Establishment of multiple shoot clumps from maize (Zea mays L.) and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene als (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene als has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R₁ plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Establishment of multiple shoot clumps from maize (Zea mays L.) and regeneration of herbicide-resistant transgenic plantlets

A kind of quick, efficient and season-free inducing embryoid and multiple shoot clumps system from shoot tip meristems that derived from elite inbreds of maize was established. The herbicide-resistant gene als (coding Acetolactate synthase) isolated from a mutant of Arabidopsis thaliana was transferred to tissue pieces of maize multiple shoot clumps by microprojectile bombardment. Herbicide-resistant tissue and regenerants were obtained through selections with herbicide chlorsulfuron. PCR analysis and Southern blot hybridization indicated that gene als has been transferred to some regenerants. The test of spraying chlorsulfuron displayed that the transgenic plantlets and R1 plants had favorable herbicide-resistant trait. We have established a new genotype-free system of maize which could rapidly and efficiently produce large quantities of transgenic plantlets.     

Gibberellins, Amylase, and the Onset of Heterosis in Maize Seedlings

Rood, S. B. and Larsen, K. M. 1988. Gibberellins, amylase, andthe onset of heterosis in maize seedlings.—J. exp. Bot.39: 223–233. The possible involvement of gibberellins and amylase in heterosisof maize seedlings was investigated in two parental inbreds,CM7 and CM49, and their single cross F₁ hybrid, CM7xCM49. Germinationof all three genotypes was complete within 36 h after the onsetof imbibition. By 48 h, heterosis (hybrid vigour) for increasedshoot and root length was consistently observed. The endogenousconcentration of gibberellin A₁ (GA₁) was measured in 48 h seedlingsby gas chromatography-mass spectrometry with selected ion monitoring(GC-SIM) using [²H₂]-GA₁ as an internal standard. The GA₁ concentrationwas highest in the hybrid (59 ng g^–1 dry wt.), intermediatein CM49 (9.0 ng g^–1), and lowest in CM7 (<5.0 ng g^–1).Amylase activities in all three genotypes were very low at 24h, but increased during the next 24 h, after which time amylaseactivity in the hybrid was significantly higher than that ofeither parental inbred. Inhibitors of gibberellin (GA) biosynthesis,AMO-1618 or CCC, inhibited germination, shoot and root growth,and amylase activity in all three genotypes. Conversely, exogenousgibberellic acid (GA₃) increased amylase activity, particularlyin the inbred CM7. Amylase isozymes were separated through polyacrylamidegel electrophoresis and generally similar profiles of starchdegrading enzymes were observed in the three genotypes. SinceGA is known to control a-amylase biosynthesis in some cereals,these results are consistent with the hypothesis that GAs areinvolved in the regulation of heterosis in maize. A higher endogenousGA₁ concentration in the hybrid could result in increased amylaseactivity in the hybrid seedlings and consequently, more rapidstarch hydrolysis which fuels heterosis for early growth. Key words: Amylase, germination, gibberellic acid, Gibberellin A₁, heterosis, hybrid vigour, Zea mays     

过量表达水稻细胞质型OsAPXs基因提高转基因烟草的耐盐性

为了验证水稻(Oryza sativa L.)细胞质型APXs与细胞耐盐性的关系,实验分别将OsAPXa和OsAPXb（基因登录号：D45423、AB053297）转化到烟草(Nictiana tabacum,N.plum)植株中。Southern结果表明,二基因分别整合到烟草的基因组;Northern分析表明,外源基因在转基因烟草中得到高效表达;在碳酸盐逆境下,T2代转基因植株与野生型对照相比,其APX活性呈现显著的提高,T₂代品系的H₂O₂含量和叶片受害程度显著低于野生型;T₂代品系分别在含有10 mmol·L^-1 NaHCO₃、5 mmol·L^-1 Na₂CO₃的MS培养基上生长,根的生长受到抑制,叶片产生黄化;野生型烟草则难以存活。水稻细胞质型OsAPXs基因的过量表达提高了转基因烟草的耐盐性,揭示出OsAPXa和OsAPXb在碳酸盐逆境应答过程中发挥着重要的作用。     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

