Influence of explant source, plant growth regulators and culture environment on culture initiation and establishment of Quercus robur L. in vitro

Suitable cytokinin supplements and culture environments havebeen determined for the initiation and establishment of shootcultures of Quercus robur seedling tissue. Initiation of axillaryshoot development from nodal explants required culture mediumsupplemented with BA (6-benzylamminopurine). The greatest numbersof stem segments for culture proliferation were obtained using1.0 mg I^-1 BA after 56 d culture. The frequency of shoot developmentand subsequent formation of multiple shoots at initiation wasinfluenced by the position of the nodal explant in the seedlingshoot, incubation temperature and daylength. Explants from basaland apical regions, which contained multiple axillary buds,produced the lowest frequencies of axillary shoot developmentand multiple shoot formation, many remained quiescent. Axillaryshoot development was greatest in single nodal explants excisedfrom the midstem positions, elongated regions of the shoot wherenodes were formerly associated with a leaf. Higher temperaturesstimulated shoot formation with greater numbers of stem segmentsfor culture multiplication being obtained from nodal explantsincubated at 25C. Axillary shoot development was promoted innodal explants maintained under daylengths of 16 h or more.Stem segments cut from axillary shoots which developed fromnodal explants were used to establish shoot multiplication cultureson medium supplemented with 0.4 mg I^-1 BA. Shoot formation fromstem segments was greater at higher incubation temperaturesof 25C and 30C. Multiplication coefficients for stem segmentsincreased after one subculture. Key words: Quercus robur, oak, micropropagation, cytokinin, temperature, daylength, rest, quiescence     

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.     

The effects of anther culture and plant genetic background on Agrobacterium rhizogenes-mediated transformation of commercial cultivars and derived doubled-haploid Brassica oleracea

The production of transgenic roots was scored for eight Brassica oleracea cultivars from broccoli, cabbage, cauliflower and kale following inoculation with an Agrobacterium rhizogenes cell line carrying a binary plasmid bearing the green fluorescence protein (gfp) gene in the T-DNA. Significant differences in the numbers of explants producing transgenic roots were observed between cultivars, ranging from 1.4% for Marathon F1 to 57.8% for the Green Duke F1. Three F1 cultivars were subjected to anther culture, and doubled-haploid (DH) lines were used for transformation. The DH lines produced showed considerable variation for transgenic root production with some lines showing increased efficiency compared to the parental F1 cultivar. Grouping of the DH lines into response classes with respect to transgenic root production allowed the development of potential genetic models to explain the variation in performance released from each F1 cultivar. No apparent segregation distortion for transgenic root production was observed in the DH lines following anther culture.     

Influence of stock plant pretreatment on gynogenic embryo induction from flower buds of onion

The gynogenic response of a range of onion genotypes to flower bud culture was compared using a two-step culture system. Embryogenic cultures and plantlets were produced from unpollinated ovules in whole flower bud explants 6 to 19 weeks after culture initiation. Preconditioning stock plants significantly influenced gynogenic embryogenesis. A ten-fold increase in embryogenesis was obtained when flower buds were cultured from stock plants maintained at 15 °C compared to 10 °C or the ambient temperature conditions of a glasshouse (maximum-minimum of 25–12.7 °C). A total of 49 embryos was obtained from 2660 cultured flower buds and 45% of plantlets were successfully acclimatised to glasshouse conditions. The majority of acclimatised plantlets were haploid (68%) but spontaneous double haploid plants (23%) were obtained from three genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transformation ofBrassica oleracea L.: a critical review

Brassica oleracea is a highly polymorphic species encompassing a wide range of important vegetable and fodder crops. Gene transfer into cultivated forms of this species requires reproducible and efficient methods for genetic transformation and plant regeneration. In this review, we have collated the research experience on transformation ofB. oleracea to highlight the problems encountered. Most research effort has been directed at developingAgrobacterium-mediated transformation methods with relatively little emphasis to date on direct gene transfer techniques. Common procedures for the transformation ofB. oleracea have not emerged, due to the inherent variability between and amongst genotypes. Future progress would be facilitated by the use of genetically fixed material, such as double-haploid or inbred lines, to reduce variation of response within genotypes and would avoid the need for cultivar-specific transformation protocols if responsive lines amenable to crossing with cultivated forms could be identified. The principal difficulties relate to combining efficient plant regeneration with gene transfer. Methods that enhance bacterial virulence and increase the proportion of cells susceptible to transformation and competent for regeneration are discussed. Inefficient selection is a major cause of poor transformation frequencies inB. oleracea and has resulted in the regeneration of chimeric plants uponAgrobacterium tumefaciens-mediated transformation. Promising results have been obtained withAgrobacterium rhizogenes-mediated transformation but the impact of therol genes on flowering of primary transformants has not yet been fully assessed. Strategies to reduce the deleterious effects of therol genes on flowering are discussed. Few agronomically useful characters have been introduced, the majority of research having been confined to the introduction of marker and reporter genes; possible candidate genes are discussed.     

