Search

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

Insect-resistant transgenic cotton has been commercialized for two decades. Most of the introduced cultivars express Bt gene(s) constitutively under the control of 35S promoter in whole-plant tissues. However, there have been other promoters considered by researchers to confine the toxin expression to targeted organ and tissues. We developed a triple-gene construct including GNA, cry1Ac and cp4 epsps genes. We attempted to confine cry1Ac expression to insect biting sites by cloning it to downstream of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1). Moreover, to broaden the range of resistance, GNA was driven by the 35S promoter to target the sap-sucking insects like aphids which impose large losses in cotton production. To select the transformants in selection medium and for glyphosate tolerance, GNA and cry1Ac genes were accompanied with cp4 epsps gene. Two binary vectors harboring desired genes were constructed and utilized in the study (pGTGNAoC1AC and pGTGN35C1AC). Transformation of cultivar GSN-12 was carried out by employing Agrobacterium tumefaciens strain EHA105. Plantlets were primarily screened under glyphosate (N-phosphonomethyl glycine) selection pressure and subsequently subjected to molecular and biotoxicity assays. Introduction of cry1Ac and GNA to cotton plant conferred resistance to Spodoptera littoralis and Aphis gossypii Glover. Restriction of cry1Ac toxin protein to insect biting sites along with a plant lectin attributes significantly to insect pest management strategies. © 2018, Korean Society for Plant Biotechnology and Springer Japan KK, part of Springer Nature., Acknowledgements The PhD. fellowship awarded by The Scientific and Technological Research Council of Turkey (TUBITAK)-BIDEB to Dr. S.D. Khabbazi is deeply appreciated. The authors are grateful to the Leicester University (UK) for giving permission to use AoPR1 promoter for research purposes, Dr. Selma Onarıcı (TÜBİTAK GMBE) for providing pJIT61.cry1Ac plasmid and Prof. Umut Toprak (Department of Crop Protection, Ankara University) for providing S. littoralis larvae.

The expression of insecticidal genes must be induced at appropriate time and in sufficient amount to confer protection against targeted pests. However, the increased scientific reports of resistance development in insect pest against insecticidal delta-endotoxins, produced by Bacillus thuringiensis, provide impetus for the development of alternative insect management strategies. The present study was conducted to investigate the importance of targeted expression of a hybrid insecticidal gene (SN19) in potatoes. For this purpose, two plant expression vectors were constructed by cloning hybrid SN19 gene (cry1Ba-domain I–III and cry1Ia-domain II) under the control of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1) and CaMV 35S promoter, and were transferred to Agrobacterium tumefaciens strain EHA 105. Four potato genotypes (Marabel, Innovator, Tokat 10/1 and Tokat 6/24) were transformed with EHA 105 strain harboring pTF101.1 35S–SN19 and pTF101.1 AoPR1–SN19 constructs. Phosphinothricin (PPT) was used at concentration of 1 mg/l for selection of primary transformants. PCR results showed the presence of both introduced SN19 and bar genes in 43 plants out of total 154 putative transgenics. Expression of SN19 protein in primary transformants was confirmed by Western blot assays. The mechanical wounding of transgenic plants exhibited more accumulated levels of SN19 proteins during post wounding period. Leaf biotoxicity assays with Colorado potato beetle (Coleoptera) and tomato leafminer (Lepidoptera) exhibited 100% mortality of the pests in primary transformants. Based on our mortality results with both constructs, we concluded that the potato transgenic lines exhibited targeted expression of insecticidal gene under the control of AoPR1 promoter upon insect wounding with eliminated toxicity of Cry protein and hence can be further used effectively in potato breeding programme. © 2017, Korean Society for Plant Biotechnology and Springer Japan KK. 111T816 Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK Genetic Engineering and Biotechnology Institute, Scientific and Technological Research Council of Turkey, Gebze, Kocaeli, Turkey Funding The research project was supported by a Grant from Scientific and Technological Research Council of Turkey (TÜBİTAK) Project # 111T816.

WOS:000324281500012 Plant genetic engineering has opened new avenues to modify crops and has provided a powerful tool for crop improvement. The present study reports the development of regeneration and genetic transformation protocol for the Turkish cowpea cultivar Akkiz. The immature cotyledons were cultured on Murashige and Skoog (MS) medium containing 0.25-0.75 mg L-1 6-benzylaminopurine with or without 0.25 mg L-1 alpha-naphthalene acetic acid. Shoot regeneration varied 44.4%-83.3% with 2.1-5.0 shoots per explant. Regenerated shoots were rooted in MS medium containing 0.50 mg L-1 indole-3-butyric acid and acclimatized in the greenhouse, where they flowered and set seeds. Immature cotyledons were transformed with Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pRGG containing an herbicide tolerance gene (bar) along with a uida (GUS) gene under 35S promoter. Phosphinothricin was used as a selectable marker at a concentration of 2.5 mg.L-1. Putative transformants were screened by-the histochemical GUS assay. Furthermore, molecular analysis revealed the presence of the introduced gene in the,genome of cowpea cultivar Akkiz. The selected transgenic plants showed a resistance to Basta (R) nonselective herbicide at up to 10 mL L-1 of water. Putative transgenic plants retained their pigmentation and continued to grow in the greenhouse.

