Golden rice: introgression, breeding, and field evaluation

Considerable progress has been made on the genetic engineering of rice for improved nutritional content involving micronutrients and carotenoid content. Golden Rice, developed by genetic engineering (Agrobacterium and biolistic transformation) was used in rice breeding for the transfer of high-nutritional value to the local rice cultivars. Simultaneously, commercial Asian indica rice cultivars were also developed with expression of high-carotenoid levels. The lines were developed based on POSITECH (PMI) selection system or made marker free by segregating out the marker gene from the gene of interest. Anther culture was used to develop the homozygous stable lines, which could be of much use in further introgress-breeding and in farmer’s field. Enhanced carotenoids levels (up to T3 generation) were observed in a number of lines compared to the T0-T1 seeds which could be due to transgeneration effect of growing under greenhouse versus field conditions. However, a few introgressed lines showed less carotenoid levels than the original lines used in the breeding process. Agronomic performance of introgressed lines, non-transgenic controls, and transgenic golden rice (IR64 and BR29) developed at IRRI showed acceptable and comparable data under identical limited field conditions (screenhouse data). Syngenta generated a new Golden Rice (US cultivar) containing high level of carotenoids grown in the field at Louisiana, USA is expected to be available to the public domain. Incorporation of genes for carotenogenesis in seeds by transgenesis or by introgression did not change any significant agronomic characteristics in rice plants. The ongoing and future study of bioavailability, quality, larger field testing and freedom to operate will ensure the benefit of Golden Rice to the people who need them most.     

The production of inbred lines of brussels sprouts with dominant S-alleles

Summary The best sources of Brussels sprout inbred lines with both good agronomic characters and high self-incompatibility are likely to be cultivars of reasonably good agronomic type which have not been too intensively selected. Comparison of three cultivars of different agronomic quality showed that the cultivars of poor and moderate quality had about 55% of plants with a dominant S-allele, but the most highly selected cultivar had only 25% of such plants. A programme of S-allele screening is suggested which incorporates the minimum number of tests required to determine whether or not a particular plant has a dominant S-allele. A survey of S-alleles present in commercial F₁ hybrids showed that the frequency of dominant S-alleles was only 19% in hybrids released prior to the end of 1971, but was 50% in hybrids released since 1971.     

Inbreeding depression and single cross hybrids in leeks (Allium ampeloprasum ssp. porrum)

Summary All current commercial cultivars of leeks are open-pollinated and one of the major problems with the crop is poor uniformity with much of the variation being genetic in origin. Inbred lines and single cross hybrids were produced to try to reduce the genetic variation. Inbreds were generated by single seed descent from 5400 plants taken from six commercial cultivars and performance data for uniformity, yield and quality are presented for the S₁-S₃ generations. A few relatively vigorous inbred lines were obtained but overall, inbreeding depression was very severe with no compensating increase in uniformity as measured by coefficients of variation. In contrast, the experimental hybrids gave significant uniformity, yield and quality benefits compared to open-pollinated commercial cultivars and can be used as the basis for developing a range of commercial hybrid cultivars.Abbreviations SSD Single Seed Descent - HRI Horticulture Research International     

Genetic diversity for agronomic and fiber traits in day-neutral accessions derived from primitive cotton germplasm

Upland cotton (Gossypium hirsutumL) is an important crop that is cultivated in warm climates through-out the world. Agronomic performance and fiber quality must continually be improved if cotton is to maintain economic viability. Primitive ancestors of cotton contain diversity for trait improvement; however, many of these accessions have a short-day flowering response (photoperiodic) and are not readily useable in breeding programs. In this study, 114 day-neutral derived primitive germplasm lines were evaluated in field trials for two years. Agronomic and fiber trait data were collected and analyzed. Variance components, genotypic values, and genotypic correlations were calculated. Genotypic effects for all traits studied made significant contributions to the phenotypic variation indicating genetic diversity among these lines. The predicted genotypic values showed a wide range of variation for agronomic and fiber traits. Weak genotypic correlations were found between yield and 2.5% span length and fiber strength, two important fiber traits. Although these day-neutral derived accessions had lower lint percentage, they had improved fiber length, strength, micronaire, and comparable yields with two commercial cultivars. Thus, these day-neutral derived accessions are sources of genetic variation that when used in breeding programs offer the potential to improve important traits and expand genetic diversity.Contribution of the USDA-ARS in cooperation with the Mississippi Agric. and Forestry Exp. Stn. Mention of trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA, ARS and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Morphological characterisation and agronomic evaluation of transgenic broccoli (Brassica oleracea L. var. italica) containing an antisense ACC oxidase gene

