Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

Cellular changes during the acquisition of embryogenic potential in isolated pollen grains ofNicotiana tabacum

Summary We used electron microscopical techniques to study ultrastructural changes during the acquisition of embryogenic competence in immature pollen grains ofNicotiana tabacum, isolated at the early- or mid-bicellular stage and cultured in vitro under starvation conditions. Cytoplasmic and nuclear changes during the starvation treatment are reported. Dedifferentiation of plastids, dilation of the wall of the generative cell, the appearance of a large vacuole, loss of nuclear pores in the vegetative nucleus, changes in chromatin and nucleolar structure, and a decrease in the size of the nucleolus were observed. We suggest that these events are the first step in the switch from generative to vegetative generation during pollen embryogenesis.     

Stable transformation of the food yam Dioscorea alata L. by particle bombardment

Summary A biolistic particle gun was used to deliver genetic material into intact yam cells. Cultured suspension cells of D. alata were bombarded with microprojectiles coated with pBI221.2 DNA and histochemical assays were carried out to show transient GUS expression in bombarded cells. Stably transformed D. alata cells were recovered from cultured cells after bombardment with microprojectiles coated with pRT99gus harbouring both the nptII and uidA genes. Bombarded cells were selected on a medium containing geneticin (G418). Two months after bombardment, calli resistant to G418 were assayed for GUS expression. There was a 100% correlation between resistance to G418 and GUS expression. From these calli, four cell lines were established and GUS activity in each line was determined fluorometrically. The use of a specific GUS inhibitor showed that the GUS activity was due to the introduced uidA gene rather than to any intrinsic GUS-like activity originating from the plant. Incorporation of the introduced DNA into the plant genomic DNA was confirmed by Southern analysis.Abbreviations GUS -glucuronidase - MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--D-glucuronide - PVP Polyvinylpyrrolidone - SDS Sodium dodecyl sulphate - TAE Tris-acetate-EDTA buffer - X-Gluc 5-Bromo-4-chloro-3-indolyl--D-glucuronide     

Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves

Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. Xanthi and Ky 17 which were cultured in the presence of 100 or 200 g/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.     

Transformation of soybean via particle bombardment of embryogenic suspension culture tissue

Summary Embryogenic suspension culture tissue of soybean (Glycine max Merrill.) was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance andβ-glucuronidase (GUS). One to two weeks after bombardment, embryogenic tissue was placed in a liquid proliferation medium containing hygromycin. Four to six weeks after bombardment, lobes of yellow-green, hygromycin-resistant tissue, which began as outgrowths on brown clumps of hygromycin-sensitive tissue, were isolated and cultured to give rise to clones of transgenic embryogenic material. In vivo GUS assays of hygromycin-resistant clones showed that the early outgrowths could be negative, sectored, or positive for GUS activity. Transgenic, fertile plants could be routinely produced from the proliferating transgenic embryogenic clones. Southern hybridization analyses confirmed stable transformation and indicated that both copy number and integration pattern of the introduced DNA varied among independently transformed clones. Hybridization analysis of DNA from progeny plants showed genetic linkage of multiple copies of introduced DNA. An average of three transgenic clones were obtained per bombardment making this procedure very suitable for transformation of soybean.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Genetic transformation of Linum by particle bombardment

Summary Linseed flax (Linum usitatissimum L.) was transformed by bombarding hypocotyl tissues with gold particles coated with plasmid DNA carrying the β-glucuronidase (GUS) (uid-A) and neomycin phosphotransferase II (npt-II) genes. Transient expression of the introduced β-glucuronidase gene was used to study factors influencing the DNA delivery, while progeny analyses confirmed stable transformation. The efficiency of DNA delivery, uptake and expression was significantly affected by the duration of hypocotyl preculture, bombardment distances, the level of chamber vacuum, the quantity of DNA, and the size of particles. Nineteen independent GUS-positive shoots were recovered and regenerated into whole plants, from which 10 plants successfully produced viable seeds. Analysis of T₁ and T₂ self pollinated progeny for histochemical and fluorometric GUS assays and polymerase chain reaction (PCR) analyses for uid-A, plus npt-II PCR and germination assays in progeny plants demonstrated that the transgenes were expressed in selected plants and transmitted to progeny, usually via a single Mendelian locus. The results show that particle bombardment can be used to produce transgenic Linum plants. The system is rapid, simple and offers an alternative to Agrobacterium methods.     

