Pejman Azadi

Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

Micropropagation of Lilium ledebourii (Baker) Boiss as affected by plant growth regulator, sucrose concentration, harvesting season and cold treatments

A protocol for the micropropagation in different harvesting time of Lilium ledebourii (Baker) Boiss, an endangered rare species endemic to Iran has been developed. In vitro scale culture of this species, using bulbs from three harvesting seasons (spring, summer and winter), was attempted. Among the various treatments tested, the Murashige and Skoog (MS) medium supplemented with 0.1 mg l -1 naphthaleneacetic acid (NAA) + 0.1 mg l -1 benzyladenin (BA) and 6% sucrose in all harvesting seasons proved to be superior to others. The best results for fresh weight of bulblets, rooting parameters and the survival rate after transplantation to greenhouse were obtained from early winter-harvested bulbs. Summer-harvested bulbs had the highest number of bulblets per explant. The bulblets at the end of the culture period were given cold treatment at 4oC for 2-8 weeks at a 2-weeks interval and then transplanted to a potting mixture of sand, leaf mold and peat moss (1:1:1 v/v). The best emergence rate (90%) was achieved at 8 weeks cold treatment for winter harvested bulbs.

Current status and biotechnological advances in genetic engineering of ornamental plants

Cut flower markets are developing in many countries as the international demand for cut flowers is rapidly growing. Developing new varieties with modified characteristics is an important aim in floriculture. Production of transgenic ornamental plants can shorten the time required in the conventional breeding of a cultivar. Biotechnology tools in combination with conventional breeding methods have been used by cut flower breeders to change flower color, plant architecture, post-harvest traits, and disease resistance. In this review, we describe advances in genetic engineering that have led to the development of new cut flower varieties.

Botrytis cinerea-resistant marker-free Petunia hybrida produced using the MAT vector system

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacteriumtumefaciens strain EHA105. Infected leaf explants of P.hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida.

Biotechnological advances in Lilium

Modern powerful techniques in plant biotechnology have been developed in lilies (Lilium spp., Liliaceae) to propagate, improve and make new phenotypes. Reliable in vitro culture methods are available to multiply lilies rapidly and shorten breeding programs. Lilium is also an ideal model plant to study in vitro pollination and embryo rescue methods. Although lilies are recalcitrant to genetic manipulation, superior genotypes are developed with improved flower colour and form, disease resistance and year round forcing ability. Different DNA molecular markers have been developed for rapid indirect selection, genetic diversity evaluation, mutation detection and construction of Lilium linkage map. Some disease resistance-QTLs are already mapped on the Lilium linkage map. This review presents latest information on in vitro propagation, genetic engineering and molecular advances made in lily.

Shoot organogenesis from cotyledons of sunflower

SUMMARY The experiment was conducted in order to study the regeneration potential of cotyledon explants from some Iranian inbred lines of sunflower (Helianthusannuus L.) and their F1 hybrids. Cotyledons of 2-day-old seedlings of three cytoplasmic male sterile inbred lines used as female (CMS 24, CMS 31 and CMS 60/52) and five inbred lines used as male (R-81.1, R-92, R-97, R-201 and R-232) and finally their 15 F1 hybrids were plated on shoot induction medium, Murashige and Skoog (MS) containing 3% sucrose, 0.5% agar-agar and different concentrations of BAP and NAA. Analysis of variance showed significant differences for all organogenesis traits studied among parental genotypes and F1 hybrids, hormone combination treatments and genotype × hormone combination treatment interaction. Shoot organogenesis was optimized on MS medium with 1 mg l-1 BAP and 1 mg l-1 NAA. Interaction “(CMS 24 × R-201) × T2” showed the highest values for number of explants shooting per 100 explants plated (55%). Interaction “R- 92× T2” showed the highest value for average number of shoots per explants plated (8.65). Interaction “R-81.1 × T2” showed the highest value for average number of shoots per explants shooting (23). Interaction “CMS 24 × T2” showed the highest value for number of explants rooting per 100 explants plated (35%). RESUMEN E experimento realizado por estudio la regeneración potential de explantes cotiledones a las líneas inbreda Irano de girasol (Helianthus annuus L.) y los híbridos F1. Cotiledones de la semillas dos días la 3 lineas utilizado para maternal (CMS 24, CMS 31 y CMS 60/52) y cinco lineas inbredas paternal (R- 81.1, R-92, R-97, R-201 y R-232) y 15 híbridos F1 se transfirieron a un media de Murashige and Skoog (MS) agüé contiene 3% sucrose, 0.5% agar-agar y concentración diferentes BAP y NAA. E1 análisis de varianza ilustro diferentes significados para todos los rasgos oranogamesis genotipos paren tal y híbridos F1, y combina tío tratamientos y combinación genotipo y hormaon organogenesis se optimo en la medio MS auge contiene 1 mg 1-1 BAP y 1 mg l-1 NAA. Interacción, “(CMS 24 x R-201) x T2”, indico alta valscion por el numero de germoglio cada (55%), interacción “R-92 x T2” indico alta valuación para la mediano numero de la germoglio cada explants (8.65). interacción “R-81.1 x T2” indica la alta valuación para Mediano germoglio cada explanta germoglio (23). Interacción “CMS 24 x T2” indica la alta valuación para la numeros de explantas rooting cada 100 explantas (35%). RESUME Une experimentación a été réalisée afin d étudier l’aptitude a la règèneration à partir de cotylédons de tournesol (Helianthus annuus L.). Letude porte sur trois lignées pures cytoplasme male stérile (CMS 24, CMS 31 y CMS 60/ 52), cinq lignées males (R-81.1, R-92, R-97, R-201 y R-232), et leurs 15 hybrides F1. Les cotylédons prélevés a partir de graines germées et cultivés sur le milieu MS contenant 3% de saccharose, 0.5% d agar-agar et les différents concentrations de BAP et de NAA. Les résultats d analyse de la variance montrent une différence significative parmi les génotypes, les différentes concentrations de BAP et de NAA et leurs interactions, pour tous les caractères étudies. L’utilisation de 1 mg l-1 de BAP et 1 mg 1-1 de NAA a permis une augmentation des pousses formées sur les cotylédons. L’interaction “(CMS24 x R-201) x T2” a présente le taux maximum (55%) pour le nombre de cotylédons donnant au moins une pouse/100 cotylédons cultivés. L’interaction “(R-92 x T2)” a présente le meilleur résultat (8.65) pour le nombre moyen de pousses formées. Pour le nombre moyen de pousse formées, cotylédons donnât de pousse, la plus important est obtenue par l’interaction “R-81 x T2” (23). En ce qui concerne le nombre cotylédons enracinées pour 100 cotylédons cultivés la valeur la plus fort (35%) a été obtenue par l’interaction “CMS24 x T2”.