Morphological characterisation and agronomic evaluation was conducted on 12 transgenic broccolilines containing a tomato antisense1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene. Plants of three cultivars: Shogun (Sh), Green Beauty (Gy) and Dominator (D), were regenerated from hairy root cultures after co-cultivation with Agrobacterium rhizogenes strain A4T harbouring the binary vector pLN35. The T-DNA of pLN35 contains genes encoding a tomato antisense ACC oxidase gene (35S-ACC-5′7′) and a neomycin phosphotransferase II gene (NOS-NPTII-NOS) for kanamycin resistance. The transgenic plants were transferred to a greenhouse and fertile plants obtained. Integration of the foreign DNA into the broccoli genome was confirmed by the polymerase chain reaction and Southern analyses. Transgenic plants showed evidence of hairy root (HR)-induced morphological changes to varying degrees. Of the 12 characterised transgenic lines, three lines(Gy/7, D/1 and D/2) performed within the limits of acceptability for all head quality parameters analysed (size, density, colour, shape and leafiness). The ethylene production from stalks of four field-grown transgenic lines of Green Beauty broccoli showed significant reductions in activity relative to the control 98 h after harvest. The Dominator transgenic lines D/1 and D/2 showed significant improvements in head colour relative to the control from 48 h after harvest. These results are consistent with the ethylene production patterns determined previously for these lines. The head colour results are consistent with previous results suggesting that two enzyme systems may be involved in broccoli senescence, giving two bursts of ethylene production, with only the second burst inhibited by the antisense ACC oxidase gene used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Agronomic and qualitative traits of T. turgidum ssp. dicoccum genotypes cultivated in Italy

Summary The increasing popularity of organic agriculture and health food products has led to a renewed interest in hulled wheat species such as emmer (Triticum turgidum ssp. dicoccumSchubler). Knowledge on agronomic and quality traits is required for effective and efficient use of germplasm collections in breeding programs. The objective of this study was to estimate agronomic and grain quality traits of emmer wheat cultivated in Italy. A total of 20 emmer accessions consisting of landraces, breeding lines or cultivars selected from landraces and modern cultivars were examined under low input conditions. The study was conducted for three successive years (2002–2004) at one location of Southern Italy (Foggia). The entries were characterized for agronomic and qualitative traits [grain yield (t ha⁻¹), thousand grain weight (g), test weight (kg hl⁻¹), grain protein content (%), HMWG composition, dry gluten content (%), gluten index and yellow index, alveograph indices and Total Organic Matter (TOM) on cooked pasta]. The results showed a large genetic variability for most of the traits measured and, even if most of the accessions showed inferior bread- and pasta-making performance, modern cultivars exhibited improved quality traits with some potential to perform healthy and tasty food.     

Sources of variation for sustainable field pea breeding

The pea crop (Pisum sativum L.) is a convenient source of plant protein for animal feeding. The objective of this research was to evaluate field pea breeding lines for agronomic performance and seed quality focussed to their use in a sustainable production. Thirty-five field pea breeding lines and six elite cultivars were evaluated for their agronomic value in field in Spain upon 20 traits related to flower, cycle, plant architecture, productivity and seed quality. The lines showed significant differences in all the quantitative traits evaluated and three of them, namely MB-0307, MB-0308, and MB-0319 were chosen to be evaluated, together with the advanced cultivar ZP-1233, in field and in growing chamber for agronomic performance, seed quality and ability for sustainable production. The four accessions displayed high seed protein content that had significant effect of cropping density with averages of 253.6 g kg⁻¹ under low cropping density of 60 plants m⁻² and 259.1 g kg⁻¹ under high cropping density of 90 plants m⁻², therefore, the low cropping density should be regarded as the most convenient. Average yield of the lines MB-0307, MB-0308 and MB-0319 and the cultivar ZP-1233 was fair (197.5 g m⁻²), probably due to the absence of fertilizers and irrigation, aiming for the sustainability of the crop. Intercropping with rye and herbicide application resulted in no differences on the seed yield; therefore, the ability of the breeding lines to grow without herbicide seems to be demonstrated, while the germination of the seeds at low temperature was very good. These results indicate that field pea could be a new protein crop in the North of Spain to satisfy the demand of plant protein for animal feed, based upon adapted breeding lines that combine the ability for growing under sustainable conditions with other desirable agronomic traits maintaining an adequate productivity.     

Comparison of near-isogenic lines for anthracnoseAre are genes in common bean,Phaseolus vulgaris L.