Cowpea is an important grain legume crop. The study reports an efficient in vitro multiplication and shoots regeneration protocol from preconditioned embryonic axes of the Turkish cowpea cultivar Akkiz. The embryonic axes were preconditioned with 10 mg/l BA on agar solidified MS medium for 5 days. Thereafter they were cultured on MS medium containing 0.25, 0.50, 0.75 and 1.00 mg/l BA with or without 0.10 mg/l NAA. Mean frequency (%) of shoot regeneration, number of shoots per explant and shoot length decreased with each increase in BA concentration used singly. However, a positive increase was recorded in all parameters in the presence of 0.10 mg/l NAA in the regeneration medium. A maximum mean number of 10.33 shoots per explant was recorded on an MS medium containing 1.00 mg/l BA -0.1 mg/l NAA. Regenerated shoots were rooted on an MS medium containing 0.50 mg/l IBA. Rooted plants were acclimatized at room temperature in soil mix contained in pots where they were subjected to an intermittent mistwater spray for 24 h that maintained 90% relative humidity during the first few days which was gradually reduced to 40% for 10 days. All plants flowered and set seeds in a greenhouse after 3 months.

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.

WOS: 000389771500008, In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T-0 progeny were further raised under greenhouse conditions to obtain progeny (T-1). The introduced gene was properly inherited and expressed in T-1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized., TUBITAK (The Scientific and Technological Research Council of Turkey) [2216], The corresponding author is grateful to TUBITAK (The Scientific and Technological Research Council of Turkey) for providing postdoctoral fellowship Code: 2216) to carry out the research activities at University of Ankara. The authors acknowledge Dr. Umut Toprak, Associate Professor, Department of Plant Protection, Faculty of Agriculture, University of Ankara for providing Helicoverpa armigera for our lab bioassays experiment

WOS: 000373886900005, Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept 'less intrusive' expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L-1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T (1) transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns., Scientific and Technological Research Council of Turkey TUBITAK [111O254], The work on development of transgenic cotton in our laboratory is being supported by grants from Scientific and Technological Research Council of Turkey TUBITAK (Project No. 111O254). The authors acknowledge contribution and support of TUBITAK. The authors are also thankful to Leicester University (UK) for giving permission to use AoPR1 promoter for research purposes.

Cotton (Gossypium hirsutum L.) is the most significant cash crop and backbone of global textile industry. The importance of cotton can hardly be over emphasized in the economy of cotton-growing countries as cotton and cotton products contribute significantly to the foreign exchange earnings. Cotton breeders have continuously sought to improve cotton’s quality through conventional breeding in the past centuries; however, due to limited availability of germplasm with resistant to particular insects, pests and diseases, further advancements in cotton breeding have been challenging. The progress in transformation systems in cotton paved the way for the genetic improvement by enabling the researchers to transfer specific genes among the species and to incorporate them in cotton genome. With the development of first genetically engineered cotton plant in 1987, several characteristics such as biotic (insects, viruses, bacteria and fungi) resistance, abiotic (drought, chilling, heat, salt), herbicide tolerance, manipulation of oil and fiber traits have been reported to date. Genetic engineering has emerged as a necessary tool in cotton breeding programs, strengthening classical strategies to improve yield and yield contributing factors. The current review highlights the advances and endeavors in cotton genetic engineering achieved by researchers worldwide utilizing modern biotechnological approaches. Future prospects of the transgenic cotton are also discussed.

Habitat destruction has resulted in the extinction of many plant species from the earth, and many more face extinction. Likely, the annual endemic Mediterranean knapweed (Centaurea tchihatcheffii) growing in the Golbasi district of Ankara, Turkey is facing extinction and needs urgent conservation. Plant tissue culture, a potentially useful technique for ex situ multiplication, was used for the restoration of this ill-fated plant through seed germination, micropropagation from stem nodes, and adventitious shoot regeneration from immature zygotic embryos. The seeds were highly dormant and very difficult to germinate. No results were obtained from the micropropagation of stem nodes. However, immature zygotic embryos showed the highest adventitious shoot regeneration on Murashige and Skoog (MS) medium, containing 1 mg l(-1) kinetin and 0.25 mg l(-1) NAA. Regenerated shoots were best rooted on MS medium containing 1 mg l(-1) IBA and transferred to the greenhouse for flowering and seed set. As such, the present work is the first record of in vitro propagation of critically endangered C. tchihatcheffii, using immature zygotic embryos, and is a step forward towards conservation of this indigenous species.