Deployment of disease resistance genes by plant transformation - a 'mix and match' approach

Breeding for disease resistance has often resulted in the evolution of a matching virulence within the pathogen population, leading to an apparent 'breakdown' of resistance. In general, plant breeders have responded by introducing new genes for resistance, with similar consequences. This has led to 'boom-bust' cycles, where varieties possessing effective resistance are grown on an expanding acreage (boom) until matching virulence evolves and spreads within the pathogen population (bust). A variety of resistance genes have recently been identified and characterized in model systems. Together with the development of efficient plant transformation systems these genes offer an alternative means to introduce specific resistance into a crop improvement programme. However, unless the resistance genes are deployed with care, the boom-bust cycle is likely to be perpetuated.     

Variation of γ-glucuronidase activity in cauliflower plants with <Emphasis Type="Italic">gus</Emphasis> gene introduced by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation

In this paper we show the effect of leaf tissue sampling on estimation of β-glucuronidase activity. Pieces of leaves taken from Agrobacterium-mediated transformed T₀ plants of cauliflower were sampled and the GUS activity was measured fluorometrically. Whole leaf tissue and samples of small pieces representing various leaf zones were compared. A great variation of GUS activity within leaf was observed, for which coefficient of variation reached up to 70%. The GUS activity was nearly symmetrical for the left and right side of a leaf blade, with the highest values along the top and middle parts of a leaf blade edge. The relible and repeatable estimation of GUS activity was obtained only if a whole leaf tissue, except the midrib, was used, which allow to reduce the variation to about 10%.     

Tissue specific expression of β-glucuronidase gene driven by heterologous promoters in transgenic cauliflower plants

In this paper we compare five heterologous promoters fused to β-glucuronidase gene in their influence on localization of GUS activity in cauliflower (Brassica oleracea var. botrytis) tissues: roots, leaves, petioles and curds. A constitutive promoter CaMV 35S and four tissue specific promoters were used: extAP from rape, PsMTAP from pea, RBCS3CP from tomato and SRS1P from soybean, and introduced into cauliflower seedling explants using Agrobacterium rhizogenes mediated transformation. Quantitative and histochemical GUS assays confirmed tissue specific gus expression. It was found that extAP promoter was the most active in petioles but also caused a significant gus expression in curds. GUS activity was hardly observed in curd and restricted only to its epidermis when PsMTAP promoter drove the gene. RBCS3CP and SRS1P promoters controlled similar expression of the gus gene throughout the plant except for curd where RBCS3CP was almost inactive.     

QTLs controlling the production of transgenic and adventitious roots in Brassica oleracea following treatment with Agrobacterium rhizogenes

Brassica oleracea can be genetically engineered using Agrobacterium rhizogenes. The initial stage of this process is the production of transgenic (hairy) roots; shoots are subsequently regenerated from these roots. Previous work using gus and gfp reporter genes has shown that genotypes of B. oleracea vary in their performance for transgenic root production. Quantitative trait loci (QTLs) controlling this trait have been located in one mapping population. The current study provides evidence that performance for transgenic root production is associated with performance for adventitious (non-transgenic) root production in B. oleracea across a second mapping population. This is shown by regression analyses between performance for the two traits and the demonstration that QTLs controlling the two traits map to the same positions within the genome. Since the rate of adventitious root production does not differ significantly in the presence and absence of A. rhizogenes, there is no evidence that the expression of Agrobacterium genes induces adventitious root production. It is apparent that genotypes exhibiting high adventitious root production in the absence of A. rhizogenes will also tend to show high transgenic root production, thereby allowing the selection of lines that are more efficiently transformed.