`Pingyitiancha' has good agricultural traits and is an important rootstock for apples. It was studied for Kam, Cef and Carb concentrations, precultural times, active medium of A.tum, Ca++ concentration, mixed inoculation times of AgNO3, and PVP delay selection times for getting transgenic `Pingyitiancha' stock with more resistance to drought and salt. It was demonstrated that the highest transformation frequency was obtained with 200 mg·L-1 Cef as an A.tum restraining antibiotic, 8 mg·L-1 Kam as a selection antibiotic, GCJ8 active medium, a precultural of 2 days, 5 Ca++ concentrations in preculture and coculture medium compared with standard MS medium, immersed in A. tum as OD600 for 0.5 to 1 min., 4 mg·L-1 AgNO3 and 0.7% PVP as an anti-oxidation compound to reduce hydroxybenzene oxidation and delay selection for 5 days by using `Pingyitiancha' apex shoots as explants. Using mediate of A. tum strain LBA4404 under conditions mentioned above and same explants, the HVA1 gene were transformed into `Pingyitiancha' under the control of CaMV35S promoter and obtained Kam resistance regeneration plants. Six transgenic plants were confirmed by PCR and subsequent dot blot methods.
Xiang Shen*, Zhenlin Wei, and Ling Guo
Xian Shen*, Ling Guo, and Zhenlin Wei
Malus hupenensis var. `Pingyitiancha' is an important apple stock with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. The three group gene-HVA1 come from barley was transformed into `Pingyitiancha' mediated by Agrobacterium tumefaciens and transformed regeneration plants were obtained in this research. The HAV1 gene cloned from plasmid containing it (offered by Dr. Guo Weidong) by PCR with high fidelity pfu Taq DNA polymerase. It was ligated between BamH 1 and Sac 1 site in PUC118 vector, and identified by electrophoresis after digested with BamH 1 and Sac 1. Through nuclear sequence detecting, it is confirmed that the HAV1 gene cloned in this research is 703bp.This fragment was ligated with 11kb fragment from pB121 plasmid and constructed pBHA vetor. The pBHA vector was introduced in A.tum LBA4404 by triparental mating and the binary vector was obtained. It is cinfirmed that HVA1 gene had been insert in T-DNA by in situ hybirdization. Using `Pingyitiancha' shoot apex, mediated by A. tum. System, the HAV 1 gene was transformed into the plant. Kam resistance regeneration plants were obtained, 6 of them were confirmed as transformation plants by PCR and dot blot.
B.J. Ahn, J.S. Choi, K. Kamo, and J.W. King
Biolistic transformation methods for zoysiagrasses (Korean lawngrass) were developed and used to introduce a herbicide-resistant trait. Embryogenic calli were induced from mature caryopses on MS medium supplemented with 2 mg/L of 2,4-D, and used to establish liquid agitation cultures. The cultures have been maintained over a year without loss of the embryogenic competence. A particle bombardment method was optimized for zoysiagrass based on transient gusA gene expression. The most transient GUS expression upon bombardment treatments occurred at 1100 psi of helium pressure with 10 cm of particle flying distance and 0.125 M sorbitol preculture treatment. Promoters suitable for zoysiagrass were compared, and actin and ubiquitin promoters were found effective in expressing gusA gene. Vector DNAs containing a herbicide resistant gene (bar), pBY505, were introduced into embryogenic cells of zoysiagrass using the optimized method. Total 194 putatively transformed plants were regenerated from 60 biolistic plates for over 6 months through selection culture containing 4-10 mg/L of phosphinothricin. Regenerants were grown in potting soil in greenhouse and sprayed with 1.7 g/L of Ignite herbicide. The transgenic plants showed various levels of resistance to the herbicide, while untransformed control plants were all dead. Recombination of the bar gene into the genomes of the transformants were confirmed through PCR and Southern blot analysis.
Marceline Egnin and C.S. Prakash
This study aimed to optimize factors for the efficient delivery of foreign genes into sweetpotato using Agrobacterium tumefaciens and develop transgenic plants. Disarmed Agrobacterium C58 carrying a binary vector pBI 121C2H with gusA, nptll, and the nutritional protein asp-l genes was used to cocultivate (4 days) petiole explants of the sweetpotato genotype P1318846-3. Pre-incubation of petioles for 3 days on MS medium with 2,4-D (0.2 mg·liter–1) before infection resulted in higher transformation. Putative transgenic shoots were obtained by transfer of petioles to MS medium with TDZ (0.2 mg·liter–1) and kanamycin (80 to 140 mg·liter–1). The PCR amplification of gusA, nptll, and asp-1 genes in the 37 putative transgenic shoots showed that six plants contained the three genes. However, none of these plants showed histochemical expression of the gusA gene. The introduced gene may have been methylated resulting in the lack of its expression. DNA blot hybridization studies are underway to verify the presence and integration of the transgenes.