Summary This study was undertaken to determine whether the Are gene controlling anthracnose resistance had an adverse effect on maturity and yield because backcross-derived cultivars/lines had numerically lower yield and later maturity than the respective recurrent parent. Three pairs of common bean lines near-isogenic for theAre gene were developed fromAre are F₂ plants of the sixth backcross of Seafarer, Fleetwood, and Ex Rico 23. The near-isogenic lines were tested along with the recurrent cultivars and backcross-derived cultivars/lines. The results showed no evidence of any adverse effect of theAre gene on the agronomic characteristics in 11 trails at 5 locations in 3 years.     

Characterisation of transgenic oilseed rape expressing pea lectin in anthers for improved resistance to pollen beetle

In order to evaluate pea lectin as a resistance factor against the pollen beetle (Meligethes aeneus), transgenic oilseed rape (Brassica napus) has been produced wherein the pea lectin gene expression is driven by a pollen-specific promoter. The aim of the present study was to identify and characterise non-segregating transgenic and non-transgenic lines to be compared in various future tests for benefits and risks associated with such transgenic crop plants. Three doubled haploid (DH) populations (1436, 1440 and 1451) expected to include lectin-producing lines and two DH populations (1443 and 1449) expected to be free of lectin were produced. All five populations originated from different transformation events in the cultivar Westar. The relative amounts of DNA from the marker gene cassette were quantified by conventional as well as real-time PCR analyses and lectin concentrations were estimated by western blot analysis. Two populations with high lectin concentrations, 1436 and 1451, contained higher amounts of the marker DNA and thus more lectin gene copies as compared with 1440 which had lower concentrations of the lectin. As expected all DH lines from 1443 and 1449 were free of lectin. Maximum pea lectin concentration obtained corresponded to 3% of total soluble protein in the anthers. There were significant differences between the populations with respect to bud and flowering stage phenology as well as seed yields, but the differences were not related to their transgenic status. All in all there were 171 lines tested for phenology and transgenic status, out of which 89 with similar phenology were subjected to tests for lectin concentration and seed yield, and 20 of these lines were retested in the next generation. Finally two lines with high lectin concentrations and one with an intermediate level along with two matching non-transgenic lines were selected for future benefit/risk experiments.     

Comparison of phenotypic and molecular marker-based classifications of hard red winter wheat cultivars

Genetic diversity is the basis for successful crop improvement and can be estimated by different methods. The objectives of this study were to estimate the genetic diversity of 30 ancestral to modern hard red winter wheat (Triticum aestivum L.) cultivars adapted to the Northern Great Plains using pedigree information, morphological traits (agronomic measurements from six environments), end-use quality traits (micro-quality assays on 50 g grain or milled flour samples for the six environments), and molecular markers (seed storage proteins separated using SDS-PAGE, 51 SSRs, and 23 SRAP DNA markers), and to determine the relationships of genetic distance estimates obtained from these methods. Relationships among diversity estimates were determined using simple (Pearson) and rank (Spearman) correlation coefficients between distance estimates and by clustering cultivars using genetic-distances for different traits. All methods found a wide range in genetic diversity. The genetic distance estimates based on pedigree had the highest values due to possible over-estimation arising from model assumptions. The genetic diversity estimates based on seed storage protein were lowest because they were the major determinants of end-use quality, which is a highly selected trait. In general, the diversity estimates from each of the methods were positively correlated at a low level with the exceptions of SRAP diversity estimates being independent of morphologic traits (simple correlation), SDS-PAGE, and SSR diversity estimates (rank correlation). However, SSR markers, thought to be among the most efficient markers for estimating genetic diversity, were most highly correlated with seed storage proteins. The procedures used to accurately estimate genetic diversity will depend largely upon the tools available to the researcher and their application to the breeding scheme.     

Agronomic and quality effects in winter wheat of a gene conditioning resistance to wheat streak mosaic virus

Wheat streak mosaic virus (WSMV) is one of the most important diseases limiting winter wheat (Triticum aestivum L.) production in the western Great Plains of North America. There is no known effective WSMV resistance within the primary gene pool of wheat. However, a resistance gene (Wsm1) has been transferred to wheat from a perennial relative, intermediate wheat-grass [Thinopyrum intermedium (Host) Barkworth & DR Dewey]. Nebraska-adapted winter wheat lines carrying Wsm1 were used to characterize the effects of this alien introgression on agronomic and quality traits. Sister-lines from six breeding populations were evaluated under virus-free conditions, and under a naturally occurring viral infection. In uninfected locations, no significant difference for grain yield was detected between resistant (R) and susceptible (S) lines, when averaged over populations, but resistant lines had significantly higher test weights. Within populations, significantly higher grain yield was observed only in population 1, while significantly higher test weights occurred in populations 1, 2, 5 and 6. At the infected location, resistant lines were significantly higher in yield in five of six populations. In two of six populations, susceptible lines were significantly higher in bread loaf volume and bake mix time, while in the remaining populations, no significant quality differences were observed. As the Wsm1 gene provided yield advantages under viral infection, and there was no yield detriment in the absence of the virus, its deployment in hard winter wheat cultivars merits consideration.Joint contribution of the United States Department of Agriculture, Agricultural Research Service and the Department of Agronomy, University of Nebraska-Lincoln as Journal Series Paper No. 15066. Mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA or the University of Nebraska over other firms or products not mentioned.     

