Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

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.     

Improved frost tolerance and winter survival in winter barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by <Emphasis Type="Italic">in vitro</Emphasis> selection of proline overaccumulating lines

Embryogenic calli derived from anther cultures of the two-rowed winter barley cultivar Igri were plated on solid L3 medium containing the proline analogue hydroxyproline (Hyp), 10–20 mmol l^–1. Exposure to Hyp caused severe degeneration of most of the calli. Hyp resistant calli, distinguishable by their lighter colour and higher growth rate, and control calli not exposed to Hyp were plated on L3 regeneration medium. From 22,500 anthers exposed to Hyp 46 Hyp resistant regenerates were obtained, which were transferred to soil. After cultivation for 5–10 weeks at normal growth conditions they were cold hardened at 2 C under short day conditions together with control regenerates. Frost tolerance assays with segments of fully grown leaves of unhardened and cold hardened plants revealed that Hyp resistant regenerants were significantly more frost tolerant than the control regenerants. Improved frost tolerance was found also in the progenies R₁ to R₉, and genotypic segregation in the R₁ generation in a 1:2:1 ratio was indicated. Increased proline content was observed in the R₂ generation and in subsequent generations and was significantly (P 0.001) correlated with increased frost tolerance in the Hyp lines. Comparative studies of R₉ progenies from homozygous R₂ plants with the wild type Igri under field conditions in winter at three locations in Europe as well as crossing experiments confirmed the heritable improvement of frost tolerance and winter survival, respectively, in the Hyp lines. The results support the hypothesis that proline accumulation in cold acclimated winter barley plants is causally related to the acquisition of frost tolerance. Moreover, the described biotechnological procedure may be applicable in breeding programs for improved winter hardiness and possibly also for other stress tolerances.     

Genetic and histological characterization of a novel recessive genic male sterile line of <Emphasis Type="Italic">Brassica napus</Emphasis> derived from a cross with <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

In a previously made cross Brassica napus cv. Oro (2n = 38) × Capsella bursa-pastoris (2n = 4x = 32), one F₁ hybrid with 2n = 38 was totally male sterile. The hybrid contained no complete chromosomes from C. bursa-pastoris, but some specific AFLP (amplified fragment length polymorphism) bands of C. bursa-pastoris were detected. The hybrid was morphologically quite similar to ‘Oro’ except for smaller flowers with rudimentary stamens but normal pistils, and showed good seed-set after pollination by ‘Oro’ and other B. napus cultivars. The fertility segregation ratios (3:1, 1:1) in its progenies indicated that the male sterility was controlled by a single recessive gene. In the pollen mother cells of the male sterile hybrid, chromosome pairing and segregation were normal. Histological sectioning of its anthers showed that the tapetum was multiple layers and was hypertrophic from the stage of sporogenic cells, and that the tetrads were compressed by the vacuolated and disaggregated tapetum and no mature pollen grains were formed in anther sacs, thus resulting in male sterility. The possible mechanisms for the production of the male sterile hybrid and its potential in breeding are discussed.     

Genetic analysis reveals a dominant <Emphasis Type="Italic">S</Emphasis> locus and an <Emphasis Type="Italic">S</Emphasis> suppressor locus in natural self-compatible <Emphasis Type="Italic">Brassica napus</Emphasis>

A self-incompatible (SI) line, S-1300, and its maintainer 97-wen135, a self-compatible (SC) line, were used to study the inheritance of maintenance for self-incompatibility in B. napus. The ratio of SI plants to SC plants from S-1300 × 97-wen135 F₂ and (S-1300 × 97-wen135) × 97-wen135 was 346:260 and 249:232, fitting the expected ratio of 9:7 and 1:1, respectively. Based on these observations, here we propose a genetic model in which two independent loci, S locus and S suppressor locus (sp), are predicted to control the inheritance of maintenance for self-incompatibility in B. napus. The genotypes of S-1300 and 97-wen135 are S ₁₃₀₀ S ₁₃₀₀ sp ₁₃₀₀ sp ₁₃₀₀ and S ₁₃₅ S ₁₃₅ sp ₁₃₅ sp ₁₃₅ , respectively. S ₁₃₅ is dominant to S ₁₃₀₀ , but coexistence of sp ₁₃₀₀ and sp ₁₃₅ fails to suppress S locus. Both S ₁₃₀₀ and S ₁₃₅ can be suppressed by sp ₁₃₅ , while sp ₁₃₀₀ can suppress S ₁₃₅ but not S ₁₃₀₀ . The model contains two characteristics: that a dominant S locus exists in self-compatible B. napus, and that co-suppression will occur when sp loci are heterozygous. The model has been validated by the segregation of S phenotypes in the (S-1300 × 97-wen135) × S-1300, the progenies of SC S-1300 × 97-wen135 F₂ plants and DH population developed from S-1300 × 97-wen135 F₁. This is the first study to report co-suppression of S suppressor loci in B. napus. The genetic model will be very useful for developing molecular markers linked to maintenance for self-incompatibility and for dissecting the mechanism of SI/SC in B. napus.     

