High frequency direct plant regeneration,micropropagation and Shikonin induction in <Emphasis Type="Italic">Arnebia hispidissima</Emphasis>

The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg l⁻¹ KIN, 0.25 mg l⁻¹ BAP, 0.1 mg l⁻¹ IAA and 100 mg l⁻¹ CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg l⁻¹ IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg g⁻¹ fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing, food, and pharmaceutical industries.     

Transformation of <Emphasis Type="Italic">Campanula</Emphasis> by wild type <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>

Compact growth is an important quality criterion in horticulture. Many Campanula species and cultivars exhibit elongated growth which is suppressed by chemical retardation and cultural practice during production to accommodate to the consumer’s desire. The production of compact plants via transformation with wild type Agrobacterium rhizogenes is an approach with great potential to produce plants that are non-GMO. Efficient transformation and regeneration procedures vary widely among both plant genera and species. Here we present a transformation protocol for Campanula. Hairy roots were produced on 26–90% of the petioles that were used for transformation of C. portenschlagiana (Cp), a C. takesimana × C. punctata hybrid (Chybr) and C. glomerata (Cg). Isolated hairy roots grew autonomously and vigorously without added hormones. The Cg hairy roots produced chlorophyll and generated plantlets in response to treatments with cytokinin (42 µM 2iP) and auxin (0.67 µM NAA). In contrast, regeneration attempts of transformed Cp and Chybr roots lead neither to the production of chlorophyll nor to the regeneration of shoots. Agropine A. rhizogenes strains integrate split T-DNA in T_L- and T_R-DNA fragments into the plant genome. In this study, regenerated plants of Cg did not contain T_R-DNA, indicating that a selective pressure against this T-DNA fragment may exist in Campanula.     

Over-expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">FT</Emphasis> gene reduces juvenile phase and induces early flowering in ornamental gentian plants

Ornamental gentian plants are perennial and have a juvenile period of over 1 year before flowering. We transformed gentian plants with a construct comprising the Arabidopsis FLOWERING LOCUS T (FT) gene (encoding a major component protein of the flowering hormone ‘florigen’) under the control of the rolC promoter from Agrobacterium rhizogenes, which is known to induce vascular-specific expression. The resultant rolCpro-FT transgenic gentian plants showed early flowering in vitro and the earliest line formed floral buds within 4 months after transformation. Regeneration experiments from leaf explants of these rolCpro-FT transgenic plants also confirmed the early flowering phenotype. After acclimatization, these transgenic plants showed normal floral development in a closed greenhouse. There is no effective method to induce early flowering by cultivation management in gentian, therefore these lines might be very useful as annual early season cultivars.     

High-frequency transgenic plant regeneration and plumbagin production through methyl jasmonate elicitation from hairy roots of <Emphasis Type="Italic">Plumbago indica</Emphasis> L.

High-frequency transgenic plant regeneration and production of plumbagin were accomplished from hairy roots induced by Agrobacterium rhizogenes strain A₄M70GUS on Plumbago indica L. Of the two types of hairy roots developed on Murashige and Skoog (MS) basal medium, Type I was long and thick with lesser branches and root hairs, and Type II was highly-branched short and slender with tufts of root hairs. Of the different lines grown in half-strength MS liquid basal media, root line 5 (R5) of the Type I yielded the highest plumbagin (0.92% DW) and was significantly different to that of the in vitro control. R5 showed a stable production of plumbagin (1.09% DW) in subsequent cultures. Elicitation of R5 with 50 μM methyl jasmonate for 48 h increased the yield of plumbagin to 5.0% DW, and was superior to 100 μM acetylsalicylic acid (3.8% DW). Plumbagin yield was five times that of twoyear-old ex vitro roots. Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the transformation. Hairy root segments cultured on MS medium containing 8.8 μM benzyladenine and 2.5 μM indole-3-butyric acid induced a mean of 9.1 shoots. Subsequent culture of the isolated shoots developed more than 50 normal shoots per culture. The root-free shoots were rooted on half-strength MS basal medium. The plantlets transferred in field conditions grew normally and exhibited 90% survival. Transgenic plant regeneration and hairy root induction in P. indica serves as reliable source of plumbagin which in turn cut off the mass destruction of the plant species.     

High frequency plantlet regeneration from nodal segment culture of female <Emphasis Type="Italic">Momordica dioica</Emphasis> (Roxb.)

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L⁻¹ 6-Benzylaminopurine (BAP) + 0.1 mg L⁻¹ Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L⁻¹ Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.     

