Extracellular Formation of O-Butylhomoserine by Anaerobes

Clostridium BB–264, Cl. acetobutyricum 314–48, Cl. kaneboi, Cl. saccharoperbutylacetonicum and other two strains of Cl isolated recently produced an unidentified ninhydrin-positive compound in medium containing 5 % glucose, 1 % ammonium acetate, 0.1 % potassium dihydrogen phosphate, 0.04 % magnesium sulfate, 0.001 % ferrous sulfate, 0.1 % yeast extract, 10 μg/liter of biotin and 1 % calcium carbonate.

This ninhydrin-positive compound was eluted with solvent composed of butanol: acetic acid: water (4: 1: 2) by chromatography on cellulose powder column. It was crystallized from ethanol and then identified as an amino acid, O-butylhomoserine (Abbrev. as O-BHSer). Yield of this amino acid increased by adding homoserine or butanol to the medium. The increase was also recognized with addition of glycine, lysine, serine, threonine or valine. The formation of this amino acid was repressed by adding methionine to the medium.

Gas pressure to the culture is one of the important factors that make the amino acid formation by anaerobes possible.     

In vitro regeneration of long spur barrenwort (Epimedium grandiflorum Morr.) from rachis explants

Summary A system for micropropagation of Epimedium grandiflorum Morr. from rachis explants was developed. Explants were cultured onto Murashige and Skoog (MS) basal salts medium supplemented with (per L) 100 mg myo-inositol, 2 mg pyridoxine-HCl, 2 mg nicotinic acid, 0.40 mg thiamine-HCl, 30 g sucrose, and 2 g Phytagel. The medium also contained 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.2, or 0.25 mg/L (0.5, 0.9, or 1.1 μM) combined with either N⁶-benzyladenine (BA) or 2-isopentenyl adenine (2ip) at 2.5, 5, or 10 mg/L (11.1, 22.2, or 44.4 μM BA or 12.3, 24.6, or 49.2 μM 2iP). Cultures were maintained at a 16-h photoperiod (40 μmol/m²/s) and 23±2° C. Callogenesis preceded shoot regeneration. Callus formation increased with higher 2,4-D concentrations. The highest percent regeneration, 83% of explants, was obtained on 10 mg BA per L (44.4 μM) combined with 0.25 mg 2,4-D per L (1.1 μM). The maximum number of shoots, 15 per explant, was obtained from explants cultured on a medium containing 0.1 mg 2,4-D per L (0.45 μM) combined with 2.5 mg BA per L (11.1 μM). Maximum shoot length, 0.4 cm, was obtained on 5 mg BA per L (22.2 μM) combined with 0.2 mg 2,4-D per L (0.9 μM). To produce whole plants, shoots were separated and rooted on hormone-free medium containing 1 g activated charcoal per L. Rachises provided an excellent source of explants for Epimedium micropropagation and proved suitable for callus production.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

The effects of silver nitrate,colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat (Triticum turgidum)

Anther culture of four tetraploid wheat (Triticum turgidum) genotypes was studied using ten different culture medium treatments in a randomized block design with four replicates. Each replicate consisted of 2 pots with 3 plants. Anther donor plants were grown in a greenhouse with a 16 h day/8 h night at 25°C and 15°C, respectively. The first treatment which was considered as the control, was potato 2 medium modified by adding 0.5 g l^–1 glutamine and solidified by gelrite (4 g l^–1). The nine test treatments differed from the control by addition of 3 different concentrations of silver nitrate (1, 2.5 and 5 mg l^–1), colchicine (10, 100 and 200 mg l^–1) or cupric sulfate (2,5 and 10 mg l^–1). The study of about 2000 anthers per genotype and treatment showed that both genotype and treatment affected embryoid formation. The presence of cupric sulfate (10 mg l^–1) and silver nitrate (2.5 and 5 mg l^–1) usually increased the frequency of embryoid formation in 3 genotypes out of the 4 studied. On the contrary, colchicine had a significant and enegative effect on anther culture responses for three out of the four genotypes studied. Because of the large genotype x medium interaction, it is very difficult to identify the best medium for embryo production by all genotypes studied.     

Morphological evaluation and antioxidant activity of <Emphasis Type="Italic">in vitro</Emphasis>- and <Emphasis Type="Italic">in vivo</Emphasis>-derived <Emphasis Type="Italic">Echinacea purpurea</Emphasis> plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L⁻¹ 6-benzylaminopurine, 0.1 mg L⁻¹ α-naphthalene acetic acid and 0.2 mg L⁻¹ gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L⁻¹ 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L⁻¹ α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L⁻¹ indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.     