Production and characterization of inter-sectional hybrids between <Emphasis Type="Italic">Kalanchoe spathulata</Emphasis> and <Emphasis Type="Italic">K. laxiflora</Emphasis> ( = <Emphasis Type="Italic">Bryophyllum crenatum</Emphasis>)

The genus Kalanchoe is currently divided into section Kalanchoe and section Bryophyllum, and there has been no successful report on the production of inter-sectional hybrids. Therefore, reciprocal crosses were made between Kalanchoe spathulata (sect. Kalanchoe) and K. laxiflora (sect. Bryophyllum) in order to obtain basic information on the reproductive barriers between these two sections. The seeds were aseptically germinated in vitro and the plants were grown in greenhouse till flowering. When K. spathulata was used as a maternal donor, 39 out of 80 plants showed intermediate characteristics between K. spathulata and K. laxiflora. In contrast, no plants were obtained in the reverse crosses. Hybridity of these plants was confirmed by flow cytometric analysis, chromosome numbers and RAPD analysis. Bulbil formation on the leaf margin as one of the conspicuous characteristics of K. laxiflora was not observed in the hybrids. Some of the hybrid lines showed some pollen fertility, but failed to yield viable seeds by self-pollination or backcross-pollination. Successful production of the inter-sectional hybrid between the two species suggests that they are not so distantly related as considered previously.     

Development and characterisation of a <Emphasis Type="Italic">Brassica carinata</Emphasis> inbred line incorporating genes for low glucosinolate content from <Emphasis Type="Italic">B. juncea</Emphasis>

The presence of high levels of sinigrin in the seeds represents a serious constraint for the commercial utilisation of Ethiopian mustard (Brassica carinata A. Braun) meal. The objective of this research was the introgression of genes for low glucosinolate content from B. juncea into B. carinata. BC₁F₁ seed from crosses between double zero B. juncea line Heera and B. carinata line N2-142 was produced. Simultaneous selection for B. carinata phenotype and low glucosinolate content was conducted from BC₁F₂ to BC₁F₄ plant generations. Forty-three BC₁F₄ derived lines were selected and subject to a detailed phenotypic and molecular evaluation to identify lines with low glucosinolate content and genetic proximity to B. carinata. Sixteen phenotypic traits and 80 SSR markers were used. Eight BC₁F₄ derived lines were very close to N2-142 both at the phenotypic and molecular level. Three of them, with average glucosinolate contents from 52 to 61 micromoles g⁻¹, compared to 35 micromoles g⁻¹ for Heera and 86 micromoles g⁻¹ for N2-142, were selected and evaluated in two additional environments, resulting in average glucosinolate contents from 43 to 56 micromoles g⁻¹, compared to 29 micromoles g⁻¹ for Heera and 84 micromoles g⁻¹ for N2-142. The best line (BCH-1773), with a glucosinolate profile made up of sinigrin (>95%) and a chromosome number of 2n = 34, was further evaluated in two environments (field and pots in open-air conditions). Average glucosinolate contents over the four environments included in this research were 42, 31 and 74 micromoles g⁻¹ for BCH-1773, Heera and N2-142, respectively. These are the lowest stable levels of glucosinolates reported so far in B. carinata.     

