpels, indument of corolla and testa, appendages of anthers, height of the intrastaminal disc, and exine of the pollen argue for the recognition of three genera. Erythrochiton s. str., characterized by often perennating inflorescences, connate, usually glabrous petals, free carpels, tomentulose seeds, and spinulose exine, consists of seven species of which four are new: E. fallax from the eastern flanks of the Andes from Colombia to Bolivia, E. odontoglossus from western Ecuador and adjacent Peru, E. trichanthus from eastern Peru, and E. gymnanthus from Costa Rica. The assignment to Toxosiphon of four species with woolly, coherent petals, connate carpels, glabrous seeds, and reticulate exine necessitates three new combinations: T. carinatus, T. macropodus, and T. trifoliatus. Recognition of a third unispecific genus with opposite simple leaves, sparsely pubescent, coherent, clawed petals, and spinulose exine requires a new genus name, Desmotes, and a new combination, D. incomparabilis.     

Temperature regulation of floral buds and floral thermogenicity in Magnolia denudata (Magnoliaceae)

Key message

The gynoecium in M. denudata was thermogenic, and the first peak in the female phase lasted longer than the second peak in the male phase during flowering.

Abstract

The floral biology of Magnolia denudata, including the thermogenesis of floral buds and blooming, were investigated using a portable infrared thermal imaging radiometer and digital infrared thermometer. We found that M. denudata buds have extremely dense trichomes that maintain internal temperatures above external temperatures. The pattern of thermogenesis in M. denudata anthesis consisted of two distinct episodes corresponding to apparent receptivity of the stigmas in the female phase and incipient shedding of pollen in the male phase: one begins in the female phase and lasts about 6 h and another occurs synchronously 24 h later and lasts about 4 h in the male phase. In addition, we found that the temperature was significantly elevated in the inner petals upon flowering, indicating that they may play an important role in producing a warm floral chamber. These results increase our understanding of the strategies used by Magnoliaceae blossoms to maintain an optimal microclimate at low temperatures in the early spring.     

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P SAG12 -ipt gene delays leaf senescence and enhances resistance to exogenous ethylene

Key message

The P _SAG12 -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions.

Abstract

Transgenic plants of Rosa hybrida ‘Linda’ were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P _SAG12 -ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P_35S-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1–6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 μl l^?1) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.     

Gene discovery and functional marker development for fragrance in sorghum (Sorghum bicolor (L.) Moench)

Key message

Sequence analysis and genetic mapping revealed that a 1,444 bp deletion causes a premature stop codon in SbBADH2 of sorghum IS19912. The non-function of SbBADH2 is responsible for fragrance in sorghum IS19912.

Abstract

2-acetyl-1-pyrroline (2AP) is a potent volatile compound causing fragrance in several plants and foods. Seeds of some varieties of rice, sorghum and soybean possess fragrance. The genes responsible for fragrance in rice and soybean are orthologs that correspond to betaine aldehyde dehydrogenase 2 (BADH2). Genotypes harboring fragrance in rice and soybean contain a premature stop codon in BADH2 which impairs the synthesis of full length functional BADH2 protein leading to the accumulation of 2AP. In this study, we reported an association between the BADH2 gene and fragrance in sorghum. An F₂ population of 187 plants developed from a cross between KU630 (non-fragrant) and IS19912 (fragrant) was used. Leaves of F₂ and F₃ progenies were evaluated for fragrance by organoleptic test, while seeds of F₂ plants were analyzed for 2AP. The tests consistently showed that the fragrance is controlled by a single recessive gene. Gene expression analysis of SbBADH1 and SbBADH2 in leaves of KU630 and IS19912 at various stages revealed that SbBADH1 and SbBADH2 were expressed in both accessions. Sequence comparison between KU630 and IS19912 revealed a continuous 1,444 bp deletion encompassing exon 12 to 15 of SbBADH2 in IS19912 which introduces a frameshift mutation and thus causes a premature stop codon. An indel marker was developed to detect polymorphism in SbBADH2. Bulk segregant and QTL analyses confirmed the association between SbBADH2 and fragrance.     

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene

Key message

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ).

Abstract

Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T₁ generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T₀ lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.