Nicotiana alata Link and Otto (Jasmine tobacco) was transformed with an autoregulated senescence-inhibition gene construct PSAG12-IPT encoding isopentenyl transferase via Agrobacterium-mediated transformation. Transformation was confirmed by polymerase chain reaction. Transgenic plants exhibited up to 2- to 4-fold fewer senesced leaves, 29% longer in situ flower life, 26% more shoot dry weight, and a 32% to 50% reduction in flowers per branch. Additionally, transgenics were 28% shorter and had up to 174% more branches, indicative of cytokinin overproduction and a lack of tight autoregulation of PSAG12-IPT. Variation among independent transgenics suggests selection for enhanced PSAG12-IPT is feasible. Our observations of increased branching and in situ flower longevity, as well as reduced plant height and flowers per branch provide new information on PSAG12-IPT and its potential value for biological study and horticultural application.
Kenneth R. Schroeder, Dennis P. Stimart, and Erik V. Nordheim
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.
Taly Trainin, Alexander Lipsky, Avraham A. Levy, and Doron Holland
The maize transposable element Activator (Ac) has been shown to be active in a number of dicots, including arabidopsis [Arabidopsis thaliana (L.) Heynh.], tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L.), and aspen (Populus tremuloides Michx.). However, no information is available on somatic transposition in any plant during several years of growth and development. It is not known how transposition affects genetic variability among vegetative parts that have developed during a long period of growth. In order to explore the possibility of using somatic Ac transposition for gene tagging and mutagenesis in fruit trees, a derivative of the maize Ac transposable element was introduced into `Duncan' grapefruit (Citrus paradisi Macf.) by Agrobacterium tumefaciens (Smith & Towns.) Conn.-mediated stable transformation. Genetically identical 4-year-old sibling trees were established by grafting one of the transformants on Troyer citrange [Citrus sinensis (L.) Osbec. × Poncirus trifoliate (L.) Ras.] rootstocks. We demonstrated that the Ac element was active upon transformation in citrus (Citrus L.) trees and that transposition can create genetic variability among tree siblings and among leaves collected from different parts of the same tree. Ac was still active among propagated plants 4 years after transformation, clearly indicating that it is capable of maintaining itself in citrus trees for a long period of time. The observation of different integration patterns in different parts of the same tree and within tree siblings originating from the same transformant suggests that an Ac-based mutagenesis system could be very useful in creating somatic mutations in citrus trees.
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.
Narumi Matsuda*, Kanji Isuzugawa, Mei Gao, Tadashi Takashina, and Koichi Nishimura
The transformation of pears such as `Conference', `Doyenne du Comice' and `Passe-Crassane' has been attributed to the high regeneration frequency from leaf discs (71% to 97%; Leblay et al. 1991). However, it has been difficult to transfer desirable genes into cultivars with low-regeneration frequency such as `Silver bell' (35.4%) and `La France' (10.7%), which are the two major pear cultivars in Japan. In this study, we developed an Agrobacterium-mediated transformation system for `Silver bell' and `La France'. For `Silver bell', leaf discs derived from in vitro shoots were used as explants. The antibiotics for selection of transformants and elimination of Agrobacteria were investigated. In the most optimum condition, which is 30 mg·L-1 Kanamycin and 500 mg·L-1 Sulbenicillin, a 3.2 % transformation efficiency was obtained. However, no success was recorded in an effort to transform `La France' using leaf disc explants because of very poor regeneration frequency. Therefore, axillary shoot meristems were used as explants for transformation of `La France'. The conditions for antibiotic selection and elimination of Agrobacteria were also investigated. In 5 mg·L-1 Kanamycin and 375 mg·L-1 Carbenicillin, transformed shoots were produced at 4.8% efficiency. No chimera was observed in the transgenic shoots during a 2-year subculture period. Since the inoculated explants developed into multiple shoots during selection, it was thought that the problem of chimera might have been overcome. Therefore, this transformation method using axillary shoot meristem may be applicable to pear cultivars recalcitrant to regeneration from leaf disc. To the best of our knowledge, this is the first report of a transformation system in pear cultivars with low regeneration efficiency.
Chifumi Nagai, Zhuping Mai, and Jerek Jong
Somatic embryogenesis of coffee has been studied for the purpose of obtaining target tissues for stable genetic transformation through use of a particle gun. Eight cultivars of C. arabica were selected for callus induction from leaves. Primary calli were induced within two weeks in over 98% of the leaf disks explanted on MS medium with 2,4-D and kinetin prior to treatment on a secondary culture medium. Somatic embryos were obtained from `Catuai' and `Blue Mountain' after six months from explanting. Somatic embryos were germinated in MS media and developed into plants. Somatic embryos and embryogenic calli are being used for gene transformation experiments with the helium-driven particle gun.