Efficient Micropropagation Protocol for <Emphasis Type="Italic">Jatropha</Emphasis> Curcas Using Liquid Culture Medium

Although several studies have been made on the micropropagation of Jatropha curcas using agar base mediums, none of them have been by using liquid medium systems. The effects of explant type and temporary immersion system (test tube, jar with filter paper boat, and growtek bioreactor) on the micropropagation of J. curcas were studied. The explant type influenced shoot quality, multiplication coefficient (MC), and rooting. Leaf explant produced more and longer shoots than nodal explant. Use of filter paper (FB) boat prevented hyperhydricity and allowed proliferation of nodal explants cultured in liquid MS (Murashige and Skoog) medium supplemented 6-benzylaminopurine (BAP) and Kinetin (KN). The best shoot bud induction (92.1±3.1%) was achieved in liquid MS medium supplemented with 2.0 mg/L KN. Leaf regeneration efficiency was compared in growtek bioreactor and in jar containing liquid MS medium supplemented with 0.5 mg/L Thidiazuron (TDZ). The best shoot bud regeneration (78.7±2.1%) was obtained in growtek bioreactor. Shoot buds achieved from nodal segment and leaf were subcultured on filter paper boats in jar and bioreactor containing liquid MS medium supplemented with BAP, Indole butyric acid (IBA), Indole-3-acetic acid (IAA), and KN. Best shoot proliferation and elongation was obtained in filter paper boats containing liquid MS medium supplemented with 1.5 mg/L BAP, 0.5 mg/L IAA, and 0.2 mg/L KN. The number of multiple shoot buds was higher in leaf explants as compared to nodal explants and the highest number of multiple shoot buds was recorded from leaf explants. Up to 76.4% rooting efficiency was obtained when the shoots were ex vitro rooted. The generated plants well established in the nursery and grew normally in outdoor conditions. The protocol has good potential for application in large-scale propagation of J. curcas using liquid medium.     

Stable resistance against the leaf miner <Emphasis Type="Italic">Leucoptera coffeella</Emphasis> expressed by genetically transformed <Emphasis Type="Italic">Coffea canephora</Emphasis> in a pluriannual field experiment in French Guiana

Summary A pluriannual field trial of transgenic clones of Coffea canephora (the Robusta coffee tree) transformed for resistance to the lepidopteran coffee leaf miner Leucoptera coffeella was installed in French Guiana. Fifty-eight transformed clones produced by transformation of the C. canephora clone 126 were planted. They were harbouring the pEF1α constitutive promoter of Arabidopsis thaliana controlling either the Bacillus thuringiensis native gene for the cry1Ac insecticidal protein (eight clones) or a synthetic cry1Ac gene (53 clones). The vectors for the transformation were a strain of the bacterium Agrobacterium tumefaciens and one of Agrobacterium rhizogenes. The transformed clones were generally independent, presenting different integration patterns of the genetic construct. Four randomly distributed groups of five plants per transformed clone were planted along with 60 untransformed control trees. Over a 4-year period after plantation six releases of L. coffeella were performed. Mines on the leaves are the marks of larvae development and were counted on plants. A majority of the independent transformed clones harbouring the synthetic gene and transformed by the strain of A. tumefaciens displayed constantly much less mines than the control, therefore expressing a stable resistance. The need for complementary research is presented.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Trichosanthus dioica</Emphasis> Roxb. for nutritional security

The pointed gourd (Trichosanthes dioica Roxb.) is an important cucurbit reported for its medicinal value, therapeutic potential, and as a popular delicacy (especially in Indian cuisine). Being nutritive and desirous, it has potential to feed the nations and addresses their nutritional security and economic prosperity. The plant is usually vegetatively propagated and cultivated for fruits during summer and rainy seasons. The limited supply of planting material, limits cultivation and production. The present study was in anticipation for direct organogenesis, callus induction, and somatic embryo formation from leaf and node explants of T. dioica Roxb. In this study, the MS medium supplemented with 0.5 mg/L BA and 0.5 mg/L 2,4-D was found to be most efficient for callus induction, followed by 0.5 mg/L Kn and 0.5 mg/L 2,4-D. The embryogenic callus was developed by sub-culturing of node callus in the same media. The scanning electron microscopic (SEM) analysis revealed the presence of embryogenic cell clusters having globular embryos, which were found irresponsive to develop further. Through direct organogenesis, the node explants have responded to produce true-to-type plants for propagation. It was observed that MS supplemented with 1.0 mg/L BA was efficient for shoot proliferation, and 0.5 mg/L IAA was found more efficient for root development. Notably, the plant remains unexplored in its potential for improvement involving molecular breeding and tissue culture. These results may be effective to produce genetically stable plants on a large scale and aid the genetic improvement of pointed gourds.     