Plant transformation: a simple particle bombardment device based on flowing helium

We observed that flowing helium at moderate pressures accelerated DNA-coated microprojectiles to velocities suitable for penetration of cells in intact plant tissues. The flowing helium principle permitted the construction of a simple and inexpensive transformation device that was easier to use than those previously described. This device provided efficient transformation of cells in soybean seedlings and other plants.     

Comparison of different techniques for gene transfer into mature and immature tobacco pollen

The particle gun, cocultivation withAgrobacterium tumefaciens, and imbibition in DNA solutions were compared as methods to transfer DNA into mature and immature pollen ofNicotiana tabacum. Bombardment of mature pollen with the β-glucuronidase gene cloned behind the pollen-specific PA2 promoter of the chalcone isomerase gene ofPetunia hybrida resulted in the expression of the β-glucuronidase gene in 0.025% of the pollen grains. Bombardment of younger stages followed byin vitro maturation also resulted in the formation of mature pollen that expressed β-glucuronidase, although at a lower frequency. Cocultivation of pollen duringin vitro maturation orin vitro germination withAgrobacterium tumefaciens did not yeild β-glucuronidase-expressing pollen. In these cases, an intron-containing β-glucuronidase gene was used which effectively prevented β-glucuronidase expression in the bacteria. Imbibition of mature, dry pollen in various DNA solutions of the same constructs also did not lead to the formation of β-glucuronidase expressing pollen.     

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

Methotrexate is a new selectable marker for tobacco immature pollen transformation

We report here a new selectable marker for tobacco immature pollen transformation based on the expression of dihydrofolate reductase (dhfr) gene which confers resistance to methotrexate (Mtx). Two immature pollen transformation approaches, i.e., male germ line transformation and particle bombardment of embryogenic mid-bicellular pollen have been used for the production of stable transgenic tobacco plants. In the first method, two methotrexate-resistant plants were selected from a total of 7161 seeds recovered after transformation experiments. In the second method, four methotrexate-resistant plants were obtained from 29 bombardments using 3.7×10⁵ pollen grains per bombardment. Southern analysis confirmed the transgenic nature of T₀ and T₁ candidate transgenic plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations.     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

Dynamics of inorganic ions in microspore and pollen grain during development of the tobacco male gametophyte

We studied the dynamics of mobile potassium, chloride, and nitrate ions during development of the microspore and differentiation of the pollen grain inNicotiana tabacum L. by measuring their concentration in aqueous extracts from cells destroyed by freezing-thawing using ion-selective electrodes. Stage-specific changes in the ion content and intracellular concentration in the male gametophyte were found. A relationship of the dynamics of ions to growth processes and changes in metabolic activity during gametophytogenesis has been discussed. The changes in the potassium and chloride ion concentrations have been interpreted as regulatory changes controlling protein synthesis in the pollen grain vegetative cell. Deceased.     

Integrated bombardment andAgrobacterium transformation system: An alternative method for soybean transformation

Described here is a new method for soybean transformation, based on microwounding of embryonic clumps by particle bombardment prior to inoculation with anAgrobacterium suspension. The method combines the advantages of somatic embryogenesis and gene transfer through an integrated transformation system. This is the first report of application of this technique to transformation ofGlycine max.     

Genetic transformation of Alstroemeria using particle bombardment

Transgenic plants were obtained after particle bombardment of embryogenic callus derived from stem segments of two tetraploid Alstroemeria genotypes with plasmids containing different selection/reporter genes. Firstly, a plasmid containing a firefly luciferase reporter gene driven by the maize ubiquitin promoter (Ubi1), was bombarded into both friable embryogenic callus and proembryos. Transient and stable expression of luciferase was visually detected by a luminometer. This selection method is non-destructive and can be applied over the whole developmental process from callus to embryo and plantlet. Molecular proof of transformation was obtained both by PCR analysis and Southern hybridization. Secondly, a plasmid containing the bar gene together with an uidA gene coding for -glucuronidase both driven by the Ubi1 promoter was bombarded into proembryos. The transgenic callus was effectively selected from the callus clumps four months after bombardment on a medium containing 5 mg/l phosphinotricin (PPT). Selection by PPT was efficient and labour-saving. Stable expression of GUS was confirmed by the histochemical staining assay and molecular proof was obtained by PCR analysis.     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Stable transformation ofArabidopsis with thebar gene using particle bombardment

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T₀) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T₂ generation. Some of the T₁ progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.     

High frequency stable transformation ofArabidopsis thaliana by particle bombardment

Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl⁻¹ hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots.