Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis

We have identified quantitative trait loci (QTL) for transgenic and adventitious root production using an Agrobacterium rhizogenes-mediated co-transformation system in conjunction with a Brassica oleracea double haploid (DH) mapping population. Three QTL for green fluorescent protein (GFP)-fluorescent root production and four QTL for adventitious root production were identified as accounting for 26% and 32% of the genetic variation in the population, respectively. Two of the QTL regions identified were common to both transgenic and adventitious root production. Two different methods of QTL analysis were employed (marker regression and interval mapping) and with the exception of one region on linkage group O7 for transgenic root production, both techniques detected the same regions of the genome. The regions we identified to be associated with the control of transgenic root production following A. rhizogenes-mediated transformation are the first to be detected using a QTL mapping approach. In addition, this is the first study to identify genetic regions that co-regulate both transgenic and adventitious root production within the constraints of an A. rhizogenes-mediated transformation process. We have identified plant genotypes that do not produce any transgenic roots that may be deficient for T-DNA integration via illegitimate recombination and that may also be potentially important for the development of homologous recombination protocols. Conversely, we have also identified plant genotypes with high rates of transgenic root production that will be critical in the development of high throughput transformation systems.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

In vitro selection and regeneration of carnation (Dianthus caryophyllus L.) plants resistant to culture filtrate of Fusarium oxysporum f.sp. dianthi

Callus cultures derived from internodal segments of two cultivars of carnation susceptible to Fusarium oxysporum f.sp. dianthi were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant lines were selected by culturing calli on growth medium containing various concentrations of the culture filtrate of F. oxysporum f.sp. dianthi. Resistant calli obtained after two cycles (25 days/cycle) of selection were used for plant regeneration. About 32% of the plants regenerated from the resistant calli had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus

Commercial barley cultivars are difficult to transform because of the lack of an efficient regeneration system. By modifying certain components in the standard culture medium, we have developed a reproducible and more efficient regeneration system. Herbicide-resistant transgenic plants from barley (Hordeum vulgare L. cv. Conlon) were obtained using this medium. Embryo-derived callus was bombarded with pAHC25, which contains the screenable marker gus (#-glucuronidase) and the selectable marker bar (bialaphos resistance gene), both driven by the maize ubiquitin promoter (Ubi1) and followed by the nos terminator. Following bombardment, callus was transferred to callus-induction medium supplemented with 5 mg/l bialaphos for selection. Resistant calli were subsequently transferred to maintenance medium containing 5 mg/l bialaphos for further selection and finally transferred to regeneration medium with 5 mg/l bialaphos. Green shoots that developed on the regeneration medium were transferred to rooting medium containing 3 mg/l bialaphos. Eighty-five transgenic plants were obtained from 13 independent transformation events. Progeny tests showed Mendelian inheritance for the transgenes. This is the first report of the production of large numbers of transgenic plants from a commercial cultivar adapted to Midwestern US barley production.     

Nitrogen addition has a stronger effect on stoichiometries of non-structural carbohydrates,nitrogen and phosphorus in <Emphasis Type="Italic">Bothriochloa ischaemum</Emphasis> than elevated CO<Subscript>2</Subscript>

The current study attempted to obtain candidate doubled haploid (DH) wheat lines by serially combining two approaches: conventional chemical mutagenesis and anther culture. Additionally, the salt tolerance levels were examined between stress-treated (100 mM NaCl) and non-treated DH groups. For the molecular analysis, IRAP markers were used to characterize retrotransposon insertion polymorphisms induced by haploidization, chromosome doubling, and/or mutagenesis in the DH lines. Various sodium azide (NaN₃) concentrations (from 0 to 5 mM) were applied to seeds of the Pehlivan wheat cultivar to obtain an M₁ generation mutant population. Anther culture was set up from the M₁ mutant population. Green plant regeneration, the frequency of selected candidate mutants within the DH form and the levels of salt tolerance between samples were screened. A total of eight thousand anthers were cultured, and sixteen candidate salt-tolerant DH mutant lines, twenty-seven candidate DH mutant lines with different characteristics and one hundred and two candidate DH lines with morphologically normal appearances were obtained from the NaN₃-mutagenized population. The IRAP patterns were quite similar between the control DH lines, and the genetic differences between the controls and DHs originating from possible mutants showed close relatedness. According to previous studies, chemical mutagenesis and anther culture were combined for the first time to detect candidate salt tolerant genotypes at the DH stage. This approach might also be useful for determining the threshold dose and efficiency of wheat mutagens.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.     

