Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

Improved genetic transformation protocol for cork oak (<Emphasis Type="Italic">Quercus suber</Emphasis> L.)

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus suber L. embryogenic masses is reported. In this work several variables were tested. Plant genotype, explant type and time elapsed between the last subculture and inoculation, i.e. the explant preculture period, were found to be very important. Interaction between inoculum density and cocultivation period influenced the transformation efficiency as well. A transformation efficiency (i.e. percentage of the inoculated explants that yielded independent transgenic embryogenic lines) of up to 43% was obtained, greatly improving the previously described method for plant transformation of adult-selected cork oak. It was also shown that this protocol could be applied to various genotypes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cryopreservation of embryogenic tissue of a range of genotypes of sweet potato (Ipomoea batatas [L] Lam.) using an encapsulation protocol

Embryogenic tissue of nine sweet potato [Ipomoea batatas (L.) Lam] genotypes from Asia, Africa and the Americas was established from in vitro axillary buds on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. Embryogenic aggregates, 1.0–2.0 mm in diameter, were encapsulated in alginate gel, precultured on medium containing elevated levels of sucrose and dehydrated prior to rapid freezing in liquid nitrogen. The maximum survival of embryogenic tissue ranged from 4% to 38%, depending on the genotype. With the incorporation of a slow-cooling step, survival was generally much higher than that obtained after rapid freezing alone. Five of eight genotypes tested with this protocol gave survival percentages in excess of 55%, and a further two in excess of 33%, all after evaporative dehydration. The most effective sucrose treatment(s), however, varied with the genotype. Received: 7 October 1996 / Revision received: 16 December 1996 / Accepted 27 January 1997     

Genetic transformation of <Emphasis Type="Italic">Torenia fournieri</Emphasis> using the PMI/mannose selection system

Transgenic torenia plants were obtained using the selectable marker gene phosphomannose isomerase (manA), which encodes the enzyme phosphomannose isomerase (PMI) to enable selection of transformed cells on media containing mannose. We found that shoot organogenesis in torenia leaf explants was effectively suppressed on medium supplemented with mannose, which indicated that torenia cells had little or no PMI activity and could not utilize mannose as a carbon source. Leaf pieces from in vitro-germinated plants were inoculated with Agrobacterium tumefaciens EHA105 containing the binary vector pKPJ with both hpt and ManA genes, and subsequently selected on shoot induction (SI) medium (half strength MS basal + 4.4 μM BA + 0.5 μM NAA) supplemented with 20 g l⁻¹ mannose and 5 g l⁻¹ sucrose as carbon sources. Transformed plants were confirmed by PCR and Southern blot. The transgene expression was evaluated using Northern blot and the chlorophenol red assay. The transformation efficiency ranged from 7% to 10%, which is 1–3% higher than that obtained by selection with hygromycin. This system provides an efficient manner for selecting transgenic flower plants without using antibiotics or herbicides.     

<Emphasis Type="Italic">Echinacea</Emphasis> biotechnology: Challenges and opportunities

Echinacea, better known as purple coneflower, has received a global attention because of its increasing medicinal value. There is enormous potential for the discovery of new medicinal compounds in this species and an immediate need for techniques to facilitate the production of high quality, chemically consistent plant material for drug development and clinical trials. In vitro tissue culture of Echinacea can play a vital role in the development of novel germplasm, rapid multiplication, and genetic modifications for an enhanced phytochemical production. Recent establishment of liquid culture techniques, large-scale bioreactors, and Agrobacterium-mediated transformation are changing the production parameters of the Echinacea species. This review provides an overview of the recent developments in in vitro technologies and challenges that remain in the Echinacea biotechnology.     

