A protocol for efficient transformation and regeneration of Carica papaya L.

Summary A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya. Based in part on a presentation at the 1997 SIVB Congress on In Vitro Biology held in Washington, DC, June 14–18, 1997.     

Stable transformation of papaya via microprojectile bombardment

Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1^–1 2,4-D, 400 mg 1^–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1^–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - X-gluc 5-Br-4-Cl-3-indolyl--D-glucuronic acid - CaMV cauliflower mosaic virus - NOS nopaline synthase - NPTII neomycin phosphotransferase II Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Potential for introducing cold tolerance into papaya by transformation with C-repeat binding factor (CBF) genes

Papaya (Carica papaya L.) production is affected by low temperatures that occur periodically in the subtropics. The C-repeat binding factor (CBF) gene family is known to induce the cold acclimation pathway in Arabidopsis thaliana. Embryogenic papaya cultures were induced from hypocotyls of “Sunrise Solo” zygotic embryos on semisolid induction medium. The CBF 1/CBF 3 genes along with the neomycin phosphotransferase (NPT II) gene were placed under the control of the CaMV 35 S promoter and introduced into a binary vector pGA 643. Embryogenic cultures were transformed with Agrobacterium strain GV 3101 harboring pGA 643. After selection of transformed embryogenic cultures for resistance to 300 mg l⁻¹ kanamycin, somatic embryo development was initiated and transgenic plants were regenerated. The presence of the CBF transgenes in regenerated plants was confirmed by Southern blot hybridization. The papaya and the related cold-tolerant Vasconcella genomes were probed for the presence of cold inducible sequences using polymerase chain reaction (PCR). Possible cold inducible sequences were present in the Vasconcella genome but were absent in the Carica genome.     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

Optimizing Agrobacterium-mediated transformation of grapevine

Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl⁻¹ of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl⁻¹ kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.     

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l^–1 or on geneticin within 12.5–50 mg l^–1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l^–1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l^–1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l^–1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l^–1) to avoid the adverse effects of kanamycin on embryogenesis.     

Evaluation of key factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of somatic embryos of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l⁻¹ kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l⁻¹ partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ timentin for 2 months. Then, kanamycin level was increased to 100 mg l⁻¹ for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA₃ for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l⁻¹ agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

Production ofAgrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos ofDatura innoxia

This work describes a new method to obtain transgenic somatic embryos fromAgrobacterium-infected immature zygotic embryos ofDatura innoxia. It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency. Critical steps for optimal transformation were the embryo stage and a short preculture treatment. The marker gene -glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development. The transgenes were transmitted to the progeny in a Mendelian fashion. The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform. We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency forAgrobacterium infection was developmental stage-specific. This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.     

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3–6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.V.G. Sunnichan and R. Kumria contributed equally to this investigation     

Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens

Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.     

超声波辅助农杆菌介导CP基因转化番木瓜（Carioca papaya L.）的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PT—NP—YKT)插入双元栽体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1／2MS 6％蔗糖 1％葡萄糖,pH5．7)调整至光密度OD600nm=15-0．20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26．3％),而在对照处理64块胚性愈伤中仅获得1个转化系(1．6％);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16．7％),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP基因转化上均表现出相似的效果。试验还表明：120mg／L是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定。     

Induction of somatic embryogenesis and genetic transformation of ohio buckeye (Aesculus glabra willd.)

Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment, four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred to fresh medium for germination.     

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

Summary An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15–20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4–5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and -glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7–8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.     

Transgenic papaya plants from Agrobacterium-mediated transformation of petioles of in vitro propagated multishoots

Summary In order to establish a model system for introduction of foreign genes into papaya (Carica papaya L.) plants by Agrobacterium-mediated transformation, petioles from multishoots were used as explant source and bacterial neomycin phosphotransferase II (NPT II) gene and -glucuronidase (GUS) gene were used as a selection marker and a reporter, respectively. Cross sections of papaya petioles obtained from multishoots micropropagated in vitro were infected with A. tumefaciens LBA4404 containing NPTII and GUS genes and co-cultured for 2 d. The putative transformed calluses were identified by growth on the selective medium containing kanamycin and carbenicillin, and consequently regenerated to plants via somatic embryogenesis. Thirteen putative transgenic lines were obtained from a total of 415 petiole fragments treated. Strong GUS activity was detected in the selected putative transgenic calli or plants by fluorogenic assay. Western blot analysis using GUS antiserum confirmed that the GUS protein was expressed in putative transformed papaya cells and transgenic plants. The presence of the GUS gene in the papaya tissues was detected by PCR amplification coupled with Southern blot.     

The sensitivity of embryogenic tissue of Picea omorika (Panč.) Purk. to antibiotics

In an attempt to develop a transformation procedure for somatic embryos of Picea omorika (Pan.) Purk. using Agrobacterium, the sensitivity of non-transformed embryogenic tissues of P. omorika to antibiotics was measured. Two groups of antibiotics were tested: antibiotics commonly used to eliminate Agrobacterium from tissue culture (carbenicillin and cefotaxime), and antibiotics for the selection of transformed tissue (kanamycin, paromomycin, neomycin and hygromycin). Carbenicillin (500 mg l^–1) reduced proliferation of P. omorika embryogenic tissue by 50% as compared to the control. Cefotaxime (500 mg l^–1) was less toxic to embryogenic tissue growth (93% of the control) and is thus more suitable for the elimination of A. tumefaciens from embryogenic tissue of P. omorika. Embryogenic tissue showed severe susceptibility to kanamycin and hygromycin. Induction of secondary embryogenesis was blocked by 10 mg l^–1 of kanamycin or 2 mg l^–1 of hygromycin. The aminoglycoside analogs of kanamycin, paromomycin and neomycin inhibited embryogenic tissue induction at concentrations of 5 and 100 mg l^–1, respectively. The higher tolerance of embryogenic tissue to neomycin indicated that of the antibiotics tested neomycin may be most useful for selection of nptII-transformed embryogenic tissue of Picea omorika.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.