Factors affecting the genetic engineering of plants by microprojectile bombardment

Since its development in the mid-1980s, microprojectile bombardment has been widely employed as a method for direct gene transfer into a wide range of plants, including the previously difficult-to-transform monocotyledonous species. Although the numerous instruments available for microprojectile-mediated gene delivery and their applications have been widely discussed, less attention has been paid to the critical factors which affect the efficiency of this method of gene delivery. In this review we do not wish to describe the array of devices used for microprojectile delivery or their uses which have already been definitively described, but instead wish to report on research developments investigating the factors which affect microprojectile-mediated transformation of plants.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 μM) 2,4-D, 0.5 mg/l (2.32 μM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T₁ progeny. However, no β-glucuronidase (GUS) activity could be detected in T₁ plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.     

Transgenic tobacco plants expressing a rice cysteine synthase gene are tolerant to toxic levels of cadmium

Plants tolerate heavy metals through sequestration with cysteine-rich peptides, phytochelatins. In this reaction, the rate limiting step is considered to be the supply of cysteine, which is synthesized by cysteine synthase (CS, EC 4.2.99.8) from hydrogen sulfide andO-acetylserine. In this study, we transformed tobacco (Nicotiana tabacum) plants withRCS1, a cytosolic cysteine synthase gene of rice (Oryza sativa), and examined their sensitivity to cadmium. The transgenic plants had up to 3-fold higher activity of cysteine synthase than wild-type plants. Upon exposure to cadmium, they exhibited obvious tolerance with much greater growth than wild-type plants. The level of phytochelatins in shoots was higher in transgenic than in wild-type plants after cadmium treatment, suggesting that cadmium was actively trapped by phytochelatins. However, the cadmium concentration per g fresh weight of whole transgenic plants was 20 percnt; lower than that of wild-type plants, suggesting cadmium to be either actively excreted or diluted by fast growth. Genetic analysis of progenies clearly showed segregation of cadmium tolerance, indicating that the trait resulted from the introduced gene. These results suggest that introduction of a cysteine synthase gene into tobacco plants resulted not only in high level production of sulfur-containing compounds that detoxify cadmium, but also in active elimination of cadmium toxicity from plant bodies.     

Transgenic tobacco plants overexpressing cotton glutathione S-transferase (GST) show enhanced resistance to methyl viologen

A GST (EC 2.5.1.18) gene (Gst-cr 1) from cotton was introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants overexpressing Gst-cr1 were normal in growth and mature compared with control, but had much higher levels of GST and GPx activities and showed an enhanced resistance to oxidative stress induced by a low concentration of methyl viologen (MV). Six antioxidant enzymes, glutathione S-transferase, glutathione peroxidase (EC 1.11.1.9), superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11) were monitored in transgenic lines and non-transgenic control during MV treatments. When they were treated with 0.03 mmol/L of MV, both transgenic lines and control showed a rapid increase in the activities of GST, GPx, SOD, POD, APx, while the activity of CAT seemed to be irregular. The percent of the increase in SOD and POD activities was much higher in control than in transgenic plants. When treated with 0.05 mmol/L of MV, both control and transgenic plants were severely damaged, and the activities of the six enzymes decreased sharply.     

Field performance of derived generations of transgenic tobacco

Two inbred cultivars of Nicotiana tabacum (tobacco), Samsun and Xanthi, were transformed with the plasmid pBI 121 using Bin 19 in Agrobacterium tumefaciens. The plasmid carries the nptII gene conferring kanamycin resistance and the uidA gene encoding -glucuronidase (GUS). Progeny carrying the genes in the homozygous condition were identified and selfed over several generations. One line homozygous for the introduced genes and one untransformed control from each cultivar were then selected and crossed reciprocally to give four families per cultivar. Seeds from each family were grown in a replicated field trial and all plants scored for a range of morphological and agronomic characters. In addition, leaf samples were taken and GUS activity measured. In the Samsun material, which contained one copy of the introduced gene at a single locus and showed high levels of GUS expression, the transformed homozygote showed twice the level of GUS activity as the hemizygotes, wheareas in the Xanthi line, which had a lower level of GUS, the hemizygotes showed the same level of GUS activity as the transformed homozygote. The agronomic data showed differences between the families, but the source of such differences could not be ascribed unambiguously. The results are discussed in the light of related information on gene expression and field performance from other transgenic material.     