Micrografting and <Emphasis Type="Italic">ex vitro</Emphasis> grafting for somatic embryo rescue and plant recovery in avocado (<Emphasis Type="Italic">Persea americana</Emphasis>)

To improve plant regeneration from oat (Avena sativa L.) anther culture, the effects of induction medium supplements and culture conditions were studied. Significantly better plant regeneration rates were obtained with cultivars Lisbeth (naked type) and Aslak when a medium containing W₁₄ salts and vitamins, supplemented with 2,4-d, BAP, Ethephon, l-cysteine and myo-inositol, was used for induction in the dark compared with a medium containing only 2,4-d and kinetin. Genotypes reacted differently on the light during the induction phase. Use of dim light significantly decreased the green plant regeneration rates in cv. Lisbeth, while in cv. Aslak the difference was not so clear. Up to 30 green plants per 100 anthers were recovered from Aslak × Lisbeth progeny and in total, over 500 oat regenerants were produced. With these numbers, acceptable rates of DH-production for cultivar breeding and genetic study purposes are approached. The agronomic performance of some DH lines was compared with that of the plants derived from commercial seeds of the same cultivars in the field experiment. A few differences were found, but generally DH lines yielded the same or more as the commercial cultivars.     

Plasmodiophora brassicae-induced Cell Death and Medium Alkalization in Clubroot-resistant Cultured Roots of Brassica rapa

Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10⁴, 10⁵ or 10⁶ spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca²⁺ in P. brassicae‐induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.     

Root Dynamics of Cultivar and Non‐Cultivar Population Sources of Two Dominant Grasses during Initial Establishment of Tallgrass Prairie

Dominance of warm‐season grasses modulates tallgrass prairie ecosystem structure and function. Reintroduction of these grasses is a widespread practice to conserve soil and restore prairie ecosystems degraded from human land use changes. Seed sources for reintroduction of dominant prairie grass species include local (non‐cultivar) and selected (cultivar) populations. The primary objective of this study was to quantify whether intraspecific variation in developing root systems exists between population sources (non‐cultivar and cultivar) of two dominant grasses (Sorghastrum nutans and Schizachyrium scoparium) widely used in restoration. Non‐cultivar and cultivar grass seedlings of both species were isolated in an experimental prairie restoration at the Konza Prairie Biological Station. We measured above‐ and belowground net primary production (ANPP and BNPP, respectively), root architecture, and root tissue quality, as well as soil moisture and plant available inorganic nitrogen (N) in soil associated with each species and source at the end of the first growing season. Cultivars had greater root length, surface area, and volume than non‐cultivars. Available inorganic N and soil moisture were present in lower amounts in soil proximal to roots of cultivars than non‐cultivars. Additionally, soil NO₃–N was negatively correlated with root volume in S. nutans cultivars. While cultivars had greater BNPP than non‐cultivars, this was not reflected aboveground root structure, as ANPP was similar between cultivars and non‐cultivars. Intraspecific variation in belowground root structure and function exists between cultivar and non‐cultivar sources of the dominant prairie grasses during initial reestablishment of tallgrass prairie. Population source selection should be considered in setting restoration goals and objectives.     

GFP accumulation controlled by an auxin-responsive promoter as a non-destructive assay to monitor early auxin response

We have constructed transgenic Arabidopsis lines that contain a gene for green fluorescent protein (GFP) under the control of auxin-responsive domains A and B of the promoter from the pea PS-IAA4/5 gene. The chimeric transgene was named BA-mgfp5-ER. GFP was detected after the application of indole-3-acetic acid at concentrations as low as 100 nM in epidermal cells in the root elongation zone. The induction of the reporter gene was highly specific to auxin and was correlated with the auxin-induced change in epidermal cell shape. No GFP accumulation was observed in the lateral root meristem that was formed as a result of exogenous auxin application. These results suggest that auxin signals were transmitted through several distinct pathways depending on the cell type. The intensity of the GFP signal was strong enough to be observed through the plastic lid of the culture dish using a dissecting microscope, thereby enabling GFP expression to be monitored in an aseptic environment. Thus, the BA-mgfp5-ER transgenic plant can be a powerful tool for screening mutants that are defective in auxin signaling and the expression of early auxin-response genes.     