Tobacco leaf disc explants were inoculated with Agrobacterum tumefaciens strain GV2260 carrying p35S GUS-INT to determine the influence of different co-cultivation temperatures (18 – 26 °C), periods (24 – 96 h) and media (solid and liquid) on transformation efficiency. Kanamycin-resistant shoots developed on leaf discs inoculated with Agrobacterium after 4 weeks of culture initiation. Regenerated shoots were excised and rooted in the basal medium supplemented with 100 mg dm −3 kanamycin and 250 mg dm −3 augmentin. The rooted plantlets were finally transferred to compost and confirmed by GUS assay and PCR analysis. The highest transformation frequency was achieved from the explants co-cultivated with A. tumefaciens in liquid medium for 48 h at 22 or 24 °C.

WOS:000356358200017 Hemianthus callitrichoides 'Cuba' is an ornamental aquatic plant used in water gardens and phytoremediation. A protocol for its rapid and reproducible shoot organogenesis from apical shoot explants was developed. Optimum culture conditions for shoot proliferation were tested in MS media containing different concentrations of 6-(gamma,gamma-dimethylallylamino)purine (2iP), 6-benzylaminopurine (BAP), 1-phenyl-3-1,2,3-thiadiazol-5-yl urea (TDZ), and gibberellic acid (GA(3)). Plant growth regulators significantly (P

Hypocotyl and stem explants excised from in vitro and greenhouse-grown seedlings of Madaras, 1886 Sel. and Omega flax cultivars were compared in terms of percentage of explants producing shoots, shoot number per explant and total shoot number per petri dish. Hypocotyl explants resulted in better shoot regeneration. In vitro-grown seedlings were found to be more suitable than greenhouse-grown seedlings as an explant source.

Seven genotypes of winter durum wheat (Triticum durum Desf.) were cultured to establish an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of immature and mature embryo cultures. Immature embryos were aseptically dissected from seeds and placed, with the scutellum upwards, in dishes containing Murashige and Skoog's (MS) mineral salts and 2mg 2,4- dichlorophenoxyacetic acid (2,4-D) per litre. Calli and regenerated plants were maintained on 2,4-D-free medium. Mature embryos were moved slightly on the imbibed seeds. For callus formation, the seeds with moved embryos were placed, furrow downwards, in dishes containing 8 mg 2,4-D per litre. The developed calli and regenerated plants were maintained on the MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Variability was observed among the wheat genotypes tested for various culture responses in both explant cultures. Callus induction rate and regeneration capacity of callus were independent of each other. Mature embryos have a low frequency of callus induction but a high regeneration capacity. Considering availability, rapidity and reliability, this form of mature embryo culture can be used as an alternative method for immature embryo culture.

Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.

WOS:000303080400007 Vigna unguiculata is an important food legume crop in the semiarid tropics. It suffers from a host of agricultural constraints including damage due to diseases and pests. Judicious application of biotechnological methods can lead to considerable improvement in this important crop. Shoot regeneration from unsliced and longitudinally sliced cotyledon node explants obtained from 3-day-old to 5-day-old seedlings grown in vitro was achieved on Murashige and Skoog (MS) medium containing 1.11, 2.22, 3.33, and 4.44 ?M benzylaminopurine (BA) supplemented with 1.0 mg/L polyvinylpyrrolidone and 500 mg/L bacteriostatic Augmentin. Callus induction was recorded in all culture media on both explants. Th e shoot regeneration frequency (%) of longitudinally sliced cotyledon node explants was 2-fold to 3-fold higher than that of unsliced cotyledon node explants. A maximum number of 9.92 shoots per longitudinally sliced cotyledon node explant was recorded on MS medium containing 3.33 ?M BA. Each increase in the BA concentration of the culture medium proportionally decreased the mean shoot length on both explants. Maximum mean shoot lengths of 2.80 cm on unsliced cotyledon nodes and 3.04 cm on longitudinally sliced cotyledon node explant were recorded on MS medium containing 1.11 ?M BA. Regenerated shoots were rooted on MS rooting medium containing 2.45 ?M indole-3-butyric acid. In vitro regenerated plants were acclimatised at room temperature in growth rooms, where they produced viable seeds. Börülce yarı kurak tropik bölgelerin önemli yemeklik tane baklagil bitkisi olup, bazı zararlara maruz kalmaktadır. Bu bitkinin ıslahı için biyoteknolojik yöntemlerin geliştirilmesi son derecede önemlidir. Bu çalışmada, in vitro koşullarda 3-5 günlük çimlenmiş fi deciklerden elde edilen dilimlenmemiş ve dikey dilimlenmiş kotiledon boğum eksplantları 1,11, 2,22, 3,33 ve 4,44 μM benzylaminopurine (BA), 1,0 mg/L PVP ve 500 mg/L Augmentin içeren MS ortamda kültüre alınarak sürgün rejenerasyonu elde edilmiştir. Tüm ortamlarda her iki eksplant üzerinde de kallus oluşumu gözlenmiştir. Dikey dilimlenmiş kotiledon boğum eksplantlarında dilimlenmemiş kotiledon boğum eksplantlarına göre daha fazla sürgün oluşum görülmüştür. Dilimlenmiş kotiledon boğumunda, en fazla sürgün (9,92 adet) 3,33 μM BA içeren MS ortamından elde edilmiştir. Tüm kültür ortamlarında, BA dozların artışı oransal olarak sürgün uzunluğunun azalmasının sebebi olmuştur. En uzun sürgün oluşumu (2,80 cm ve 3,04 cm), dilimlenmiş ve dilimlenmemiş kotiledon boğumundan 1,11 μM BA içeren MS ortamında görülmüştür. Elde edilen sürgünler 2,45 μM IBA içeren MS ortamda köklendirilmiştir. In vitro koşullarda gelişen bitkilerin adaptasyonu oda sıcaklığında sağlanmış olup, tohum elde edilmiştir

