We also demonstrate the importance of resin selection and thorough chromatography operation optimization by evaluating the cost benefit of maximizing resin binding capacity to target product. Of course, further work is needed to verify whether the use of column chromatography is needed. Transient production of thaumatin in the edible crop Spinacia oleracea was also economically competitive and captures the benefits of obviating the need for an intensive DSP. According to this analysis, the cost to produce a kg of fresh weight of spinach is $0.10, as opposed to a cheaper price for tobacco . This is attributed to the higher cost of the seeds of spinach, the longer turnaround time assumed for spinach, and the higher plant density assumed for tobacco. It is evident that field operation is very labor intensive, due to the low recipe cycle time of 2 days, which is different than the traditional time frame of growing those crops. The potential for high intra-batch variations in product yield and quality due to meteorological factors is one of the concerns of using field grown plant material for this application. These variations in turns cause inconsistency in key facility performance parameters that should be quantified using a probabilistic approach and communicated to stakeholders and will be addressed in a follow-up communication. The cost of obtaining a more controlled supply of product is reflected in the indoor upstream facilities CAPEX and COGS. This should facilitate decision making when assessing the risk and reward of each scenario.
The large-scale recombinant production of thaumatin can address the growing market need for natural,nft hydroponic safe, non-caloric sweeteners. Like stevia, the advent of thaumatin as a sugar substitute is contingent on the feasibility of its large-scale manufacturing which was addressed in this work. However, there are also social, cultural, and behavioral factors impacting sugar consumption habits that were not considered. Consumer’s preference of such products will open the door for more plant-made bio-logics for food and beverage applications, which could drive the adoption of cost-effective solutions to rising challenges through environmentally friendly and sustainable processes.While the T-DNA approach is applicable to the model plants Arabidopsis and rice, where effective transformation methods are available, it may not be feasible in many other plant species whose transformation is inefficient. Transposon can be alternatively used for insertional mutagenesis in those plants, since the generation of new insertions occurs through crossing or propagation rather than through transformation. Supported by the United States National Science Foundation-Plant Genome Program . S.Q. was supported by the Research Start-up Grants of Zhejiang Academy of Agricultural Sciences , China. P.B.F.O. was supported by EU FP5 and FP6 projects Cereal Gene Tags and CEDROME . J.-S. J. was supported by the World Class University and the Crop Functional Genomics Center projects, Korean Ministry of Education, Science and Technology, Korea.The maize Ac-Ds transposable element has been shown to be active in the plant kingdom widely .
A number of important plant genes have been cloned using the Ac-Ds element . Ds insertion libraries have been generated in Arabidopsisand rice . However, the current strategies of transposon tagging are usually slow and labor intensive and have several drawbacks. For example, in the presence of Ac transposase , transposed Ds elements may continue secondary transpositions. Unstable Ds insertions and serial transposition events may cause untagged mutations because imprecise excision or a transposition footprint can result in a mutation that is no longer associated with the transposon . Another problem is that the Ac-Ds transposable elements are highly active in rice and can transpose early in newly transformed callus cells , which results in many sibling plants carrying the same Ds insertions and consequently decreasing gene tagging efficiency. In the present study, we constructed 12 Ac-Ds transposon tagging vectors based on three approaches: AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain chemical inducible expression system; deletion of AcTPase via Crelox site-specific recombination that was initially triggered by Ds excision; and suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element. We have tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful in functional genomics of rice and they will be useful for other crop plants as well.We constructed Ac-Ds transposon tagging vectors using a GVG-inducible expression system . The vectors pJJ86 and pDs-Ac-GVG carry an in cis two-element system that consists of Ds, 35S:GVG that expresses the chimeric GVG transcription activator, and AcTPase controlled by a GVG-inducible promoter.
