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.
Marceline Egnin and C.S. Prakash
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.
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.
Xiang Shen*, Zhenlin Wei, and Ling Guo
`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.
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.
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.
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
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).
Shuhua Zhan, Zhongfang Lu, and Y. Huang
Mung bean (Vigna radiata L.) is one of the economically important crops in Southeastern Asia and also grows in USA. Genetic transformation of mung bean has been achieved using an Agrobacterium -mediated transformation system. Two transformation methods were used in this study. With the leaf-disk transformation method, freshly cut leaf strips from young seedlings of mungbean were co-cultivated for 72 hours with either a wild-type A. rhizogenes strain 11325 or the strain containing an additional binary vector carrying the npt gene. In another method, agrobacteria were applied to wounded hypocotyls of aseptically germinated seedlings. After infection, the explants were placed on shoot induction medium. Within 3-4 weeks, shoots developed from the edges of leaf disks as well as from the inoculated sites on hypocotyls. Putatively transformed shoots were selected in vitro based on their ability to root in the kanamycin-containing medium. The npt gene fragment and a-few of T-DNA fragments from the wild-type Ri plasmid were detected in regenerated mungbean plants by Southern blot analysis. These results suggested that foreign DNAs from both the Ri plasmid and the binary vector had integrated into the genome of mung bean. These transformation systems for mung bean can now be used to introduce agronomically desirable traits into this crop for its genetic improvement.
Essie T. Blay, Anant Porobo-Dessai, and C. S. Prakash
Explants of sweet potato (Ipomoea batatas) and garden egg plant (Solanum integrifolium) were cocultivated with disarmed strains of Agrobacterium tumefaciens containing binary vectors with gusA, gusA-nptll fusion or gusA-intron genes. We examined whether the addition of vir gene inducers during cocultivation would improve the transformation in both crops. Acetosyringone and galacturonic acid were tested individually and in combination. A very high GUS expression was detected histochemically in both plant species. The frequency and extent of transformation varied with the type of explant, petioles being the most responsive. The presence of the vir inducing substances in the medium influenced the percent explant area transformed but did not appreciably affect the frequency of transformation. The selective proliferation of the transformed tissue and organogenesis was achieved by the culture of explants on MS medium supplemented with antibiotics.