Agrobacterium tumefaciens– mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos

Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l^–1 picloram and 50 mg l^–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l^–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively. Received: 2 June 1997 / Revision received: 26 September 1997 / Accepted: 11 October 1997     

Micropropagation of Heracleum candicans wall: A rare medicinal herb

Summary A protocol was developed for micropropagation of Heracleum candicans Wall. by axillary shoot proliferation. Maximum shoot proliferation was obtained on Murashige and Skoog medium supplemented with 0.5 mg (2.2 μM) 6-benzyladenine per 1 and 0.1 mg (0.4 μM) 1-naphthaleneacetic acid per 1. Regenerated shoots were rooted on MS medium fortified with 1 mg (4.9 μM) indole-3-butyric acid per 1. Complete plants were transferred to soil and all of these plants were morphologically and cytologically identical to the mother plant.     

Elicitation of volatile compounds in photomixotrophic cell culture of Petroselinum crispum

Photomixotrophic cells of Petroselinum crispum accumulated >500 mg chlorophyll per kg wet weight and grew well in a broad range of phytoeffector conditions. Autoclaved fungal cells were lethal for photoheterotrophic cells, but induced in photomixotrophic cells the formation of volatile n-alkanes, phthalides, coumarins, and elemicine. Most of the compounds elicited reached a concentration maximum between 20 and 30 h after addition of the mycelium, whereas the group of n-alkanes increased steadily during the 90 h monitored. Maximum concentrations were: 12 mg of graveolone, 1 mg of bergapten, 0.5 mg of sedanenolide, and 0.5 mg of n-tetradecane per 1 nutrient medium. A dose/effect relationship was found; 10 to 25 g of fungal wet weight per 1 culture medium resulted in maximum accumulation of volatiles. The formation of volatiles by photomixotrophic in vitro cells is discussed as an integral part of plant responses to ecological stress.     

Age and Physiological Condition of Donor Plants Affect in vitro Morphogenesis in Leaf Explants of Passiflora edulis f. flavicarpa

The regeneration potential of D.alata L. germplasm preserved in vitro was compared with the micropropagation of fresh material. Nodal cuttings were conserved for 9 months in different treatments based on D-571 culture medium modified, using several variable components (mannitol, benzylaminopurine and activated charcoal). Regeneration at 8 weeks, assessed by means of percentage of explant regenerating and the multiplication at 5 weeks through the shoot length and de novo bud count formation per explant were determined. The results showed high rates (100 and 98%) of explant regeneration and micropropagation from in vitro material maintained in D-571 medium with 1.5% of mannitol + 0.1 or 1 mg l^–1 of benzylaminopurine + 2 g l^–1 of activated charcoal, respectively.     

Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693

The effects of four salt nutrients (ammonium citrate, sodium phosphate, magnesium sulfate, and manganese sulfate) on the production of mannitol by Lactobacillus intermedius NRRL B-3693 in a simplified medium containing 300 g fructose, 5 g soy peptone, and 50 g corn steep liquor per liter in pH-controlled fermentation at 5.0 at 37°C were evaluated using a fractional factorial design. Only manganese sulfate was found to be essential for mannitol production. Added manganese sulfate concentration of 0.033 g/l was found to support maximum production. The bacterium produced 200.6±0.2 g mannitol, 61.9±0.1 g lactic acid, and 40.4±0.3 g acetic acid from 300 g fructose per liter in 67 h.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

A New Synthesis of (E,E)-8, 10-Dodecadien-1-ol,Sex Pheromone of Codling Moth

A methanol-utilizing bacterium, which produced red to pink pigments and assimilated methanol via icl^- serine pathway, was isolated from soil and tentatively designated as Pseudomonas MS 31. This bacterium produced L-serine when glycine was added to the growth medium at the late exponential phase of growth. The cells showed high L-serine degradation activity. Chelating agents and some metal ions, which inhibited L-serine degradation, stimulated the L-serine accumulation. In the presence of 0.1 ? 1 mM EDTA, o-phenanthroline, 8-hydroxyquinoline, α,α'-dipyridyl, cobalt sulfate or nickel sulfate, this bacterium produced 0.7?2.1mg L-serine from 4mg glycine per ml culture.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

High intracellular expression of giant catfish growth hormone under the control of PGK promoter in Saccharomyces cerevisiae

Different yeast plasmid systems containing different promoters such as ADH1, PGK, GAPDH and GAL1, and different selectable markers, such as URA3, TRP1 and leu2-d were compared to obtain the yeast expression system that provides high intracellular expression of giant catfish growth hormone (gcGH). The highest level of gcGH expression was observed in a recombinant yeast under the control of PGK promoter (17.1 mg/l or 1.4 g/0.1 OD). The amount of gcGH was increased six-fold (102.5 mg/l) when cells were grown in a rich medium (YEPD) with the inoculum and medium ratio of 1:1, although the amount of gcGH expression per cell density did not increase (1.0 g/0.1 OD). This indicated that the increased yield of gcGH in rich medium was due to the increased cell density. The aim of the study was to produce high level gcGH in the cells of S. cerevisiae in order to use the yeast cells as potential feed additives to promote growth in giant catfish.     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

Growth the Wolinella succinogenes on H2S plus fumarate and on formate plus sulfur as energy sources

Wolinella succinogenes was found to grow on H₂S plus fumarate with the formation of elemental sulfur and succinate. The growth rate was 0.18 h^-1 (t _d=3.8 h) and the growth yield was estimated to be 6.0 g per mol fumarate used. Growth also occurred on formate plus elemental sulfur; the products formed were H₂S and CO₂. The growth rate and estimated growth yield were 0.58 h^-1 (t _d=1.2 h) and 3.5 g per mol formate used, respectively. These results suggest that certain chemotrophic anaerobes may be involved in both the formation and reduction of sulfur.     

Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm² per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of³⁵SO₄, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10⁻⁵ M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10⁻⁹ M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations. This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart, Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD.     

A new medium for spore production of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> using response surface methodology

Optimization of the medium components which enhance sporulation of the two mating types of the fungus Blakeslea trispora ATCC 14271 and ATCC 14272 (a heterothallic Zygomycota producing carotene) was achieved with the aid of response surface methodology (RSM). Glucose, corn steep liquor, yeast extract, and ammonium sulfate were investigated as carbon and nitrogen sources in a basal medium. RSM was adopted to optimize the medium in order to obtain a good growth of the fungus as a prerequisite for enhanced sporulation. In the second step, the basal medium was supplemented with different trace elements which significantly affect sporulation (i.e. CuSO₄·5H₂O, FeCl₃·6H₂O, Co(NO₃)₂·6H₂O, and MnCl₂·4H₂O). Central composite design proved to be valuable in optimizing a chemically defined solid medium for spore production of B. trispora. The composition of the new solid medium to enhance spore production by B. trispora (ATCC 14271) is as follows (per liter): 7.5 g glucose, 3.2 g corn steep liquor, 1.7 g yeast extract, 4.1 g ammonium sulfate, 6 mg CuSO₄·5H₂O, 276 mg FeCl₃·6H₂O, 2 mg Co(NO₃)₂·6H₂O, and 20 g agar (pH 6.0). Practical validation of this optimum medium gave spore number of 1.2 × 10⁸ spores/dish which is 77% higher than that produced in Potato Dextrose Agar (PDA). In the case of B. trispora (ATCC 14272) the new solid substrate for enhanced sporulation consists of (per l) 6.4 g glucose, 3.3 g corn steep liquor, 1.4 g yeast extract, 4.3 g ammonium sulfate, 264 mg CuSO₄·5H₂O, 485 mg FeCl₃·6H₂O, 223 mg MnCl₂^.4H₂O, and 20 g agar (pH 6.0). Spore numbers of 2 × 10⁷ spores/dish were obtained on the new medium by B. trispora (ATCC 14272), which is 95% higher than that produced on PDA. The results corroborated the validity and the effectiveness of the models. The new media considerably improved sporulation of both strains of B. trispora compared to the production of spores on PDA, which is the medium usually used for sporulation of the fungus.     

雷竹体细胞胚诱导的初步研究

以雷竹（Phyllostachys violascens）种胚为外植体, 脱分化产生愈伤组织, 愈伤组织诱导产生体细胞胚, 并发育成苗。实验结果表明： 愈伤组织诱导体细胞胚基本培养基为MS无机盐＋20 g·L^-1葡萄糖（glucose）＋10 mg·L^-1腺嘌呤（adenine sulfate）＋ 0.5 g·L^-1麦芽抽提物（malt extract）＋0.1 mg·L^-1 6-BA＋0.01 mg·L^-1氨氯吡啶酸（picloram）＋10 g·L^-1type A agar; 将基本培养基中的氨氯吡啶酸浓度升高至0.1 mg·L^-1即为愈伤组织诱导的最佳培养基, 早期子叶胚是愈伤组织诱导的最佳胚体; 愈伤组织在添加0.001 mg·L^-1TDZ和0.3 mg·L^-1ABA的愈伤组织最佳培养基上光照培养4周后, 去除其中的ABA, 并添加0.1 mg·L^-1NAA继续培养1个月, 体细胞胚数量最多可达87.43%, 该培养基是体细胞胚发生的最佳培养基; 将上述体细胞胚发生培养基中的6-BA浓度升高至1 mg·L^-1, 继代培养2个月后有小苗出现。组织学观察显示, 体细胞胚细胞核大、质浓且多数呈球形原胚状。     

Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

Summary To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N⁶-benzyladenine (BA) per L, and 0.8% Noble agar). The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3–4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.