Prem Anand Rajagopalan and Rafael Perl-Treves
Agrobacterium-mediated transformation of Cucumis sativus L. cotyledons was investigated, to identify important factors that affect transformation efficiency. The factors evaluated were initial explant preparation, including preculture, pricking of the explant, inoculation time and co-cultivation regime. We also modified the selection regime, compared to previously published protocols. Our results show that dissecting the proximal half of the cotyledon by a V-shaped cut resulted in a higher transformation rate, compared to two other methods of dissection, as indicated by transient β-glucuronidase gene expression. Selection on 100 mg·L-1 kanamycin resulted in the early development of nontransformed shoots, whereas a gradual increase of kanamycin concentration up to 200 mg·L-1 resulted in the subsequent formation of transgenic shoots on the same medium. The overall transformation frequency in these experiments, expressed as the number of rooted, confirmed transgenic plants per initial number of explants, was 1.7%. The stable integration of T-DNA was confirmed in the primary transformants and their progeny. Abbreviations: ABA = abscisic acid; AdS = adenine sulfate; AS = acetosyringone; BA = 6-benzylaminopurine; GA3 = gibberellic acid; IBA = indolebutyric acid.
Zhanyuan Zhang, A. Mitra, and D.P. Coyne
Factors influencing Agrobacterium–mediated DNA transfer of P. vulgaris were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains, A2760 and EHA105, were used with more emphasis on the former due to its overall higher transformation rate. Ten bean entries including breeding lines and cultivars from both Meso-American and Andean origins were compared for compatibility with the two bacterial strains under different pre- and coculture conditions. Pinto `Othello' was extensively used in testing different transformation conditions. Factors found to have significant effects on transformation rate included Agrobacterium-host interactions, explant maturity, preculture and cocultivation conditions, as well as selection schemes, based on transient expression. Some factors, such as the effect of explant maturity and dark preconditioning of explants on gene transfer, have not been reported before. The best transformation conditions included the use of susceptible genotypes and mature explants, preconditioning of explants in darkness, followed by a maximum cocultivation period in the presence of cytokinin, and the use of high selection pressure.
John M. Sherman, James W. Moyer, and Margaret E. Daub
An efficient, high-frequency regeneration and Agrobacterium-mediated transformation system was developed allowing the genetic engineering of three chrysanthemum (Dendranthema grandiflora Tzvelev) cultivars: the formerly recalcitrant and economically important cut-flower mum `Polaris' and two potted mums, `Hekla' and `Iridon'. The regeneration protocol used leaf explants on a sequence of media with four hormone regimes. Explants were first cultured on an embryogenesis-type medium containing a high concentration of 2,4-D, which promoted callus formation. Shoot primordia were induced by culture on medium lacking 2,4-D, followed by shoot elongation on a high-cytokinin plus gibberellic acid medium. Finally, elongated shoots were rooted on a low-auxin rooting medium. Transformed plants of the three cultivars were obtained following co-culture of leaf explants with A. tumefaciens strain EHA 105 harboring the plasmid pBI121 containing genes for neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS). Stable transformation of the three cultivars was verified via GUS assays and Southern analysis.
Shin Je Kim, Kyung-Hee Paek, and Byung-Dong Kim
A cDNA clone of cucumber mosaic virus (CMV) 117 N-satellite RNA driven by the cauliflower mosaic virus (CaMV) 35S transcript promoter, was stably integrated into the genome of Petunia hybrida `Bluepicoti' tissues by Agrobacterium tumefaciens Ti plasmid-mediated transformation. Transgenic plants producing CMV satellite RNA showed delayed disease development when inoculated with CMV-Y, a helper virus for the I17N-satellite RNA. Furthermore, transgenic petunia plants showed delayed disease development against tobacco mosaic virus (TMV), a tobamovirus not related to CMV. Northern blot analysis revealed that large amounts of unit length satellite RNA (335 bp) were produced in CMV-infected transgenic petunia plants; whereas, mainly transcripts driven by the CaMV 35S promoter (approximately 1 kb) were produced in TMV-infected transgenic plants. SDS-PAGE and Western blotting showed that symptom reduction was correlated with a reduction in the amount of viral coat protein in transgenic plants.