Transformation of sweetgum via microprojectile bombardment of nodule cultures

Summary A sweetgum (Liquidambar styraciflua) nodule culture system was developed and integrated with genetic transformation by microprojectile bombardment. Nodule cultures were established from seedling hypocotyls and proliferated in liquid medium containing 0.1 mg (0.45 μM) thidiazuron (TDZ) per 1 and 0.01 mg (0.045 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1. Shoots differentiated from the nodules in liquid media containing (per 1) 1 mg (4.4 μM) benzyladenine (BA), 0.5 mg (2.2 μM) BA, and 0.01 mg (0.054 μM) naphthaleneacetic acid (NAA), or 0.5 mg BA, 0.01 mg NAA, and 0.05 mg (0.23 μM) TDZ under the light. Differentiating shoots required 4 wk of dark treatment for further development on semisolid medium containing 1 mg BA per 1. Elongated shoots were harvested and the basal ends were soaked in a solution containing 10 mg (49.2 μM) indole-3-butyric acid (IBA) per 1 before being planted in potting mix for ex vitro rooting. Roots formed and leaves expanded in 2 wk. Sweetgum nodules were stably transformed by microprojectile bombardment with a 7.4-kb plasmid, pTRA 140, harboring CaMV 35S-HPH and CaMV 35S-GUS. Evidence that nodules growing in the presence of hygromycin B were stably transformed was provided by polymerase chain reaction analysis and β-glucuronidase activity. Sweetgum shoots differentiated in liquid medium in the presence of hygromycin B. Shoots transferred to solid medium lacking hygromycin B elongated and displayed β-glucuronidase activity in their expanding leaves and stems. Southern analysis confirmed the presence of the GUS gene in nodules and shoots. Transgenic shoots initiated roots and showed leaf expansion 2 wk after being planted in potting mix.     

Transgenic tobacco plants producing caffeine: a potential new strategy for insect pest control

Caffeine (1,3,7–trimethylxanthine) is one of the most widely used plant secondary metabolites, primarily as a stimulant and an ingredient in drugs. In nature, caffeine is believed to function in chemical defense, acting as an antiherbivory and allelopathic agent, and therefore it might be employed to protect agriculturally important crop plants. In coffee plants, caffeine is synthesized from the precursor xanthosine in four steps, three N-methylations and removal of ribose. We had previously isolated genes encoding three distinct N-methyltransferases, and we demonstrated production of recombinant enzymes that yielded caffeine in in vitro reconstitution experiments. When these caffeine biosynthetic pathway genes were simultaneously expressed in tobacco plants (Nicotiana tabacum), caffeine was successfully produced up to 5 μg/g fresh weight in leaves. The leaves were unpalatable to tobacco cutworms (Spodoptera litura). This repellent action appeared to be more widely␣applicable to lepidopteran caterpillars as observed with small white (Pieris rapae) fed on Chinese cabbages that had been top-treated with caffeine. Our recent results suggest a novel approach to strengthen anti-herbivore traits by producing caffeine in crop plants.     

Production of transgenic maize plants and progeny by bombardment of hi-II immature embryos

Summary Production of transgenic maize (Zea mays L.) callus, plants, and progeny from microprojectile bombardment of 2–5-d cultured Hi-II immature embryos is described. Histological evidence indicates that these tissues are amenable to transformation due to surface layer cell division of the scutellum. Two out of every 100 bombarded embryos produced transgenic callus and R₀ transgenic plants were both male and female fertile. Expected segregation of transgenes was observed in progeny. The primary advantage of bombarding these tissues is increased male and female fertility of transgenic plants compared with those produced using long-term callus or suspension cultures.     

Transgene inheritance in plants genetically engineered by microprojectile bombardment

Microprojectile bombardment to deliver DNA into plant cells represents a major breakthrough in the development of plant transformation technologies and accordingly has resulted in transformation of numerous species considered recalcitrant toAgrobacterium- or protoplast-mediated transformation methods. This article attempts to review the current understanding of the molecular and genetic behavior of transgenes introduced by microprojectile bombardment. The characteristic features of the transgene integration pattern resulting from DNA delivery via microprojectile bombardment include integration of the full length transgene as well as rearranged copies of the introduced DNA. Copy number of both the transgene and rearranged fragments is often highly variable. Most frequently the multiple transgene copies and rearranged fragments are inherited as a single locus. However, a variable proportion of transgenic events produced by microprojectile bombardment exhibit Mendelian ratios for monogenic and digenic segregation vs events exhibiting segregation distortion. The potential mechanisms underlying these observations are discussed.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.     

Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for β-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.     

Rapid,efficient production of homozygous transgenic tobacco plants withagrobacterium tumefaciens: A seed-to-seed protocol

An optimized complete protocol was developed forAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cultivar SR1), producing T₁ flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T₂ seedlings in 6 to 7 months from the time of cocultivation withAgrobacterium. Previous protocols require up to 9 to 12 months to obtain similar results. Procedures unique and important to this protocol include; a modified “whole-leaf” transformation coupled with a long duration of cocultivation, resulting in high rates of transformation, high levels of kanamycin in selection media resulting in few escapes, and extensive rooting of regenerants prior to a greenhouse hardening procedure. Once in the greenhouse, primary regenerants were maintained in small containers with long day photoperiod and high light levels, greatly shortening the time to seed set. Flowers from primary transformants were bagged to allow self pollination, and seed capsules harvested and dried prior to normal maturation on the plant. T₁ and T₂ seeds were plated and selected on kanamycin media by an improved seed plating technique which eliminates the need for the placement of individual seeds, saving time and improving selection homogeneity. Using this protocol, over 130 independent tobacco lines from six separate gene constructs have been generated in a very short time period. Of these 130, nearly 60 percent segregated 3∶1 for kanamycin resistance: susceptibility, indicating single transgene insertion events.     

A quantitative and histological comparison of gus expression with different promoter constructs used in microprojectile bombardment of peanut leaf tissue

Summary β-glucuronidase (GUS) expression driven with different promoter constructs was quantitatively and histologically compared in peanut leaf tissue following microprojectile bombardment. X-Gluc staining patterns varied with the construct used. Tissues bombarded with the pAC2GUS construct had larger foci and a greater percentage of area staining blue. pEmuGN exhibited the greatest numbers of blue spots compared to pAC2GUS and pTRA140. Histological evaluations of blue staining foci showed a diffusion gradient of blue precipitate from a central, prominently-staining cell outward to as many as seven cell layers. The intensity of X-gluc product in centrally-staining cells varied. Gold microprojectile particles were usually located within the three surface cell layers. Depending on the plasmid construct, 72–90% of the centrally-staining cells had at least one gold particle. However, the presence of GUS expression did not appear to require a microprojectile within the nucleus, which was observed in 37% or fewer of the centrally-staining cells. With the pAC2GUS construct, staining patterns varied with location within leaflets and had an “edge effect,” i.e., blue spots were frequently larger at the margin versus central regions. This enhanced activity could be anticipated with an actin promoter in the more mitotically active marginal leaf cells. Total GUS activity as determined by fluorometric analyses was correlated with the percentage of X-gluc stained area. The pAC2GUS construct exhibited the highest total GUS activity among the three constructs.     

Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat

Using the PDS-1000/He Biolistic® Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term β-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five μl pAHC25 (0.75 mg ml^-1 in TE buffer) was precipitated onto 25 μl gold particles (60 mg ml^-1 in sterile water), using 20 μl spermidine (0.1 M) and 50 μl CaCl₂ (2.5 M). The particles were centrifuged and resuspended in 75 μl absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29′′ Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 ± 17 GUS-positive foci/MDE (mean ± s.e.m, n=3), with 17 ± 4 foci/MDE at 15 days, giving a 3.0-fold increase (p<0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.     

Effect of boron supply on nitrate concentration and its reduction in roots and leaves of tobacco plants

Shoot and root mass of tobacco plants treated with only 0.05 μM boron was decreased by 25 and 50 %, respectively, when compared to plants sufficiently supplied with B (2 and 5 μM). Leaf B content of 0.05 μM B-treated plants decreased (about 80–90 %) when compared to 2 μM B treated plants; this drop of B content were not as marked (about 25–45 %) in roots. Leaf and root nitrate contents in B-deficient plants were 45–60 % and 35–45 % lower, respectively, than those from 2 and 5 μM B treated plants. It is suggested that B deficiency might decrease nitrate uptake rather than nitrate reductase activity in tobacco plants.     

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb²⁺) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 μM to 200 μM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 μM Pb²⁺. In tobacco plants growing for 6 weeks in soil polluted with Pb²⁺ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb²⁺-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb²⁺, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb²⁺ accumulation in the above-ground parts may explain the lower levels or the absence of Pb²⁺-induced DNA damage in leaves.