The effect of transfers of alien genes for leaf rust resistance on the agronomic and quality characteristics of wheat

Summary Nine transfers of leaf rust (Puccinia recondita Rob. ex Desm.) resistance to wheat (Triticum aestivum L.) from Agropyron elongatum Host. Beauv., Triticum speltoides Tausch and rye (Secale cereale L.) were backcrossed up to 10 times to commercial wheat cultivars. The objective was to study the effect of the transfers on agronomic and quality characters and to make them available in desirable genetic backgrounds. The results varied greatly for different transfers. In four cases no promising material was obtained even after nine backcrosses. However, for the remaining five transfers material with potential as a new cultivar was obtained.     

Transgenic fertile plants of soybean [Glycine max (L.) Merrill] obtained from bombarded embryogenic tissue

This work describes the production of transgenic, fertile plants of soybean [Glycine max (L.) Merrill]. The transformation method combines the advantages of somatic embryo genesis with the efficiency of particle bombardment of tissues that have a great capacity for in vitro proliferation and regeneration. The results described here represent the first report of transformation of soybean cultivars recommended for commercial growing in South Brazil using somatic embryogenesis, and may open the field for the improvement of this crop in this country by genetic engineering. This revised version was published online in August 2006 with corrections to the Cover Date.     

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T₁ lines, derived from 80 T₀ plants of 9 rice varieties. On average, about 33% T₁ lines had either morphological or agronomic variant plants. Most of the variations in T₁ plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T₃ or T₄ generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Constitutive expression of Cry proteins in roots and border cells of transgenic cotton

Transgenic cotton plants expressing Cry1Ac and Cry2Ab, from the soil bacterium Bacillus thuringiensis (Bt), provide effective control of certain lepidopteran pests, however, little is known about the proteins below ground expression. We used ELISA to quantify in vitro expression of the Cry1Ac and Cry2Ab proteins in mucilage, root border cells and root tips in five transgenic cultivars of cotton compared to conventional cultivar Sicot 189. Expression of Cry proteins in roots and border cells of the transgenic cotton cultivars was constitutive and at detectable levels, with Cry1Ac and Cry2Ab protein expression ranging from <20 ppb to >100 pbb. To determine if genetically modified cotton demonstrated simple differences in properties of the root, when compared to an elite parental line (cv. Sicot 189), we enumerated border cells on seedling radicles. Border cell counts of 14 cultivars ranged from 0.2 to 1.1 × 10⁴ cells per root tip with an average of 5 × 10³ border cells. Border cell production in the transgenic cultivars was generally similar to that of both donor and elite parents, the exception being the cultivar Sicot 189, which had substantially more border cells than all of its transgenic derivatives. Comparison of border cell number with varietal disease resistance ranking found a limited relationship (r ² = 0.65, n = 7) between border cell numbers and the commercial resistance rank against Fusarium wilt of cotton. The implications of differences in cotton cultivar border cell number and root tip expression of Cry proteins for plant–microbe interactions in the rhizosphere and the soil ecosystem are yet to be resolved.     

Estimating agronomic traits of transgenic tobacco lines

Tobacco transgenic lines belonging to four cultivars modified with three genetic constructions providing the resistance to Potato virus Y (PVY) had been described as regards their agronomic and biological traits. The constructions included viral genes: the coat protein gene of Lettuce mosaic virus (LMV) and the replicase gene of PVY used in sense and antisense orientation. Evaluation of the chosen farm traits of the transgenic tobacco lines was performed by comparing them with their nontransgenic equivalents. The traits such as growth and development rate, height of stalks, leaf area, morphology of plants, course of meiosis and pollen viability were described. The cured leaves were analysed as regards to their physical traits and chemical composition. The individual transgenic lines differed from control cultivars to a little extent. Only insignificant changes, mainly in the quality of raw material were observed. Genetic modification did not cause any changes in the course of meiosis and pollen viability.     