Light-enhanced protein synthesis in gravitropically stimulated root caps of corn

Light stimulates gravitropic bending (downward growth) in roots of many cultivars of corn (Zea mays). In this work, using the cultivar Merit, we show that light stimulates protein synthesis in the root cap, with protein levels increasing 1.3 to 1.6 times that recorded for tissues maintained in continuous dark. Light enhances protein levels both in intact caps (attached to the root) and in caps in culture. Protein synthesis is optimal in cultured caps when 1 nanomolar indole-3-acetic acid is included in the culture medium. If cap tissue is illuminated and subsequently returned to the dark, in the 2-hour period following illumination protein levels decline to that observed in dark controls. It is proposed that light-stimulated protein synthesis mediates in part downward bending in roots of these cultivars of corn.     

Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation

. Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.     

Cultivar and cultivar x environment effects on the development of callus and polyhaploid plants from anther cultures of wheat

Summary Plants of three common wheat (Triticum aestivum L. em. Thell) cultivars and one randomly selected doubled-haploid line derived by anther culture from each of the three cultivars were each grown in three environments, a field environment, a greenhouse environment, and a growth chamber environment. Anthers containing largely miduninucleate to late uninucleate microspores were cultured and calli were induced to regenerate plants in order to assess the effects of cultivar, cultivar family (cultivar and corresponding doubled-haploid derivative), anther-donor plant environment, and cultivar X environment interaction on androgenic responses. Large differences in response were observed among cultivars as well as between cultivars and doubled-haploids. Differences between cultivar and doubled-haploid within cultivar family usually resulted from higher frequency of response in the cultivar, contrary to the hypothesis that anther culture per se constitutes a general selective device for superior androgenic responses. Also, in a second experiment, anther callusing frequency was greater in the cultivar Kitt than in any of five unique doubled-haploid lines derived from Kitt. Significant effects were also observed in the first experiment for the interactions of cultivar family X environment as well as doubled-haploid vs. cultivar X environment, although the effect of environment itself was less significant than these interactions.Contribution from the USDA, SEA, AR, Beltsville, Md, and the Department of Agronomy, University of Maryland, College Park, Md, as scientific article No. A-3413, contribution No. 6486     

The effect of Lr19-translocation on in vitro androgenesis and inheritance of leaf-rust resistance in DH3 lines and F2 hybrids of common wheat

Leaf-rust resistance and androgenesis were studied in the anther cultures of Triticum aestivum L., which included Saratovskaya 29 cultivar, the isogenic line Ps29, and three F1 hybrids (L503/S55, L504/S58, ATS7/L1063) with 7DS-7DL-7Ae#1L translocation of Lr19 gene (Lr19 translocation) from Agropyron elongatum (Host) P.B. The Lr19 translocation was shown to affect the induction of embryogenesis and green plant regeneration. The frequencies of Lr19 translocation differed in F2 hybrids obtained by traditional hybridization and in sets of DH lines obtained in F1 anther cultures derived from the same combinations of T. aestivum parental forms. The number of leaf-rust resistant genotypes tended to decrease. The frequency of Lr19 translocation in the set of DH3 lines derived from F1 L504/S58 was significantly lower than in other sets of DH3 lines and F2 hybrid populations.     

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)–type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport. Received October 15, 1998; accepted May 12, 1999     

籼稻花粉无性系变异的研究

对７个籼稻品种通过花药培养获得的１６１个花粉植株进行了花粉无性系变异的研究。结果表明：（１）所有花粉无性系在遗传上都是纯合的,其整齐度与起始亲本相近或超过起始亲本;（２）花粉无性系的变异主要在数量性状上有一定变幅,变异方向有负向也有正向的。同一来源的无性系间的变异系数略高于起始亲本,仅千粒重达到显著标准。以所考查的性状为基数,变异的频率为１２．５％,同时有３－４个性状发生变异的无性系数占总数的４．３％;（３）少数变异体性状有重大变异,并具有育种价值;（４）根据１０种同工酶和ＲＦＬＰ分析,仅从３个性状发生重大变化的变异体中检测出与起始亲本的差异。     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.