Novel and potential application of cryopreservation to plant genetic transformation

The progress of genetic transformation in rice was reviewed and the factors influencing Agrobacterium tumefaciens-mediated target gene in rice was analyzed through the procedure,which include Agrobacterium tumefaciens strains and Vir gene activation,rice genotypes and medium types,infection time,co-culture patterns,co-culture time,marker gene and reporter gene selection,selection pressure,and finally the problems existing in genetic transformation in rice and countermeasures were pointed out.

Agrobacterium Tumefaciens-Mediated Transformation in Rice

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

/pdf/selection-of-suitable-endogenous-reference-genes-for-4hsu8i1aie.pdf

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

Papaya ( Carica papaya L.) is an important tropical fruit crop. The fruit is consumed fresh and used in the pharmaceutical, rayon and food industries. Papaya improvement for stress tolerance and qualitative traits using conventional breeding has been difficult due to the narrow germplasm pool in the Carica genus and sexual incompatibility problems encountered during intergeneric hybridization with other genera in the Caricaceae family. Genetic engineering is an important tool in papaya improvement for modifying one or more traits in elite cultivars without altering existing characteristics. Advances in genetic engineering have been facilitated by concerted efforts for genome sequencing of papaya, development of papaya regeneration systems and efficient gene insertion techniques for transfer of desirable traits. Papaya regeneration via organogenesis and somatic embryogenesis has been refined during the past 3 decades. Early efforts to optimize gene insertion protocols utilized a number of reporter and selectable marker genes, viral- and bacterial-derived regulatory sequences and functional genes for biotic and abiotic stress tolerance. Transgenic plants were routinely produced with several cultivars. One of the best success stories in the commercialization of a genetically modified fruit crop has involved the development of transgenic papaya ring spot virus (PRSV) resistant Rainbow and SunUp cultivars, which saved the Hawaiian papaya industry. Additionally, genetically modified papayas with traits for disease resistance and extended shelf life have been extensively screened in field tests. The papaya genome sequence was published in 2008 and has opened new avenues for papaya improvement by precision breeding, which involves the use of regulatory and functional gene sequences from related genera of the Caricaceae family, and is a logical extension of conventional breeding and genetic transformation. The application of precision breeding technology for papaya can pave the way for the development of consumer and eco-friendly cultivars that would be developed in ways similar to conventional breeding while causing fewer GMO-related concerns.

Advances in papaya biotechnology

The transgenic papaya is a valuable strategy for creating plants resistant to papaya ringspot virus (PRSV) infection and increasing production. This study was further performed to evaluate the comparative toxicity effects of the newly developed transgenic line of the fruits of two backcross transgenic papaya lines (2210 and 823) and one hybrid line (823-2210) and compare to their parent non-transgenic (TN-2) counterparts. The stability analysis of coat protein (CP) of PRSV was investigated using the digestion stability assays in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and bile salts to detect the CP fragments. Results revealed that the CP fragments were rapidly hydrolyzed in SGF and were undetectable in organs and gastrointestinal contents in rats. For the genotoxicity, three in vitro assays were conducted and exhibited that non-transgenic and backcross transgenic papaya fruits were negative. Moreover, a repeated animal feeding study was conducted by feeding 2 g/kg of body weight ...

Toxicity Assessment of Transgenic Papaya Ringspot Virus of 823-2210 Line Papaya Fruits

The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene, and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus.

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Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus.