<Emphasis Type="Italic">ASYMMETRIC LEAVES2-LIKE38</Emphasis> gene,a Member of AS2/LOB family of <Emphasis Type="Italic">Arabidopsis</Emphasis>, causes leaf dorsoventral alternation in transgenic cockscomb plants

ASYMMETRIC LEAVES2-LIKE38/LBD41 gene of Arabidopsis is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. To explore ASL38 function, we transformed 35S:ASL38 constructs into cockscomb (Celosia plumosus) plants via Agrobacterium tumefaciens and obtained T1 35S:ASL38 plants. The extremely folded or crinkly leaves were seen in these T1 cockscomb plants. The anatomical analysis of these malformed leaf blades indicated that adaxial cells revealed abaxialized traits, which were never seen in those of wild-type plants. These results suggested that ectopic expression of ASL38 might lead to alternations of dorsoventrality in folded or crinkly leaves of 35S:ASL38 cockscomb. In general, all data showed that ASL38 might be involved in dorsoventral determination in lateral organ development of plants.     

<Emphasis Type="Italic">ASYMMETRIC LEAVES2</Emphasis> gene,a member of LOB/AS2 family of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>, causes an abaxializing leaves in transgenic cockscomb

The leaf primordium derives from the peripheral zone of shoot apical meristem. During the formation of leaf primordia, they need to establish the proximodistal, mediolateral, and ab/adaxial axes. Among these axes, the ab/adaxial axis might be the most important. ASYMMETRIC LEAVES2 (AS2) gene is a member of AS2/LATERAL ORGAN BOUNDARY (LOB) family of Arabidopsis thaliana. In this work, we transformed 35S:AS2 transgene constructs to cockscomb (Celosia cristata) via Agrobacterium tumefaciens. All primary transformants subsequently obtained were placed into phenotypic categories and self-pollinated. As a whole, a total of 44 T1 35S:AS2 cockscomb plants obtained were grouped into two major categories: (I) slightly wrinkled leaves (28/44), (II) extremely curved leaves (16/44), on the basis of their leaf phenotypes. Furthermore, we characterized the anatomical features of these malformed leaves; and found the transformation of adaxial cell types into abaxial cell ones. A series of data suggest that AS2 might be involved in the determination of abaxial polarity in cockscomb plants. However, a few research teams have reported that AS2 might be involved in the determination of adaxial polarity in leaf primodia of Arabidopsis thaliana. These data above indicate that the roles of the same ab/adaxial determinant might differ between distinct species. At last, the different function of AS2 in distinct species was discussed.     

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.     

Efficient Agrobacterium-mediated genetic transformation method using hypocotyl explants of radish (Raphanus sativus L.)

To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar “Pirabikku”. Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the β-glucuronidase (GUS) gene in T₁ plants from fertile T₀ plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the medicinal plant <Emphasis Type="Italic">Centaurea montana</Emphasis>

An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T₀ and T₁ plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.     

Transformation of <Emphasis Type="Italic">PttKN1</Emphasis> gene to cockscomb

We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media supplemented with 0.5 mg l⁻¹ 6-BA (for explants of apical meristems of C. cristata) or 2.0 mg l⁻¹ 6-BA, 0.5  mg l⁻¹ NAA and 0.5  mg l⁻¹ IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula × P. tremuoides knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported.     

Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both β-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.     

GUS activity for miR165a/166b, REV, and WUS/CLV3 in in vitro direct Arabidopsis thaliana shoot regeneration

In Arabidopsis thaliana, the process of shoot regeneration in vitro requires the presence of specific miRNAs. We describe here the β-glucuronidase (GUS) expression domains for miR165a/166b, REV, and WUS/CLV3 during direct shoot regeneration. Increased GUS activity of miR166b and REV were first detected within the shoot apical meristem of explants, while no pmiR165a::GUS activity appeared there. The zone of pWUS::GUS activity covered the inner sides of developing protuberances, while that of pCLV3::GUS was more restricted. Once the primary shoot had emerged from the protuberance, pREV::GUS activity was turned on throughout the protuberance. pmiR165a::GUS activity was detected in a small number of protuberance surface cells, while pWUS::GUS activity was restricted to within a few cells beneath the protuberance surface. After the differentiation of leaf-like structures, GUS activity for miR165a and miR166b appeared largely on their abaxial surface, while pWUS::GUS activity was concentrated at the apex of the primary shoot, and no pCLV3::GUS activity was detectable. Following the formation of secondary shoots, pmiR165a::GUS activity was detected on their abaxial surface. GUS activity for miR166b, REV, and WUS/CLV3 were concentrated in the stem apical meristem. The observations suggested that each member of this set of genes might play a distinct role in both primary and secondary shoot regeneration.     

Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape,transgene inheritance,and efficiency

Public concern and metabolic drain were the main driving forces for the development of a selectable marker-free transformation system. We demonstrated here the production of transgenic tobacco plants using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens-infected leaf explants were allowed to produce shoots on a shoot induction medium (SIM) containing no selective compounds. Up to 35.1% of the A. tumefaciens-infected leaf explants produced histochemically GUS⁺ shoots, 3.1% of regenerated shoots were GUS⁺, and 72% of the GUS⁺ shoots were stably transformed by producing GUS⁺ T1 seedlings. When polymerase chain reaction (PCR) was used to screen the regenerated shoots, 4% of the shoots were found to be PCR⁺ for the transgene and 65% of the PCR⁺ shoots were stable transformants. Also, generation of PCR⁺ escapes decreased linearly as the number of subculture increased from one to three on SIM containing the antibiotic that kills the Agrobacterium. Twenty-five to 75% of the transformants were able to transmit transgene activity to the T1 generation in a Mendelian 3:1 ratio, and a transformation efficiency of 2.2–2.8% was achieved for the most effective binary vector. These results indicated that majority of the GUS⁺ or PCR⁺ shoots recovered under no selection were stable transformants, and only one-third of them were chimeric or escapes. Transgenes in these transgenic plants were able to transmit the transgene into progeny in a similar fashion as those recovered under selection.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

Activity of promoters of carnation etched ring virus and dahlia mosaic virus in tobacco protoplasts and transgenic plants

Promoters of carnation etched ring virus (CERV) and dahlia mosaic virus (DMV) were cloned into binary vectors pCambia 1304, pCambia 1281Z, and pCambia 1291Z with reporter GFP and GUS genes. Activities of these promoters in tobacco protoplasts and transgenic plants were determined using these constructs. Histochemical GUS analysis demonstrated the absence of tissue-specificity in transgenic plants transformed with these promoters. The quantitative analysis of these promoter activities in transgenic tobacco plants, using 4-methylumbelliferone as a substrate, showed that 35S CaMV, CERV, and DMV promoters displayed approximately similar activities in transgenic tobacco plants.     

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD₆₀₀ of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.     

Rice LIM protein OsPLIM2a is involved in rice seed and tiller development

Yield of major monocotyledonous crops including wheat, rice, barley, and sorghum is greatly influenced by tillering. However, deciphering the underlying mechanisms of the tillering has long been hindered because many changeable factors are involved in the process. Plant two LIM-domain-containing proteins bind to and stabilize actin filaments that are major constituents in the formation of higher-order actin cytoskeleton. Here, we report that rice LIM-domain protein, OsPLIM2a, is involved in rice tillering likely through actin cytoskeleton organization. OsPLIM2 genes (OsPLIM2a, OsPLIM2b, and OsPLIM2c) expressed in reproductive organs including anthers, but not in other tissues. Analysis of both OsPLIM2a and OsPLIM2c promoter fused to GUS reporter revealed that both promoters directed strong and specific GUS expression in pollens. Transient expression of OsPLIM2a-GFP and OsPLIM2c-GFP in tobacco leaves showed that OsPLIM2a and OsPLIM2c could bind to actin filaments, which is consistent with other plant LIM proteins with actin-binding property. To examine further physiological roles of rice OsPLIM2a and OsPLIM2c, transgenic rice plants with 35S:OsPLIM2a or 35S:OsPLIM2c were examined for any phenotypic changes. Transgenic plants overexpressing OsPLIM2a produced bigger seeds than wild type, whereas they exhibited reduction in tiller numbers. These results suggest that OsPLIM2a may participate positively in seed development but negatively in tiller differentiation. Protein interaction assays using OsPLIM2c proteins revealed that OsPLIM2c interacted with at least three proteins including rice Fimbrin, of which homologs in Arabidopsis play crucial roles in pollen tube growth, implying that rice OsPLIM2c and Fimbrin may exert roles together in pollen tube growth.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the genome-sequenced poplar clone,nisqually-1 (<Emphasis Type="Italic">Populus trichocarpa</Emphasis>)

The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood (Populus trichocarpa). To improve its utility for functional genomics research, having an efficient means for transformation and regeneration is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants with a strain ofAgrobacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was incorporated in the selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during the early stages of shoot induction. Histochemical assays revealed expression of theGUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol, transgenic plants were recovered within 6 mo at an efficiency of 6%, adequate to produce a large number of transgenic events with modest effort.     

Tissue specific response of Agrobacterium tumefaciens attachment to Sorghum bicolor (L) Moench

Agrobacterium mediated genetic transformation of plants have advantages over other methods, especially for making single copy transgenic plants with reduced chances of gene silencing and instability. However, monocotyledonous plant species could not utilize the full potential of this system because of possible limitations in Agrobacterium interaction with monocot plant cells. Agrobacterium attachment as a factor in genetic transformation was studied in the leaf, shoot apex, and leaf derived callus of sorghum (Sorghum bicolor (L) Moench). Pre-induction of Agrobacterium with acetosyringone was found necessary for Agrobacterium attachment to sorghum tissues. All the explants responded positively, with preferential Agrobacterium attachment and colonization around the tissues having actively dividing cells. Callus proved to be the best explant for Agrobacterium attachment as observed in scanning electron microscopy and transient GUS expression. Loss of Agrobacterium attachment was observed with an increase in the degree of tissue differentiation.Key words: Genetic transformation, Acetosyringone, Scanning electron microscopy, Transient gene expression, GUS assays, qRT-PCR