Reliable Transformation System Research Articles

Spirulina, The importance and Significance of Cloning

Many items have impacts on recombinant protein production in making it affordable, the most important of which is host with its characteristics such as compatibility, yields, expression system, costs, environmental effects, etc. Humans have been using spirulina (Arthrospira sp.) algae for centuries. It enjoys features which propose it as a golden host cell. Although several researches have been conducted to develop a stable and reliable transformation system, only a few projects meet the need. The aim of current paper was to focus on spirulina transformation researches. So reason to select the host and procedures to optimize a transformation system were given.

Shoot regeneration process and optimization of Agrobacterium-mediated transformation in Sinningia speciosa

The florist’s Gloxinia, Sinningia speciosa, which bears considerable flower trait variations, is an emerging model plants to study floral traits development. However, the investigation of the genetic information linking these floral traits is limited due to a lack of a reliable and efficient transformation system for functional studies. This study aims to optimize a stable genetic transformation system for S. speciosa. Detailed regeneration process and tissue culture parameters are also elucidated. The results show that the plant regeneration, initiated from a single perivascular parenchyma cell, can be induced from leaf and petiole explants in the presence of 1 mg/mL 6-benzylaminopurine (BA) and 0.1 mg/mL naphthalene-acetic acid (NAA) through embryogenesis. In the presence of 0.1 mg/mL NAA only, the adventitious roots form prior to the re-differentiation of shoot tissues in leaf explants. When the proximal end of the petiole is orientated upright with the distal end to the medium, it results in higher success of regeneration, suggesting that hormone supplies must follow endogenous basipetal auxin polarity. Using a glucuronidase (GUS) reporter gene construct, maximum transformation (3.13%) was obtained after a 3 day pre-culture and 5 day co-culture from cotyledons and leaves of 3-week-old seedlings inoculating Agrobacterium strain EHA105. The putative transgenic lines were validated by RT-PCR, Southern blotting and GUS activity. Our result demonstrates that young seedlings are the best material for transformation, probably because young leaves are only a few cell layers thick allowing inner perivascular cell (the origin of regeneration) to be more accessible for Agrobacterium infiltration.

Genetically Transformed Root-Based Culture Technology in Medicinal Plant Cosmos bipinnatus

Background: Cosmos bipinnatus is an important medicinal plant with antioxidative, antigenotoxic, anti-inflammatory, and anti-proliferative effects on several cancer cell lines. It has great potential for development as a promising cancer chemo-preventive agent. Hairy root-based culture technology is a new sustainable production platform for producing specific pharmaceutical secondary metabolites. Objectives: The current study developed and introduced a reliable transformation system for C. bipinnatus by optimization of aspects important in transformation frequency using Agrobacterium rhizogenes. Methods: Five bacterial strains, including ATCC 15834, ATCC 31798, A7, MAFF-02-10266, and MSU440, 2 explant types (leaf and stem), and 2 co-cultivation media (full MS and ½ MS) were examined. Genomic DNA was extracted using a modified CTAB protocol from putative transgenic root lines and the control root. Transgenic hairy root lines were approved by means of Polymerase Chain Reaction (PCR) using specific rolB gene primers. Results: The highest ratio of genetically transformed root induction was found from leaf explants using A. rhisogenes strains ATCC15834 and MSU440 (72% to 73%). When ½ MS medium was used as a co-cultivation medium, a significant increase in transformation frequency (84%) was observed. Conclusions: The MSU440 Agrobacterium strain and ½ MS co-cultivation medium could significantly improve genetic transformation efficiency for establishment of hairy root-based cultures for C. bipinnatus.

Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris

Chlorella is a green unicellular alga that has a wide range of future biotechnical applications such as production of pharmaceuticals and biodiesel. Efficient genetic transformation of Chlorella vulgaris has been difficult due to technical limitations. In this study, an efficient and reliable transformation system of electroporation was established using two different reporter genes. First, C. vulgaris cell wall was digested with enzyme mixture for preparing protoplasts. The optimal transformation efficiency was 1.67 × 104 ± 0.083 cfu μg−1 plasmid under the following conditions: 2 × 106 cells mL−1 of growing culture; 655 V pulse voltage with 3.4 ms pulse width. After transformation, green and cyan fluorescence were observed from transgenic C. vulgaris harboring gfp (green fluorescent protein) gene of pCAMBIA1302 and cfp (cyan fluorescent protein) gene of pSK397, respectively, using laser confocal microscope. RT-PCR analysis as well as Southern blot confirmed the integration of reporter gene at the molecular level. This efficient transformation system of C. vulgaris would be valuable for the production of recombinant proteins in the future.

High-efficient transgenic hairy roots induction in chicory: re-dawn of a traditional herb

Plant roots can be manipulated by Agrobacterium rhizogenes to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13) in combination with three co-cultivation media (MS, B5, LS) were examined to establish an efficient and reliable transformation system for chicory (Cichorium intybus L.) using A. rhizogenes. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l-1 in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l-1, respectively). Beta-glucuronidase (GUS) gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 %) was achieved using A13 strain and MS cultivation medium. Detection of GUS and hptII genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.

Reliable transformation system for Microbotryum lychnidis-dioicae informed by genome and transcriptome project.

Microbotryum lychnidis-dioicae is a member of a species complex infecting host plants in the Caryophyllaceae. It is used as a model system in many areas of research, but attempts to make this organism tractable for reverse genetic approaches have not been fruitful. Here, we exploited the recently obtained genome sequence and transcriptome analysis to inform our design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi. Reproducible transformation was demonstrated at the genomic, transcriptional and functional levels. Moreover, these initial proof-of-principle experiments provide evidence that supports the findings from initial global transcriptome analysis regarding expression from the respective promoters under different growth conditions of the fungus. The technique thus provides for the first time the ability to stably introduce transgenes and over-express target M. lychnidis-dioicae genes.

Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335.

Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0-6 mg l-1) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l-1 ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T1 plants. Southern analysis of T1 plants confirmed the integration of bar gene into the genomic DNA and the bar positive T1 plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot node-glufosinate system in an Indian soybean genotype.

High rates of virus-induced gene silencing by tobacco rattle virus in Populus.

Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P. × canescens. Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium-infiltrated into leaves of the two poplar species. Agrobacterium-mediated delivery of the shorter insert, TRV2-PePDS266, into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS558. The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV-VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65-73 and 83-94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P. × canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P. × canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P. × canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P. × canescens using three different statistical approaches, geNorm, NormFinder and BestKeeper. Quantitative real-time PCR showed significant reductions in PDS transcripts (55-64%) in the photobleached leaves of both VIGS-treated poplar species. Our results demonstrate that the TRV-based VIGS provides a practical tool for gene functional analysis in Populus sp., especially in those poplar species which are otherwise recalcitrant to transformation.

Grapevine (Vitis vinifera L.).

Grapevine (Vitis) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice production. Wine is by far the major product of grapes, and the focus of this chapter is on wine grape cultivars. Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer acceptance and are firmly entrenched in the market place because of varietal name branding and the association of certain wine styles and regions with specific cultivars. In light of this situation, grapevine improvement by a transgenic approach is attractive when compared to a classical breeding approach. The transfer of individual traits as single genes with a minimum disruption to the original genome would leave the traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a successful transgenic approach to grapevine improvement. There are three criteria for achieving an efficient Agrobacterium-mediated transformation system: (1) the production of highly regenerative transformable tissue, (2) optimal cocultivation conditions for both grapevine tissue and Agrobacterium, and (3) an efficient selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation system that meets these criteria. We also describe a protocol for the production of transformed roots suitable for functional gene studies and for the production of semi-transgenic grafted plants.

A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma.

Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91%) was observed when MS medium lacking NH4NO3, KH2PO4, KNO3 and CaCl2. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.