转MDMV CP基因玉米植株的再生

用基因枪法将玉米矮花叶病毒外壳蛋白基因导入玉米自交系综31幼胚诱导的愈伤组织中,在含有Bialaphos 6 mg·L^-1的选择培养基上经过3个月的抗性筛选,抗性愈伤组织在分化培养基上生成可育再生植株。PCR、PCR-Southern blot及DNA点杂交结果表明,外源基因已导入到玉米基因组中。转基因T1和T2代植株在大田表现出对MDMV的抗性,可以降低发病率,减轻发病程度。     

The Influence of Nitrate and Ammonium Nutrition on the Growth of Wheat (Triticum aestivum) and Maize (Zea mays) Plants

The effects of NO^-₃ and NH⁺₄ nutrition on hydroponically grownwheat (Triticum aestivum L.) and maize (Zea mays L.) were assessedfrom measurements of growth, gas exchange and xylem sap nitrogencontents. Biomass accumulation and shoot moisture contents ofwheat and maize were lower with NH⁺₄ than with NO^-₃ nutrition.The shoot:root ratios of wheat plants were increased with NH⁺₄compared to NO^-₃ nutrition, while those of maize were unaffectedby the nitrogen source. Differences between NO^-₃ and NH⁺₄-fedplant biomasses were apparent soon after introduction of thenitrogen into the root medium of both wheat and maize, and thesedifferences were compounded during growth. Photosynthetic rates of 4 mM N-fed wheat were unaffected bythe form of nitrogen supplied whereas those of 12 mM NH⁺₄-fedwheat plants were reduced to 85% of those 12 mM NO^-₃-fed wheatplants. In maize supplied with 4 and 12 mM NH⁺₄ the photosyntheticrates were 87 and 82% respectively of those of NO^-₃-fed plants.Reduced photosynthetic rates of NH⁺₄ compared to NO^-₃-fed wheatand maize plants may thus partially explain reduced biomassaccumulation in plants supplied with NH⁺₄ compared to NO^-₃ nutrition.Differences in the partitioning of biomass between the shootsand roots of NO^-₃-and NH⁺₄-fed plants may also, however, arisefrom xylem translocation of carbon from the root to the shootin the form of amino compounds. The organic nitrogen contentof xylem sap was found to be considerably higher in NH⁺₄- thanin NO^-₃-fed plants. This may result in depletion of root carbohydrateresources through translocation of amino compounds to the shootin NH⁺₄-fed wheat plants. The concentration of carbon associatedwith organic nitrogen in the xylem sap of maize was considerablyhigher than that in wheat. This may indicate that the shootand root components of maize share a common carbon pool andthus differences induced by different forms of inorganic nitrogenare manifested as altered overall growth rather than changesin the shoot:root ratios.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize, nitrogen, growth, photosynthesis, amino acids, xylem     

High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants

This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7-10 months.     

The Influence of NO-3 and NH+4 Nutrition on the Gas Exchange Characteristics of the Roots of Wheat (Triticum aestivum) and Maize (Zea mays) Plants

Respiratory oxygen consumption by roots was 1·4- and1·6-fold larger in NH⁺₄-fed than in NO^-₃-fed wheat (Triticumaestivum L.) and maize (Zea mays L.) plants respectively. Higherroot oxygen consumption in NH⁺₄-fed plants than in NO^-₃-fedplants was associated with higher total nitrogen contents inNH⁺4-fed plants. Root oxygen consumption was, however, not correlatedwith growth rates or shoot:root ratios. Carbon dioxide releasewas 1·4- and 1·2-fold larger in NO⁺3-fed thanin NH⁺₄-fed wheat and maize plants respectively. Differencesin oxygen and carbon dioxide gas exchange rates resulted inthe gas exchange quotients of NH^-₄-fed plants (wheat, 0·5;maize, 0·6) being greatly reduced compared with thoseof NO^-₃-fed plants (wheat, 1·0; maize, 1·1). Measuredrates of HCO^-₃ assimilation by PEPc in roots were considerablylarger in 4 mM NH⁺₄-fed than in 4 NO^-₃ plants (wheat, 2·6-fold;maize, 8·3-fold). These differences were, however, insufficientto account for the observed differences in root carbon dioxideflux and it is probable that HCO^-₃ uptake is also importantin determining carbon dioxide fluxes. Thus reduced root extension in NH⁺₄-fed compared with NO^-₃-fedwheat plants could not be ascribed to differences in carbondioxide losses from roots.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize assimilation, ammonium assimilation, root respiration     