Ploidy level studies on the <Emphasis Type="Italic">Dioscorea cayenensis</Emphasis>/<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> complex core set

The Guinea yams, Dioscorea cayenensis Lam. and D. rotundata Poir. (D. cayenensis–D. rotundata complex), represent a highly important crop, widely distributed in the humid and semi-humid tropics. The ploidy levels of 170 accessions of the core set of Guinea yams from West African countries was determined using flow cytometry with propidium iodide staining. One hundred and eight of the genotypes were found to be tetraploid, 47 were hexaploid and five were octoploid. One mixoploid individual containing tetraploid and hexaploid nuclei was also detected. A deeper analysis considering each separate taxon revealed that while for D. rotundata the majority of individuals were tetraploid, for D. cayenensis this ploidy level was not detected in any of the accessions. Also, no association between ploidy level and place of cultivation was found for the evaluated germplasm. The obtained data is highly valuable for breeding programs of Guinea yam, especially for the optimization of future hybridization experiments directed to the genetic improvement of this economically important crop.     

Molecular markers linked to <Emphasis Type="Italic">Bn</Emphasis>;<Emphasis Type="Italic">rf</Emphasis>: a recessive epistatic inhibitor gene of recessive genic male sterility in <Emphasis Type="Italic">Brassica napus </Emphasis>L.

7–7365AB is a recessive genic male sterile (RGMS) two-type line, which can be applied in a three-line system with the interim-maintainer, 7–7365C. Fertility of this system is controlled by two duplicate dominant epistatic genes (Bn;Ms3 and Bn;Ms4) and one recessive epistatic inhibitor gene (Bn;rf). Therefore an individual with the genotype of Bn;ms3ms3ms4ms4Rf_ exhibits male sterility, whereas, plant with Bn;ms3ms3ms4ms4rfrf shows fertility because homozygosity at the Bn;rf locus (Bn;rfrf) can inhibit the expression of two recessive male sterile genes in homozygous Bn;ms3ms3ms4ms4 plant. A cross of 7–7365A (Bn;ms3ms3ms4ms4RfRf) and 7–7365C (Bn;ms3ms3ms4ms4rfrf) can generate a complete male sterile population served as a mother line with restorer in alternative strips for the multiplication of hybrid seeds. In the present study, molecular mapping of the Bn;Rf gene was performed in a BC₁ population from the cross between 7–7365A and 7–7365C. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) technique was used to identify molecular markers linked to the gene of interest. From a survey of 768 primer combinations, seven AFLP markers were identified. The closest marker, XM5, was co-segregated with the Bn;Rf locus and successfully converted into a sequence characterized amplified region (SCAR) marker, designated as XSC5. Two flanking markers, XM3 and XM2, were 0.6 cM and 2.6 cM away from the target gene, respectively. XM1 was subsequently mapped on linkage group N7 using a doubled-haploid (DH) mapping population derived from the cross Tapidor × Ningyou7, available at IMSORB, UK. To further confirm the location of the Bn;Rf gene, additional simple sequence repeat (SSR) markers in linkage group N7 from the reference maps were screened in the BC₁ population. Two SSR markers, CB10594 and BRMS018, showed polymorphisms in our mapping population. The molecular markers found in the present study will facilitate the selection of interim-maintainer.     

Sources of resistance to diseases of sugar beet in related <Emphasis Type="Italic">Beta</Emphasis> germplasm: I. Foliar diseases

Resistance to four foliar diseases of sugar beet (Beta vulgaris ssp. vulgaris), virus yellows caused by Beet mild yellowing virus (BMYV) and Beet yellows virus (BYV), powdery mildew (Erysiphe betae) and Cercospora leaf spot (Cercospora beticola), was assessed in up to 600 accessions of closely related wild and cultivated Beta species. Most accessions were from the Section Beta, a taxon containing types most closely related to, and sexually compatible with, sugar beet and therefore most valuable for use in crop improvement. Between 1–12% of accessions were highly resistant (resistance scores of 2 on an international standardised resistance scale of 1–9) to these diseases. These levels, however, underestimate the potential number of resistant sources available from this section as some accessions with intermediate mean resistance scores contained a significant proportion of highly resistant plants within segregating populations. Variation in resistance to all diseases except BYV was observed within the Section Beta. Much higher levels of resistance were observed, and more frequently, in more distantly related sections of the genus Beta. Accessions of the Section Corollinae were highly resistant to both viruses (>62% of accessions tested), but less so to Cercospora (15%) and they were very susceptible to powdery mildew. Section Procumbentes accessions were highly resistant to BMYV and Cercospora (100%) but less so to powdery mildew (50%) and BYV (20%). However, sexual incompatibility between these sections and sugar beet make utilisation of these sources impractical using conventional breeding methods.     