A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum

An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated transformation and growth rate of hairy root. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, and three explants types, hypocotyls, leaves and excised shoots were examined. The highest frequency of transformation was achieved using LBA9402 strain in the excised shoots. Several inoculation and co-cultivation media and different concentration of arginine were evaluated using LBA9402 strain and the excised shoots as explant. Interestingly, a drastic increase in the frequency of transformation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH4NO3 KH2PO4, KNO3 and CaCl2 was used. The effect of sucrose concentration and the ratio of NH4 +: NO3 − on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH4 + to NO3 − on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization.

Recent Advances in Blueberry Transformation

Although Vaccinium cultivars have been generated exclusively through the traditional methods of controlled hybridization and deliberate selection, genetic transformation could provide a powerful approach to supplement conventional breeding methods for Vaccinium by introducing genes of interest. A reliable transformation system depends on efficient plant regeneration, reliable gene delivery, and effective selection. To date, in blueberry, adventitious shoot regeneration using leaf explants has been the most desirable regeneration system; Agrobacterium-mediated transformation is the major gene delivery method; and effective selection has been reported using either the nptII or the bar gene as selectable markers. In 2004, stable transformation was reported for four highbush blueberry cultivars, with a transformation frequency ranging from 5.0–15.3%. In 2006, the first field trial of transgenic blueberry with herbicide resistance was performed. In 2009, a blueberry C-repeat binding factor (CBF) gene (GenBank AF234316) was transformed into a southern highbush blueberry cultivar Legacy in order to improve cold tolerance by elucidating the CBF-regulated network in blueberry. This progress has demonstrated that blueberries can be improved through genetic engineering. In the future, as more genomic resources for Vaccinium become available, more genes of interest will be identified and isolated. Genetic transformation will allow us to evaluate these genes as to their functions. Currently, there is industry-wide concern about the application of transformation technology in blueberries, even though numerous transgenic crops have been deployed. Hopefully, new engineering strategies, such as intragenic transformation, RNAi, and marker-free technologies, will reduce public concerns about transgenic Vaccinium plants.

Regeneration and Agrobacterium-mediated transformation of mature woody species Photinia × fraseri “Red Robin”

An efficient regeneration protocol for genetic transformation was developed from the leaves of an 11-year-old Phtinia × fraseri “Red Robin” tree. A high frequency of adventitious buds (88.63 ± 1.38%) and the highest maximum mean number of adventitious buds per explant (4.65 ± 0.48) were obtained in light conditions on Murashige–Skoog (MS) medium containing 2 mg l−1 benzyladenine (BA) and 0.2 mg l−1 α-Naphthaleneacetic acid (NAA). After preculturing for 6 days, over 95% of the shoots successfully rooted on 1/2 MS medium supplemented with 0.3 mg l−1 indole-3-butyric acid (IBA) within 3 weeks. For genetic transformation, two crucial parameters (30 mg l−1 kanamycin for selection and 30-min suspension time) were optimized. Taken together, a reliable transformation system with an efficiency of more than 5% was established. This genetic transformation protocol can be utilized for further genetic manipulation of the Photinia tree.

Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) Point Mutation by Recombination with a Truncated Nonfunctional ARG7 Gene

Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a (6073)G to A transition in exon 9 of ARG7 leading to a (288)Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, (27)Leu to Phe and (285)Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5' end led to exact reversion of the mutant base (6073)A to the wild-type (6073)G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.

Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm-3 kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T0 putative transformants and their seed progenies (T1 to T3 generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T0 - T3 plants were morphologically normal and fertile.

Transgenic peach plants (Prunus persica L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an in vivo marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacterium-mediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.