The inducible promoter is transactivated through interaction between GVG and the 4xGAL4-upstream activating sequence . The transactivating activity of GVG is regulated by treatment with the steroid chemical dexamethasone . The Ds element in pJJ86 contains the 4x CaMV 35S enhancers for activation tagging , while the Ds in pDsAc-GVG does not. Excision of Ds from pJJ86 can be detected because in the resulting T-DNA fragment, the β-glucuronidase gene is driven by a CaMV 35S promoter. We also constructed a two-vector tagging system in which GVG-inducible AcTPase and Ds are in separate vectors . The strategy of the two-vector system is that transgenic plants carrying the GVG-inducible AcTPase and Ds are generated, respectively, and the AcTPase and Ds are combined in F1 by genetic crosses. In this case, Ds is mobilized in the presence of AcTPase in F1 plants, but stabilized after it is uncoupled from AcTPase in the subsequent generation. To test whether the inducible Ac-Ds system is functional in rice, we transformed rice cultivar Nipponbare with pJJ86. Independently transformed rice calli were cultured for 5 d on media with DEX to induce expression of AcTPase. Because Ds transposition can be detected by GUS activity, the DEX-treated calli and untreated controls were stained for GUS activity. DEX treatment of pJJ86-transformed calli exhibited stronger GUS staining than controls , indicating that the DEX inducible system in this vector is functional in rice. At the same time, there was low background of GUS activity in the untreated rice calli , suggesting that some background transposition occurred in the pJJ86 transformants.Because the Ac-Ds transposable elements are active in newly transformed callus cells and early transposition events lead to the same Ds insertions in sibling plants, we constructed a novel Ds element, designated HPT-Ds, and used the hygromycin resistance gene to suppress transposition. The pHPT-Ds1 vector carrying HPT-Ds and GVGinducible AcTPase in cis is shown in Figure 1E. The HPT gene in HPT-Ds has the same intron and triple splice acceptors as in the gene-trap Ds . Because HPT-Ds is immediately downstream of maize ubiquitin 1 promoter in T-DNA, the Ubi:HPT-Ds fusion confers hygromycin resistance, and transformed rice cells are thereby selected on hygromycin media. In case of transposition, HPT-Ds in the rice genome may not have a promoter nearby for transcription and the rice cells lose hygromycin resistance and can be counter-selected by hygromycin. To examine the efficacy of the HPT-Ds element,nft system we made a test construct containing Ubi:HPT-Ds and confirmed the function of the Ubi-driving HPT gene in a rice transformation experiment.
A total of 250 rice calli were transformed using a particle bombardment method and hygromycin-resistant cells were selected from 30 callus explants after 50 d of selection on hgromycin media. In constructing the pHPT-Ds1 vector, HPT-Ds was cloned between Ubi and GUS so that transposant cells canbe detected by GUS assay. The pHPT-Ds2 vector is similar to pHPT-Ds1 except that pHPT-Ds2 carries a Bar gene and transposition can be selected by herbicide resistance . pHPT-Ds1 was introduced into rice cultivar Nipponbare. A total of 26 stably transformed callus lines were obtained. In the condition without DEX treatment, five calli were randomly selected from each callus line and GUS-assayed for detection of transposant cells. Transposant cells were detected in 84.6% of callus lines but mosaic GUS patterns occurred at low frequency as compared with the GUS patterns of untreated pJJ86 calli . GUS assays were also carried out on 14 of pHPT-Ds1 transformed plantlets; 57.1% plantlets contained transposant cells that were rarely distributed in the tissue . The results of pJJ86 transformants and pHPT-Ds1 transformants indicated that there was background transposition activity in the rice calli and plantlets selected from hygromycinmedia, and that the growth of rice cells containing HPT-Ds transposition events were partially suppressed by hygromycin counter selection. To characterize the HPT-Ds excision events, rice genomic DNA of eight GUS-positive pHPT-Ds1 transformants was extracted and examined in nested polymerase chain reaction reactions using Ubi- and GUS-specific primers . Reconstructed