Assimilate Partitioning in Red and White Clover Either Dependent on N2 Fixation in Root Nodules or Utilizing Nitrate Nitrogen

During vegetative growth in controlled environments, the patternof distribution of ¹⁴C-labelled assimilates to shoot and root,and to the meristems of the shoot, was measured in red and whiteclover plants either wholly dependent on N₂ fixation in rootnodules or receiving abundant nitrate nitrogen but lacking nodules. In experiments where single leaves on the primary shoot wereexposed to ¹⁴CO₂, nodulated plants of both clovers generallyexported more of their labelled assimilates to root (+nodules),than equivalent plants utilizing nitrate nitrogen, and thiswas offset by reduced export to branches (red clover) or stolons(white clover). The intensity of these effects varied with experiment.The export of labelled assimilate to growing leaves at the terminalmeristem of the donor shoot was not influenced by source ofnitrogen. Internode elongation in the donor shoot utilized nolabelled assimilate. Whole plants of white clover exposed to ¹⁴CO₂ on seven occasionsover 32 days exhibited the same effect on export to root (+nodules),which increased slightly in intensity with increasing plantage. Nodulated plants had larger root: shoot ratios than theirequivalents utilizing nitrate nitrogen. Trifolium repens, Trifolium pratense, red clover, white clover, nitrogen fixation, nitrate utilization, assimilate partitioning     

Use of hi ii-elite inbred hybrids in Agrobacterium-based transformation of maize

Summary Hybrid embryos resulting from crosses between a highly regenerable maize germplasm (Hi II) and certain elite inbreds were treated with Agrobacterium tumefaciens containing the uidA (GUS) and pat genes under the control of different constitutive promoters. Six of the elite inbred lines were derived from a Lancaster background and three were derived from an Iowa Stiff Stalk background. Hybrid embryos from all three Stiff Stalk lines gave transgenic events at various frequencies, two of them at a comparable frequency to that observed with Hi II embryos. Embryos from only one Lancaster/Hi II hybrid were successfully transformed and the frequency was quite low. Additional Lancaster elite inbreds were then tested as a hybrid with Hi II and failed to produce a single transgenic event. The transgenic Hi II/elite events showed many characteristics of ‘hybrid vigor’ including more aggressive rooting, thicker stems, and taller stature than plants derived from Hi II events. The hybrid T₀ plants exhibited excellent tassel development in the greenhouse with abundant pollen shed. Seed set in the greenhouse was significantly (3–5-fold) higher than with Hi II transformats. Attempts to transform embryos derived from self or sibling crosses of the four inbred lines that were successful as hybrids with Hi II did not produce any transgenic events. T₀ plants having ∼50% elite genomic contribution perform nearly as well in the greenhouse as seed-derived elite inbred parents and offer a significantly reduced time line for recombinant protein product development from transgenic plants.     

Isolation of transgenic progeny of maize by embryo rescue under selective conditions

Summary Fertile transgenic maize plants (T₀) and progeny (T₁) were obtained using microprojectile bombardment and callus selection on hygromycin B. To quickly identify progeny expressing the transgene, embryos from T3 generation kernels were excised 20 days after pollination and exposed to different concentrations of hygromycin B. Surviving and non-surviving embryos were assayed for the presence of the hygromycin phosphotransferase (aphIV) gene using polymerase chain reaction. Embryos that germinated and survived on 25, 50, or 100 mg/liter hygromycin possessed theaphIV gene. Embryos that did not germinate lacked the gene. Progeny surviving selection were transferred to the greenhouse and tested for expression of the gene using a leaf disc assay. The results demonstrated that the gene construct was expressed in both embryo and leaf tissue and that selection during germination successfully eliminated progeny lacking the gene of interest. This method is also useful for rapid-cycling of maize generations.     