Identification of AFLP and SCAR markers linked to the male fertility restorer gene of <Emphasis Type="Italic">pol</Emphasis> CMS (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The pol cytoplasmic male-sterility system has been widely used as a component for utilization of heterosis in Brassica napus and offers an attractive system for study on nuclear–mitochondrial interactions in plants. Genetic analyses have indicated that one dominant gene, Rfp, was required to achieve complete fertility restoration. As a first step toward cloning of this restorer gene, we attempted molecular mapping of the Rfp locus using the amplified fragment length polymorphism (AFLP) technique combined with bulked segregant analysis (BSA) method. A BC₁ population segregating for Rfp gene was used for tagging. From the survey of 1,024 AFLP primer combinations, 13 linked AFLP markers were obtained and five of them were successfully converted into sequence characterized amplified region (SCAR) markers. A population of 193 plants was screened using these markers and the closest AFLP markers flanking Rfp were at the distances of 2.0 and 5.3 cM away, respectively. Further the AFLP or SCAR markers linked to the Rfp gene were integrated to one doubled-haploid (DH) population derived from the cross Quantum × No.2127-17 available in our laboratory, and Rfp gene was mapped on N18, which was the same as the previous report. These molecular markers will facilitate the marker-assisted selection (MAS) of pol CMS restorer lines.     

A cytoplasmic-nuclear male-sterility system derived from a cross between <Emphasis Type="Italic">Cajanus cajanifolius</Emphasis> and <Emphasis Type="Italic">Cajanus cajan</Emphasis>

Cytoplasmic-nuclear male-sterility is an important biological tool, which has been used by plant breeders to increase yields in cross-pollinated cereals and vegetables by commercial exploitation of the phenomenon of hybrid vigor. In legumes, no such example exists due to the absence of an economic way of mass pollen transfer from male to female parent. Pigeonpea [Cajanus cajan (L.) Millsp.], however, is a different legume where a moderate level of insect-aided natural out-crossing (25–70%) exists and it can be used to produce commercial hybrid cultivars, if an efficient and stable cytoplasmic-nuclear male-sterility (CMS) system is available. This paper reports the development of a stable CMS system (ICP 2039A), derived from an inter-specific hybrid of Cajanus cajanifolius, a wild relative of pigeonpea, with a cultivar ICP 11501. Using this genetic material, designated as the A₄ cytoplasm, a number of fertility restorers and maintainers have been developed. The best short-duration experimental pigeonpea hybrid ICPH 2470 produced 3205 kg ha⁻¹ grain yield in 125 days, exhibiting 77.5% advantage over the control cultivar UPAS 120. At present, all the important biological systems necessary for a successful commercial hybrid breeding program are available in pigeonpea and the package of this technology has been adopted by private seed sector in India for the production and marketing of hybrid varieties.     

Genetics of resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in <Emphasis Type="Italic">Cucumis sativus</Emphasis> var. <Emphasis Type="Italic">hardwickii</Emphasis> R. Alef

The genetics of resistance to Cucumber mosaic virus (CMV) in Cucumis sativus var. hardwickii R. Alef, the wild progenitor of cultivated cucumber was assessed by challenge inoculation and by natural infection of CMV. Among the 31 genotypes of C. sativus var. hardwickii collected from 21 locations in India the lowest mean percent disease intensity (PDI) was recorded in IC-277048 (6.33%) while the highest PDI was observed in IC-331631 (75.33%). All the four cultivated varieties (DC-1, DC-2, CHC-1 and CHC-2) showed very high PDI and susceptible disease reaction. Based on mean PDI, 8 genotypes were categorized as resistant, 13 as moderately resistant, 9 as moderately susceptible and one as susceptible. A chi-square test of frequency distribution based on mean PDI in F₂ progenies of six resistant × susceptible crosses revealed monogenic recessive Mendelian ratio 1(R):3(S) to be the best fit. This monogenic recessive model was further confirmed by 1(R):1(S) ratio as the best fit for back cross with resistant parent and no fit for either 3:1 or 1:1 in the back cross with the susceptible parent. The results revealed that CMV resistance in C. sativus var. hardwickii was controlled by a single recessive gene. Considering the cross compatibility between C. sativus var. hardwickii and cultivated cucumber, the resistance trait can be easily transferred to cultivated species through simple backcross breeding.     

High frequency direct plant regeneration from leaf and petals of Cape Primrose (<Emphasis Type="Italic">Streptocarpus</Emphasis>)

High frequency direct plant regeneration from leaf and petal explants was accomplished for the first time in Streptocarpus varieties. The shoot induction frequency varied with respect to the benzylaminopurine (BAP) concentration added to the Murashige and Skoog (MS) medium. MS medium with 0.5 mg l⁻¹ BAP exhibited the highest (69.9%) plant regeneration frequency with an average of 186 shoots per explant. A higher concentration of BAP inhibited shoot bud induction and plant regeneration along with necrosis of explants. Petal explants derived from the varieties ‘Branwen’ (pink and white) and ‘Chorus Line’ (violet and white) displayed plant regeneration frequency of 22.2–47.4% (within a total of 12 weeks) on MS medium containing 2.0 mg l⁻¹ α-naphthaleneacetic acid and 0.5 mg l⁻¹ BAP for 8 weeks followed by 4 weeks on MS medium with 1.0 mg l⁻¹ BAP. Scanning electron microscopy confirmed direct plant regeneration without callus. Regenerated plants from leaf explants with well-developed leaves and roots were hardened and successfully transferred to pots in glasshouse exhibiting 86% survival at the end of 4–6 weeks. Whereas, regenerated plants from flower petal explants upon transfer to pots in glasshouse exhibited 75–82% survival at the end of 4–6 weeks.     

Identification of RAPD markers linked to the rust (<Emphasis Type="Italic">Uromyces fabae</Emphasis>) resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Summary Two RAPD markers linked to gene for resistance (assayed as pustule number cm⁻² leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC₁F₁ [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F₁, F₂, BC₁F₁ and BC₁F₂ generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC₁F₁ individuals. Two RAPD makers, viz., SC10-82₃₆₀ (primer, GCCGTGAAGT), and SCRI-71₁₀₀₀ (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.     

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.     

<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.     

Creation of new maize germplasm using alien introgression from <Emphasis Type="Italic">Zea mays</Emphasis> ssp. <Emphasis Type="Italic">mexicana</Emphasis>

Zea mays ssp. mexicana, an annual wild relative of maize, has many desirable characteristics for maize improvement. To transfer alien genetic germplasm into maize background, F₁ hybrids were generated by using Z. mays ssp. mexicana as the female parent and cultivated maize inbred line Ye515 as the male parent. Alien introgression lines, with a large range of genetic diversity, were produced by backcross and successive self-pollinations. A number of alien introgression lines with the predominant traits of cultivated maize were selected. Genomic in situ hybridization (GISH) proved that small chromosome segments of Z. mays ssp. mexicana had been integrated into the maize genome. Some outstanding alien introgression lines were evaluated in performance trials which showed 54.6% hybrids had grain yield greater than that of hybrid check Yedan12 which possessed 50% Ye515 parentage, and 17.1, 9.9% hybrids had grain yield competitive or greater than those of Nongda108 and Zheng958, which were elite commercial hybrids in China, respectively. The results indicated that some of the introgression lines had excellent agronomic traits and combining ability for maize cultivar, and demonstrated that Z. mays ssp. mexicana was a valuable source for maize breeding, and could be used to broaden and enrich the maize germplasm.     

<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.     

Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic <Emphasis Type="Italic">cry1Ab</Emphasis> gene

Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation. PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis. The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance of the inserted transgene was confirmed in F₁ plants obtained through crossing of T₀ plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected to further bioassay analyses against other lepidopteran pests of sugar beet.