Ubi:GUS sequence containing the HPT-Ds empty donor site was confirmed by sequencing the 657-bp PCR product . These results suggested that HPT-Ds elements in the pHPT-Ds1 transformants excised from the T-DNA. To get more information about the background transposition in the GVG-inducible AcPTase system, we constructed pHPT-Ds3 and pHPT-Ds4 by removing the 35S:GVG from pHPT-Ds1 and pHPT-Ds2, respectively. According to the GUS assay results of pHPT-Ds3 transformed callus lines, 57.1% of the callus lines showed somatic transposition. The mosaic GUS patterns of pHPT-Ds3 transformants were similar to those of pHPT-Ds1 transformants and the transposition frequency was a little lower than 84.6% of the pHPT-Ds1 calli. Our explanation for the results of pHPT-Ds1 and pHPT-Ds3 is that the background transposition in the GVG-inducible Ac-Ds system was primarily due to a low-level leaky expression of 4xUAS:AcTPase .The HPT-Ds element described in the present study is a novel Ds whose HPT gene has a dual function. During plant transformation and selection, HPT expression relies on the upstream Ubi promoter to confer resistance to hygromycin in selection media. In case of transposition, the HPT gene may be inactive because the 5 flanking sequence of HPT-Ds at a new genomic site may not be able to provide promoter activity. It is conceivable that most of the transposant cells become sensitive to hygromycin. Therefore, the counter-selection nature of the HPT gene in HPT-Ds can be used to diminish transposant cells in newly transformed rice calli on hygromycin media. In testing pHPT-Ds1 and pHPT-Ds3, it was observed that early transposition events in transformed calli and plantlets were suppressed by hygromycin. Few transposant cells in the calli and plantlets were able to grow under the hygromycin selection pressure, which might be due to escaping transposant cells or because of promoter activity of the 5 transposon flanking sequence. Because transposition requires transposase, an important theme in transposon tagging research is how to efficiently control transposase activity.
It was reported that AcTPase driven by strong promoters mediated high-frequency Ds excision in several dicot plants . Strong double enhancers of CaMV 35S promoter adjacent to wild type Ac element induced high-frequency Ac excision in rice transformation . In the present study, we have used the GVG-inducible promoter to control AcTPase expression and transposition was induced to high levels by DEX treatment of pJJ86 transformed callus. However, we also observed a leaky expression of AcTPase in the GVG-inducible Ac system in the transformants of pJJ86, pHPT-Ds1 and pHPT-Ds7 based on GUS assay results. Our explanation is that the transposition background was primarily from a low level of leaky expression of 4xUAS:AcTPase. Consistently, in the pHPT-Ds3 and pHPT-Ds5 vectors that do not have 35S:GVG, 57.1% of the pHPT-Ds3 transformants and 76.6% of the pHPT-Ds5 transformants still showed transposition in somatic cells. In spite of the wild type Ac element having a weak promoter that supports only 0.2% expression of the CaMV 35S promoter , the wild type Acitself can transpose in rice with a relatively low activity for three successive generations . This indicates that a weak expression of AcTPase can cause transposition events. In Southern blot analysis of genomic DNA of pHPT-Ds7 and pHPT-Ds8 transformants, the 5.4 kb hybridizing band represented the HPT-Ds at FDS in T-DNA. For the hybridizing bands larger or smaller than 5.4 kb, we explain that some of the bands might be from transposed HPT-Ds. The pHPTDs7 transformants showed transposition in somatic cells as suggested by GUS assay results. Because the rice genomic DNA for Southern hybridization was extracted from few leaves of a transformant, transposition in other leaves might not have been detected in the results. Also, since a rice transformant may have more than one T-DNA copy and may contain rearranged T-DNA, the hybridizing bands larger or smaller than 5.4 kb might possibly be from transgene rearrangement. Nevertheless, the efficacy of the HPT-Ds element when it was brought together with the GVG-inducible-AcTPase and the Cre-lox recombination system in pHPT-Ds7 and pHPT-Ds8 was confirmed by GUS assay and Southern blot analysis.