The Combined Effects of Salinity and Root Anoxia on Growth and Net Na+ and K+-accumulation in Zea mays Grown in Solution Culture

The effects of excess salinity and oxygen deficiency on growthand solute relations in Zea mays L. cv. Pioneer 3906 were examinedin greenhouse experiments. The roots of plants 14 d old growingin nutrient solution containing additions of NaCl in the range1.0–200 mol m^–3 were either exposed to a severedeficiency of O₂ by bubbling with nitrogen gas (N₂ treatment),or maintained with a supply of air (controls), for a periodof 1–7 d. The threshold NaCl concentration resulting inappreciable inhibition of leaf extension, and shoot f. wt gainin controls was between 10 and 25 mol m^–3. At 25 mol m^–3NaCl the ratio of Na+/K+ transported to shoots was about 20times greater than in plants in 1.0 mol m^–3 NaCl. Theeffect of addition of NaCl to the nutrient solution was to enhanceNa+ movement but simultaneously depress the rate of K+ transportto shoots (per g f. wt roots). Interactions between NaCl levels and aeration treatment wereshown by analyses of variance to be statistically significantfor leaf extension, shoot and root f. wt gains, Na+ and K+ concentrationsin shoots and roots. When roots were N₂-treated, shoot and rootgrowth were depressed, the effect of aeration treatment beinggreatest at NaCl concentrations of 50 mol m^–3 or less.Additionally, N₂-treatment greatly accelerated Na- transportto shoots while depressing K+ transport still further, so thatat 10 mol m^–3 NaCl the ratio Na+/K+ acquired by the shootswas 230 times greater than in controls. Over the concentrationrange 1.0 to 50 mol m^–3 NaCl, the ratio Na+/K+ transportedto shoots by anoxic roots increased by a factor of 860. Mechanisms controlling changes in solute flux to the shoot,and the significance in relation to plant tolerance of excesssalts or oxygen deficiency are discussed. Anaerobic, corn, flooding, maize, oxygen-deficiency, salinity     

Shoot meristem: an ideal explant for Zea mays L. transformation.

We report on a rapid high-frequency somatic embryogenesis and plant regeneration protocol for Zea mays. Maize plants were regenerated from complete shoot meristem (3-4 mm) explants via organogenesis and somatic embryogenesis. In organogenesis, the shoot meristems were directly cultured on a high-cytokinin medium comprising 5-10 mg x L(-1) 6-benzylaminopurine (BAP). The number of multiple shoots produced per meristem varied from six to eight Plantlet regeneration through organogenesis resulted in just four weeks. Callus was induced in five days of incubation on an auxin-modified Murashige and Skoog (MS) medium. Prolific callus, with numerous somatic embryos, developed within 3-4 weeks when cultured on an auxin medium containing 5 mg 2,4-dichlorophenoxyacetic acid x L(-1). The number of multiple shoots varied from three to six per callus. Using R23 (Pioneer, Hi-Bred, Johnston, Iowa), the frequency of callus induction was consistently in excess of 80% and plant regeneration ranged between 47 and 64%. All regenerated plantlets survived in the greenhouse and produced normal plants. Each transgenic plant produced leaves, glumes, and anthers that uniformly expressed green fluorescent protein (GFP). The GFP gene segregated in the pollen. Based on this data it is concluded that the transgenics arose from single-cell somatic embryos. The rate of transfer DNA (T-DNA) transfer to complete shoot meristems of Zea mays was high on the auxin medium and was independent of using super-virulent strains of Agrobacterium.     

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.)

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the in-vitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277–1289; N. Sinha et al., 1993 Genes Dev. 7: 787–797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro. Received: 2 June 1997 / Accepted: 21 July 1997     

Expression and Functional Analysis of the ZCN1(ZmTFL1) Gene, a TERMINAL FLOWER 1 Homologue that Regulates the Vegetative to Reproductive Transition in Maize

TERMINAL FLOWER 1 (TFL1) homologs play critical roles in regulating flowering time and/or maintaining flowering of meristems. In this study, the gene of maize TFL1 ortholog ZmTFL1 (ZCN1) was cloned from both the tropical inbred line CML288 and temperate inbred line Huangzao 4, and the function of ZmTFL1 (ZCN1) was determined during different periods of floral development. Spatial and temporal expression patterns revealed that ZCN1 was predominantly localized in shoot apical meristems that develop into flowers, and only at low levels in leaves. To further identify the role of ZCN1 in floral development of maize, the morphology of shoot apices in maize during floral development was investigated using laser scanning confocal microscopy. Moreover, the relative levels of expression of ZCN1, ZCN8, DLF1, and ZAP1 genes were determined. Over-expression of ZCN1 partially complemented the late flowering phenotype in the tfl1-14 Arabidopsis mutant. Moreover, transgenic Arabidopsis plants exhibited indeterminate inflorescence with increased shoot length and higher numbers of trichomes on leaves. In addition, expression levels of AP1 were significantly down-regulated in 35S::ZCN1 transgenic Arabidopsis plants. These results indicated that ZCN1 as well as its homolog TFL1 in Arabidopsis are involved in the regulation of floral transition in maize.     