A simple protocol for somatic embryogenesis in Rosa hybrida L. cv. Apollo

Summary A successful regeneration system from in vitro-derived leaves of Rosa hybrida cv. Apollo, via somatic embryogenesis, has been established. The influence of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and zeatin on the induction of embryogenic calli was evaluated. The highest percentage of embryogenic callus induction (48.3%) was obtained on full-strength MS medium containing 1.0 mg l–1 2,4-D, while at higher concentrations (4.0 or 5.0 mg l–1 2,4-D) no embryogenic calli were observed. The induction of embryogenic calli also occurred on MS media containing 0.5, 1.0, 2.0, or 4.0 mg l–1 naphthaleneacetic acid (NAA). The lowest concentration of NAA (0.5 mg l–1) showed the highest rate of production of embryogenic calli. For maturation, embryogenic calli were transferred onto MS media containing different concentrations of 6-benzyladenine (BA), thidiazuron (TDZ), or abscisic acid (ABA) alone, or in various combinations. The highest rate of embryo maturation (85.0%) was observed on MS medium supplemented with 1.0 mg l–1 ABA alone. Mature embryos showed the highest rate of germination on full-strength MS medium containing 2.0 mg l–1 BA alone. After rooting, well-developed plantlets were maintained under greenhouse conditions. The morphological characteristics and flower colours of somatic embryo-derived plants were the same as the donor parent.

Elimination of macro elements from inoculation and co-cultivation media enhances the efficiency of Agrobacterium-mediated transformation in Petunia

In order to evaluate the effect of inoculation and co-cultivation media elements on transformation frequency in Petunia hybrida, modified MS media with different elements were tested on Alvan and Large Flower Alvan (LF Alvan), two local cultivars. Leaf explants of both cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 (pBI121) containing neomycin phosphotransferase (nptII) and an intron-containing β-glucuronidase (gus) genes. When medium lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used as inoculation and co-cultivation medium, a higher frequency of transformation for Alvan (22%) and LF Alvan (16%) was obtained. Kanamycin resistant plantlets were stained blue by GUS assay. Furthermore, polymerase chain reaction (PCR) analysis revealed the presence of both gus and nptII genes in all putative transformants. Finally, southern blot hybridization confirmed insertion of 1–4 copies of gus gene in transgenic plants.

Somatic Embryogenesis in Rosa hybrida

Roses are among the most economically important cut flowers throughout the world. Improving a number of rose traits, including resistance to pests and diseases, flower color and form, vase life and fragrance are some of the major goals of rose breeding programs (Bao et al. in Plant cell, tissue and organ culture 109:411–418, 2012; Ludwig et al. in III International symposium on rose research and cultivation, pp 341–347, 2000; Zakizadeh et al. in European J Hortic Sci 73:111–117, 2008).

Silver nitrate and adenine sulphate induced high regeneration frequency in the recalcitrant plant Cosmos bipinnatus using cotyledon explants

ABSTRACT Plant regeneration ability was studied in the medicinal-ornamental plant, Cosmos bipinnatus ‘Sonata white’, which is a dicotyledonous recalcitrant plant to shoot induction. Cotyledons were used as sources of explants to investigate plant regeneration. High frequency of direct shoot induction was obtained when BA (5 mg/l) and AgNO3 (5 mg/l) were used in combination with 20 mg/l adenine sulphate (73.8%) in Murashige and Skoog (MS) medium. The highest shoot number per explant (5.7) was induced on MS medium supplemented with 5 mg/l BA, 5 mg/l AgNO3, and 40 mg/l adenine. Eight week-old shoots were transferred to root induction media containing MS and half-strength MS medium with different concentration of IBA. The highest rate of root induction (70.8%) was obtained on half-strength MS medium with 1.5 mg/l IBA within four weeks. The plantlets were transferred to pot and kept in the greenhouse condition. Seventy percent of the plantlets successfully acclimatised. Abbreviations: BA, 6-benzylaminopurine; IBA, Indole-3-butyric acid; MS, Murashige and Skoog; PGRs, plant growth regulators.