Alan G. Smith and Elizabeth S. Zimmermann
Euonymus alata is an attractive landscape plant that has been reported to be an invasive species. Genetic modification through transformation is a method of reducing its invasiveness by producing sterile cultivars having limited or no seed production. A critical step in Agrobacterium-mediated gene transfer is the production of adventitious shoots. E. alata internodes and leaves from in vitro cultures were tested for adventitious shoot production on 16 plant growth regulator combinations: four levels of 6-benzylamino purine (BA) and three auxin treatments [0.5 or 0.25 mg·L-1 indole-3-butyric acid and 0.1 mg·L-1 naphthaleneacetic acid (NAA)], as well as no auxin. The optimal BA levels were found to be 0.5 or 1.0 mg·L-1 for maximizing the number of explants forming shoots and for producing the greatest number of shoots per explant. Culturing on NAA gave the greatest number of shoots per explant with both 0.5 and 1.0 mg·L-1 BA. Shoot production from internode segments was markedly superior to leaves. An initial dark treatment of 10 days did not influence shoot production. Using 1.0 mg BA with 0.1 mg·L-1 NAA, E. alata internodes were transformed with A. tumefaciens EHA105 carrying Kanamycin resistance and β-glucuronidase genes. Transformed shoots were selected on 30 mg·L-1 Kanamycin. Of the 36 shoots produced, 16 were confirmed to be transformed by β-glucuronidase histochemistry. Treatment with rooting powder containing indole-3-butyric acid did not aid rooting of shoots, but after 3 months in soil in high humidity, 21 of 24 E. alata shoots from tissue culture were rooted and acclimated.
F.A. Hammerschlag and A.C. Smigocki
Peach [Prunus persica (L.) Batsch] plants #94-1, #99-1, and #40-1, carrying a cytokinin biosynthesis (ipt) gene following transformation with the shooty mutant strain of Agrobacterium tumefaciens, were evaluated for altered growth habit and axillary shoot formation, both in vitro and in the greenhouse. After 9 weeks of in vitro propagation on four different levels of 6-benzyladenine (BA), only transformant #99-1 exhibited significantly greater axillary shoot formation (on 10 μm BA), and significantly greater fresh mass (on 3,10, and 30 μm BA) than the control #RG-3. Tolerance to a supra-optimal (30 μm) concentration of BA was indicated by fresh mass increases for #99-1 shoot cultures. Delayed senescence on 0 μm BA was exhibited by 87% of the transformants, but by only 12% of the control plants. Greenhouse-grown #99-1 and #40-1 were significantly shorter than #RG-3 plants at 6 weeks and at 1 year, but only #40-1 exhibited significantly greater branching than the controls. Chemical names used: 6-benzyladenine (BA); isopentenyl transferase (ipt).
Eric L. Zeldin, Thomas P. Jury, Rodney A. Serres, and Brent H. McCown
Corp., for their assistance with the ACCELL transformation system and PCR/Southern analysis, respectively, and Michell Sass, University of Wisconsin-Madison, for her PCR analyses. The helpful review of the manuscript by Michael Havey and Irwin Goldman
Hai-nan Liu, Jian-rong Feng, Xiao-fang Liu, Wen-hui Li, Wen-juan Lv, and Ming Luo
vectors of SFB . Fusion PCR was used to construct the ihpRNA. We wanted to influence SFB expression by genetic transformation, thereby creating a mutant SC apricot. The successful construction of sense, antisense, and RNAi expression vectors for SFB
Zhanyuan Zhang, A. Mitra, and D.P. Coyne
Optimization of parameters influencing biolistic transformation is a crucial stage towards repeatable transformation of common beans. However, there has been no published study on such optimization of this crop species in a helium particle delivery system (BioRad). Using an intron-containing β-glucuronidase (GUS) gene as a reporter, we optimized several critical parameters of biolistic PDS-1000/He delivery system for common bean transformation. The target explant tissues included cotyledons, zygotic embryos, and meristemic shoot tips suitable for organogenesis. Thus, pretreatment of target tissues with osmotic medium containing 0.15–0.25 m mannitol and 0.15–0.25 m sorbitol, positioning of target tissues in 4 cm microcarrier flying distance, the use of 1.6-μm gold particle and high concentration of coating DNA, and bombardment of young immature tissues twice at 2000 psi, etc., significantly increased transformation rate and achieved the best coverage and penetration of the meristemic areas involved in direct shoot organogenesis.
Ruigang Wu, Yi Wang, Ting Wu, Xuefeng Xu, and Zhenhai Han
negative control ( Zheng et al., 2009 ). MdMYB4 overexpression vector construction and apple calli transformation. The complete sequence of the 35S promoter was amplified from the pBI121 vector ( Supplemental Table 2 ) and cloned into the pCAMBIA1304