Influence of the Form of Nitrogen Supply on Root Uptake and Translocation of Cations in the Xylem Exudate of Maize (Zea mays L)

Concentrations of inorganic cations are often lower in plantssupplied with NH₄⁺ as compared with NO₃^–. To examine whetherthis is attributable to impaired root uptake of cations or lowerinternal demand, the rates of uptake and translocation of K,Mg, and Ca were compared in maize plants (Zea mays L.) withdifferent growth-related nutrient demands. Plants were grownin nutrient solution with either 1·0 mol m^–3 NO₃^–or NH₄⁺ and the shoot growth rate per unit weight of roots wasmodified by varying the temperature of the shoot base (SBT)including the apical shoot meristem. The shoot growth rate per unit weight of roots, which was takenas the parameter for the nutrient demand imposed on the rootsystem, was markedly lower at 12°C than at 24°C SBT.As a consequence of the lower nutrient demand at 12°C SBT,uptake rates of NO₃^– and NH₄⁺ declined by more than 50%Compared with NO₃^– supply, NH₄⁺ nutrition depressed theconcentrations of K and particularly of Ca in the shoot, bothin plants with high and with low nutrient demand. This indicatesa control of cation concentration by internal demand ratherthan by uptake capacity of the roots. Translocation rates of K, Mg and Ca in the xylem exudate werelower in NH₄⁺- than in NO₃^–-fed plants. Net accumulationrates of Ca in the shoot were also decreased, whereas net accumulationrates of K in the shoot were even higher in NH₄⁺-fed plants.It is concluded that reduced cation concentrations in the xylemsap of plants supplied with NH₄⁺ are due to the lower demandof cations for charge balance. The lower K translocation tothe shoot is compensated by reduced retranslocation to the roots.For Ca, in contrast, decreased translocation rates in NH₄⁺-fedplants result in lower shoot concentration. Key words: Nitrogen form, cation nutrition, charge balance, xylem exudate, recirculation     

Transgenic maize plants of tropical and subtropical genotypes obtained from calluses containing organogenic and embryogenic-like structures derived from shoot tips

A highly efficient system for the production of transgenic maize plants starting from tropical and subtropical genotypes was developed. The method is based on particle bombardment of organogenic calli derived from shoot tips. Six tropical maize genotypes were successfully transformed and regenerated using this protocol. Genetic transformation was confirmed by Southern blot analysis of T0 plants and segregation analysis of the resistance marker in the T1 progeny. Plant transfer into the greenhouse was 100% successful, and no problems of fertility were observed with the transgenic plants produced with this transformation protocol.     

Efficient transformation of scutellar tissue of immature maize embryos

  An efficient transformation system for maize was established by improving transformation conditions for the particle bombardment of the scutellar tissue of immature embryos. Particle bombardment was carried out using constructs containing the pat gene as the selection marker and a PDS 1000/He gun (Biorad). Transformation parameters, such as the amount of gold particles used per bombardment, particle velocity, preculture time of the scutellum prior to bombardment and osmotic treatment of the target tissue before and after bombardment, were analysed. Fertile transgenic regenerants of the maize inbred lines H99, A188 and Pa91 and the crosses A188×H99 and Pa91×H99 were selected on Basta-containing medium. The transformation frequency was between 2% and 4%. A total of 29 transgenic plant lines was obtained and verified with Southern blot analysis. All of the transgenic plants were fertile and set seeds. The R1 progeny of single plants was analysed. A Mendelian segregation of the transgenes was observed for all of the transformants tested. For 1 candidate, stable inheritance and stable expression of the transgenes were followed up to the R₄ generation. Received: 28 October 1996/Accepted: 15 November 1996