2，4_D对甘薯体细胞胚胎发生的调控

将来源于‘徐薯１８’叶片的胚性愈伤组织,接种在含有不同２,４Ｄ浓度的液体ＭＳ培养基中进行悬浮培养,悬浮细胞表现出不同的形态结构、分裂方式和发育途径：２,４Ｄ浓度为１ｍｇ／Ｌ时,细胞均等分裂,增殖迅速;不含２,４Ｄ时,细胞多进行不均等分裂,并发育成体细胞胚。不同２,４Ｄ浓度中培养的悬浮细胞,其胞外过氧化物同工酶谱及其随时间变化的方式有很大差异,并与细胞的生长、发育过程密切相关     

甘薯生物技术研究进展

新兴的生物技术为甘薯这一古老的农作物带来了新的发展契机。细胞大规模培养、体细胞融合、基因转导等技术的研究和应用 ,可望从根本上改变甘薯传统的生产和育种模式。本文综合近年来国内外甘薯体细胞胚胎发生、原生质体培养和基因工程等方面的研究进展 ,对影响甘薯体胚发生体系及原生质体再生体系建立的诸多因素进行了详细论述 ,讨论了甘薯基因工程研究的应用潜力和目前存在的一些问题。     

Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l^-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l^-1 spermine and 0.1 mg l^-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l^-1 gibberellic acid, 0.2 mg l^-1 kinetin (KIN) and 0.1 mg l^-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Somatic embryogenesis and Agrobacterium-mediated transformation system for scented geraniums (Pelargonium sp. `Frensham')

Plant regeneration via somatic embryogenesis was achieved from leaf petioles of Pelargonium sp. `Frensham' cultured on Murashige and Skoog medium containing 15 μM N⁶-benzyladenine, and 5 μM α-naphthaleneacetic acid (NAA). More than 80% of these somatic embryos converted into plants when isolated and cultured on Murashige and Skoog medium supplemented with 15 μM NAA. Stable transgenic plants were obtained by co-cultivation of the petioles (prior to culture) with Agrobacterium tumefaciens strains LBA4404 (harbouring a binary vector pBI121 carrying the nptII and gus genes) and LBG66 (harbouring a binary plasmid pJQ418 carrying the gus/int:nptII fusion gene). Transformants were selected using kanamycin and transformation was verified by β-glucuronidase histochemical assay and polymerase chain reaction. Southern analysis further confirmed the integration of these genes into the genome of transgenic plants. We report here for the first time, an Agrobacterium-mediated model transformation system coupled with regeneration via somatic embryogenesis for production of transgenics in Pelargonium sp. Received: 20 September 1996 / Accepted: 13 November 1996     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Repetitive somatic embryogenesis in carnation on picloram supplemented media

An efficient method of repetitive somatic embryogenesis and plant regeneration of two carnation cultivars (Sagres and Impulse) was established using a two-step protocol. In the first step, embryogenic tissue were induced from petal explants on MS culture medium containing 9% sucrose (w/v), 9 μM 2,4-D and 0.8 μM BA. In the second step, embryogenic tissue transferred onto the MS medium containing 3% sucrose supplemented with different concentrations of picloram (0.8, 2, 4, 8 and 16 μM) to produce primary somatic embryos. Precotyledonary clumps and cotyledonary somatic embryos were then isolated and subcultured onto the same media as the second step where they formed secondary somatic embryos in repetitive cycles. Cotyledony somatic embryos were converted into plantlets when they transferred onto the growth regulator-free half-strength MS medium followed by being acclimated to the greenhouse conditions.     

Genetic transformation of duckweed Lemna gibba and Lemna minor

Summary We developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.     

松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统.整个体细胞胚胎发生过程主要包括3个步骤:胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换.过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理.然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量.此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要.因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义.     

Construction of two vectors for gene expression in Trichoderma reesei

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.     

Chilli peppers — A review on tissue culture and transgenesis

Biotechnology techniques involving plant tissue culture and recombinant DNA technologies are powerful tools that can complement conventional breeding and expedite Capsicum improvement. The rate of progress in Capsicum is relatively slower than other members of Solanaceae because of its high genotypic dependence and recalcitrant nature. Capsicum is a recalcitrant plant in terms of in vitro cell, tissue and organ differentiation, plant regeneration and genetic transformation which makes it difficult to apply recombinant DNA technologies aimed at genetic improvement against pests, diseases and abiotic stress. Despite this, application of tissue culture and genetic transformation have led to significant development in chilli pepper plants, and studies are underway to achieve the targets of pre-harvest improvement and post-harvest characterization for value addition to this crop. This review presents a consolidated account of in vitro propagation and focuses upon contemporary information on biotechnological advances made in Capsicum.