Line selection for exploiting durum wheat (T. turgidum L. var. durum) local landraces in modern variety development program

Summary Pure line selection for grain yield applied within four durum wheat local landraces (Triticum turgidum L. var durum) was studied in an effort to determine: (i) the agronomic performance of selected line mixtures (bulks) and their value as potential new cultivars, (ii) the effectiveness of pure line selection for grain yield within local landraces and (iii) the correlated selection response of yield components and kernel quality traits. Four local durum wheat landraces were used. Two of them, Mytilini-1 and Mytilini-2 are still cropped in some rural areas of the island Mytilini whereas the other two, Limnos and Mavragani are maintained in the Greek Gene Bank. Following their preliminary evaluation for agronomic performance under current growing conditions, 100 individual plants from each landrace were randomly selected. Selected plants (pure lines) were field evaluated head to row during 1997–98 and 1998–99 growing seasons. Based on grain yield performance, bidirectional selection, high vs low, was practised within each landrace. Thus the five higher yielding lines and their corresponding lower ones from each landrace were identified. Selected lines, with no further progeny testing, were pooled using a balanced seed composite to form the high yielding five line bulk and its respective low counterpart. The resulting 16 bulks (eight high and eight low) along with their four source landraces and two commercial varieties, Mexicalli and Simmeto, used as checks, entered field testing for agronomic performance in two and one location during 1999–00 and 2000–01 growing seasons respectively. Data for grain yield and yield components (1000 kernel weight, number of kernels, and productive tillers) along with kernel quality data (protein content and vitreous) were recorded. Data for grain yield indicated that the landraces studied seemed to be line mixtures, as expected, since they responded to bidirectional phenotypic pure line selection. The high yielding bulks were of interesting agronomic performance, equivalent to or significantly better than that of the commercial checks and are worth further testing as potential cultivars. Selected high bulks of Mytilini 2 landrace significantly outyielded both the source landrace and the commercial checks maintaining the high protein content of the source landrace, which was significantly higher than checks. This performance provides evidence that the within landraces selection of lines combining high grain yield with high protein content seems to be feasible. In conclusion, evidence is presented that genetic variability seems to be available within the landraces studied which could be worth using in breeding programs. Furthermore the simple pure line selection breeding effort undertaken seemed to be effective in developing line bulks with promising performance and worth further testing as potential cultivars.     

Inheritance of resistance to black rot (Xanthomonas campestris pv. Campestris) in cauliflower (Brassica oleracea var. Botrytis)

Summary Black rot disease caused by Xanthomonas campestris pv. campestris is a limiting factor in the commercial production of the cauliflower crop. Crosses were attempted between SN 445, a mid season cultivar resistant to black rot and two highly susceptible commercial cultivars (Pusa Snowball-1 and K-1). Studies of the F₁'s, F₂'s and back crosses indicated that SN 445, carries a dominant gene imparting resistance to black rot.     

Inheritance of resistance to pea blight (Ascochyta pinodella) and induction of resistance in pea (Pisum sativum L.)

Summary Pea blight caused by Assochyta pinodella does considerable damage to the pea crop every year. To ascertain the inheritance of resistance to pea blight and incorporate resistance in the commercial cultivars, crosses were made between Kinnauri resistant to pea blight and four highly susceptible commercial pea cultivars — Bonneville, Lincoln, GC 141 and Sel. 18. Studies of the F₁'s, F₂'s, back crosses and F₃'s indicated that Kinnauri carries a dominant gene imparting resistance to pea blight.     

Comparison of selection procedures in breeding for seed yield in segregating sesame populations

Summary Four hundred sesame (Sesamum indicum L.) lines selected by five methods from five crosses were tested for yield and other agronomic characters in F₆ and F₇. More high yielding lines were isolated by the bulk method and in the cross involving two local cultivars when two seasons means and performance in each individual season were considered. The cross involving two exotic cultivars did not produce a single line superior to local recommended variety. The 80 highest yielding lines recorded a 10.8% increase in yield compared to the mean of 400 lines. Thirty five lines were among the top 20% both seasons with a 13.2% increase in yield. Highly significant seasonal and population effects were recorded. There were no significant differences among the methods of selection although the yield differences among lines were highly significant. Interactions of seasons × populations, seasons × methods, populations × methods and seasons × populations × methods were highly significant. The results indicate that simple and less expensive bulk method may be used with equal success as the pedigree, single seed descent or early generation testing methods when selecting for yield in segregating sesame populations. The importance of inclusion of a high yielding, locally adapted cultivar in the crossing programmes for yield breeding of sesame has also been revealed.Abbreviations BM Bulk method - EGT Early generation testing - G × E Genotype × environment - MSS Modified single seed descent - PM Pedigree method - SSD Single seed descent