The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

This paper describes the development of a reliable transformation system for garlic Allium sativum L. and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus sources: callus induced from both apical and non-apical root segments of in vitro plantlets, true garlic seeds and bulbils. Two different reporter genes were used in our garlic transformation experiments, namely the gusA gene coding for -glucuronidase and the gfp gene coding for green fluorescent protein. A total of seven independent transformed callus lines derived from different callus sources were obtained. The advantage of the system developed is the short time period needed for completion of the protocol about 6 months and the year-round availability of high quality callus from in vitro roots. The highest transformation frequency in a single experiment 1.47%, was obtained using garlic cv. ‘Printanor’. Differences existed between cultivars in transformation frequency but were not significant. The same was found for the plasmids used in transforming garlic. Via PCR the presence of the gusA, hpt hygromycin phosphotransferase and gfp genes could be demonstrated in putative transformed in vitro plants. Southern hybridization showed that the reporter gene gusA and the selective gene hpt were stably integrated into the garlic genome. After transfer to the greenhouse of in vitro regenerants, transgenic garlic harbouring the gusA gene survived and grew well, whereas the gfp transgenic garlic gradually died under these conditions. Using this protocol transgenic garlic resistant to beet armyworm using the cry1Ca and H04 resistance genes from Bacillus thuringiensis were developed. Via Southern hybridization it was shown that the cry1Ca sequence was stably integrated into the garlic genome. After transfer of the transgenic in vitro garlic plants to the greenhouse, the cry1Ca plants developed normally and grew well to maturity with normal bulbs. However, all transgenic in vitro H04 garlic plants did not survive after transfer to the greenhouse. Transgenic cry1Ca garlic plants proved completely resistant to beet armyworm in a number of in vitro bio-assays. This finding will facilitate the development of new garlic cultivars resistant to beet armyworm.

Gaeumannomyces graminis, the take‐all fungus and its relatives

SUMMARY Take-all, caused by the fungus Gaeumannomyces graminis var. tritici, is the most important root disease of wheat worldwide. Many years of intensive research, reflected by the large volume of literature on take-all, has led to a considerable degree of understanding of many aspects of the disease. However, effective and economic control of the disease remains difficult. The application of molecular techniques to study G. graminis and related fungi has resulted in some significant advances, particularly in the development of improved methods for identification and in elucidating the role of the enzyme avenacinase as a pathogenicity determinant in the closely related oat take-all fungus (G. graminis var. avenae). Some progress in identifying other factors that may be involved in determining host range and pathogenicity has been made, despite the difficulties of performing genetic analyses and the lack of a reliable transformation system.

Screening of Regenerable Genotypes of Italian Ryegrass (Lolium multiflorum Lam.)

We screened regenerable genotypes from 12 cultivars of Italian ryegrass (Lolium multiflorum Lam.) through tissue culture of mature seeds, shoot tips and root tips. Although three morphological types of calli, friable, watery, and compact with fine root hair were observed, green shoots regenerated only from the friable calli. The highest frequency of the formation of regenerable callus from mature seeds was 1.4% in the diploid cultivar Waseaoba and 1.1% in the tetraploid cultivar Meritra. The plants regenerated from mature seed-derived calli were maintained and propagated aseptically in vitro, and the method of regenerating plantlets via callus culture was established by using their shoot tips. Sixty-three seeds of Waseaoba and 44 seeds of Meritra were aseptically sown, each seedling (genotype) was propagated in vitro, and the shoot tip from each genotype was subjected to tissue culture. More than 22% of these genotypes formed regenerable calli. From these results, we conclude that the shoot tip of the in vitro-preserved plantlet is useful for producing the regenerable calli routinely. The information obtained in the present study is useful for establishing a reliable transformation system for Italian ryegrass.

Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi.

A reliable DNA-mediated transformation system has been developed for Pseudozyma flocculosa, a fungus that is antagonistic to powdery-mildew fungi. Plasmids harboring various selectable markers under the control of different promoters were tested. Molecular analyses demonstrated that successful transformation could be achieved using a plasmid that confers resistance to hygromycin B under the control of the Ustilago maydis hsp70 promoter and terminator sequences. On average, 1-40 (mean = 20) transformants were obtained per 10 microg of linearized DNA per 10(8) protoplasts. Southern analysis of the transformants revealed that, in each case, the vector had integrated in multiple tandem copies into the genome of P. flocculosa, and that integration events were random. Pulsed-field gel electrophoresis was employed to separate the genome of P. flocculosa into at least 11 chromosomes with sizes ranging from 0.55 Mb to 2.9 Mb. Hybridization with the plasmid indicated that integration of vector DNA had occurred in one to several chromosomes depending on the transformant examined.