Expression of <Emphasis Type="Italic">Bruguiera gymnorhiza</Emphasis><Emphasis Type="Italic">BgARP1</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

We have previously reported that expression of salt-responsive genes, including Bruguiera gymnorhiza ankyrin repeat protein 1 (BgARP1), enhances salt tolerance in both Agrobacterium tumefaciens and Arabidopsis. In this report, we further characterized BgARP1-expressing Arabidopsis to elucidate the role of BgARP1 in salt tolerance. BgARP1-expressing plants exhibited more vigorous growth than wild-type plants on MS plates containing 125–175 mM NaCl. Real-time PCR analysis showed enhanced induction of osmotin34 in the 2-week-old transformants under 125 mM NaCl. It was also showed that induction of typical salt-responsive genes, including RD29A, RD29B, and RD22, was blunted and delayed in the 4-week-old transformants during 24 h after 200 mM NaCl treatment. Ion content analysis showed that transgenic plants contained more K⁺, Ca²⁺, and NO₃ ⁻, and less NH₄ ⁺, than wild-type plants grown in 200 mM NaCl. Our results suggest that BgARP1-expressing plants may reduce salt stress by up-regulating osmotin34 gene expression and maintaining K⁺ homeostasis and regulating Ca²⁺ content. These results indicate that BgARP1 is functional on a heterogeneous background.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.     

Resistance screening of <Emphasis Type="Italic">Coffea</Emphasis> spp. accessions for <Emphasis Type="Italic">Pratylenchus coffeae</Emphasis> and <Emphasis Type="Italic">Radopholus arabocoffeae</Emphasis> in Vietnam

Coffee varieties with resistance for the plant-parasitic nematodes Pratylenchus coffeae and Radopholus arabocoffeae are limited in Vietnam. A selection of imported varieties and high yield varieties of Arabica coffee in Vietnam were evaluated for resistance to both plant-parasitic nematode species in Northern Vietnam. The same experiments were carried out with hybrid arabica coffee, three selected clones of Coffea canephora and one clone of Coffea excelsa in the Western Highland of Vietnam. The screened coffee accessions from Ethiopia (KH1, KH13, KH20, KH21, KH29, and KH31) were susceptible and good host for P. coffeae. Also accessions 90P4 (Portugal) and Oro azteca (Mexico) had a reproduction factor Rf > 1. Pluma Hidalgo (Mexico), 90/6 (Vietnam), 90P3 (Portugal), 90P2 (Vietnam), Variedad (Mexico), 90T (Portugal), and Garnica (Mexico) were poor hosts (Rf < 1) but not tolerant to P. coffeae, expressed by a reduction of root weight compared to untreated control plants. Most of the coffee accessions tested in Northern Vietnam were intolerant to R. arabocoffeae, except 90T which showed no reduction of root weight, even at high initial nematode densities (4,000/pot). Good hosts for R. arabocoffeae were Variedad, KH1, KH21, KH29, KH20, KH31, and KH13 with Rf > 1. Pluma Hidalgo, 90/6, 90P3, 90P2, 90T, Oro azteca, and Garnica were poor hosts (Rf < 1). In the Western Highland experiment, all arabica coffee accessions were susceptible for P. coffeae with Rf ranging from 1.41 to 1.59. Tolerance to P. coffeae was found in C. liberica var. Dewevrei, Hong34 and Nhuantren. Coffea excelsa, Hong34, Nhuantren, and H1C19 were tolerant to R. arabocoffeae at the highest inoculation density (4,000 nematodes/pot). The most susceptible accessions were Nhuantren and K55. Resistance (Rf < 1) to R. arabocoffeae was found in C. liberica var. Dewevrei and Hong34. This article reports on the first screening for resistance and tolerance to P. coffeae and R. arabocoffeae in coffee accessions in Vietnam and shows promising results for enhanced coffee-breeding.     

Resynthesizing <Emphasis Type="Italic">Brassica napus</Emphasis> from interspecific hybridization between <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis> through ovary culture

Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l^–1 BA+0.05 mg l^–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l^–1 BA+0.1 mg l^–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l^–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.     

A simple,rapid and reliable bioassay for evaluating seedling vigor under submergence in <Emphasis Type="Italic">indica</Emphasis> and <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Submergence is a major stress causing yield losses particularly in the direct-seeded rice cultivation system and necessitates the development of a simple, rapid and reliable bioassay for a large scale screening of rice germplasms with tolerance against submergence stress. We developed two new bioassay methods that were based primarily on the seedling vigor evaluated by the ability of fast shoot elongation under submerged conditions, and compared their effectiveness with two other available methods. All four bioassay methods using cultivars of 7 indica and 6 japonica types revealed significant and consistent cultivar differences in seedling vigor under submergence and/or submergence tolerance. Japonica cultivars were more vigorous than indica cultivars, with Nipponbare being the most vigorous. The simplest test tube method showed the highest correlations to all other methods. Our results suggest that seedling vigor serves as a submergence avoidance mechanism and confers tolerance on rice seedlings to flooding during early crop establishment. A possible relationship is discussed between seedling vigor based on fast shoot elongation and submergence tolerance defined by recovery from submergence stress.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with a fungal phytase gene improves phosphorus acquisition

A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.     

Measurement of the field response of <Emphasis Type="Italic">Musa</Emphasis> genotypes to <Emphasis Type="Italic">Radopholus Similis</Emphasis> and <Emphasis Type="Italic">Helicotylenchus Multicinctus</Emphasis> and the implications for nematode resistance breeding

Crop growth and damage parameters (plant growth and yield, root damage and nematode population densities), believed to be associated with resistance of Musa genotypes to nematodes under field conditions, were evaluated in a field trial of 24 Musa genotypes inoculated at planting with a combination of Radopholus similis and Helicotylenchus multicinctus with the objective to identify parameters with strong association with nematode resistance and high heritability. Correlation and path analysis of the association between plant growth, yield, root damage and nematode population densities showed a strong negative association between percentage dead roots, percentage root necrosis, R. similis and H. multicinctus population densities and yield. The strongest negative association was observed between percentage dead roots and yield. Broad-sense genotype heritability estimates demonstrated that heritability estimates for percentage dead roots, number of large lesions and nematode population density were most affected by inoculation with nematodes. These results indicate therefore that effective selection for nematode resistance under field conditions could be obtained by using an index, that includes percentage dead roots, the number of large lesions, and nematode population density.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration,flowering and fruiting from nodal explants of <Emphasis Type="Italic">Andrographis lineata</Emphasis> nees (Acanthaceae)

In the present study, in vitro propagation of tribal endemic medicinal plant, Andrographis lineata has been established using mature nodal explants. High frequency of regeneration (91.4%) was achieved on MS medium containing BA (3.0 mg L^–1) along with IAA (0.2 mg L^–1). Strikingly, irrespective of the season and collection period, we observed axillary flower induction and fruit formation from the above in vitro cultures supplemented with BA and NAA. Transition from the vegetative to the reproductive phase occurred within 2 months of culture, and importantly was influenced by factors such as sucrose and cytokinins. In vitro-regenerated flowers were morphologically identical to in vivo flowers. Furthermore, our scanning electron microscopic studies revealed that pollen external morphology of both in vitro and in vivo flowers were similar. The flowers self-fertilized and produced fruits in vitro. Elongated shoots rooted well on half-strength MS basal medium, and were successfully acclimatized to the garden conditions. Altogether, our established protocol can be utilized in plant breeding for the purpose of ex situ conservation, quick flowering, and fruit set.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pigeon pea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.] for resistance to legume pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L⁻¹ kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L⁻¹ sucrose and 0.5 mg L⁻¹ indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of faba bean <Emphasis Type="Italic">(Vicia faba</Emphasis> L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.     

Microsporogenesis of <Emphasis Type="Italic">Rps</Emphasis>8/<Emphasis Type="Italic">rps</Emphasis>8 heterozygous soybean lines

Phytophthora root and stem rot caused by Phytophthora sojae, is one of the most damaging diseases of soybean, for which management is principally done by planting resistant cultivars with race specific resistance which are conferred by Rps (Resistance to Phytophthora sojae) genes. The Rps8 locus, identified in the South Korean landrace PI 399073, is located in a 2.23 Mbp region on soybean chromosome 13. In eight cv. Williams (rps8/rps8) × PI 399073 (Rps8/Rps8) populations, this region exhibited strong segregation distortion. In a cross between the South Korean lines PI 399073 (Rps8/Rps8) and PI 408211B (multiple Rps genes) this region segregated in a Mendelian fashion. In this study, microsporogenesis was evaluated to identify meiotic abnormalities that may be associated with the segregation distortion of the Rps8 region. Pollen was collected from greenhouse-grown plants of the parental genotypes: Williams, PI 399073, and PI 408211B; as well as selected Rps8/rps8 RILs from Williams × PI 399073 BC₄F_2:3 and PI 399073 × PI 408211B F_4:5 populations. There were no differences for pollen viability among the genotypes. However, for PI 399073, a mix of dyads, triads, tetrads and pentads was observed. A high frequency of meiotic abnormalities including fragments, laggards, multinucleated microspores; and microcytes containing DNA was also observed in Rps8/rps8 Williams × PI 399073 BC₄F_2:3 RILs. These meiotic abnormalities may contribute to the high degree of segregation distortion present in the Williams × PI 399073 populations.