The hairy vetch (Vicia villosa Roth) is a climbing, prostrate or trailing legume grown as forage.It fixes atmospheric nitrogen, reduces soil erosion and provides an instant mulch. Multiple axillary shoot regeneration from a mature seed explant (zygotic embryo with two cotyledons) was obtained on MS medium containing 0.05 - 1.6 mg/l TDZ with or without 0.10 mg/l IBA. The frequency (%) of shoot regeneration ranged from 45.83-75.00% with a maximum number of 28.6 shoots per explant on MS medium containing 0.20 mg/l TDZ-0.10 mg/l IBA. The mean shoot length decreased proportionately with each increase in TDZ concentration irrespective of the IBA concentration in the culture medium. However, comparing the two types of regeneration media, longer shoots were recorded in the presence of IBA in the culture medium. Regenerated shoots were pulse treated with 50 mg/l IBA for 5, 10 and 20 min for rooting.

There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.

Multiple shoot formation from the plumular apices excised from mature embryos of cowpea cv. Akkiz was obtained after pulse treatment with 10 mg/l BAP for 5 days followed by culture on MS medium containing 0.25, 0.50, 0.75 and 1.00, 1.25 mg/l BAP - with or without 0.10 mg/l NAA. Callus induction and shoot regeneration was recorded on all cultures containing BAP with or without NAA. However, inclusion of 0.1 mg/l NAA had positive effect on callus diameter and shoot length. Maximum mean number of 7.11 shoots per explant were obtained on MS medium containing 1.00 mg/l BAP. Longer shoots were recorded on MS medium containing various concentration of BAP + 0.1 mg/l NAA compared to those containing various concentrations of BAP singly. All shoots cultured on MS medium containing I mg/l BAP were rooted on MS medium containing 0.50 mg/l IBA. Rooted plants were acclimatized at room temprature in soil contained in pots. All plants flowered and set seeds in the greenhouse after 3 months. (C) 2009 Elsevier B.V. All rights reserved.

Transformation of fenugreek ( Trigonella foenumgraecum) was carried out with A281 oncogenic strain of Agrobacterium tumefaciens using root, cotyledon and hypocotyl explants excised from 1-week-old seedlings, which showed that the plant was highly susceptible to transformation. Tumors ( calli) were selected on 50 mg dm(-3) kanamycin. They were analyzed for beta-glucuronidase (GUS) expression. Presence of uidA (gus) gene, was confirmed by polymerase chain reaction (PCR) amplification.

A procedure has been developed which allows high frequency adventitious shoot regeneration from immature cotyledons of pea. Prolific shoot regeneration occurred following an initial callus growth on a Murashige and Skoog (MS) medium containing 0.5 mg/l 6-benzylaminopurine (BAP) and 4 mg/l α-naphthaleneacetic acid (NAA). Cotyledon expiants proximal to the embryonic axis had the highest regeneration potential, however, the presence of an embryonic axis inhibited adventitious shoot regeneration. Addition of silver nitrate (AgNO3) to the medium did not promote the number of regenerated shoots but resulted in shoots with well developed tendrils and large stipules which had a reduced rooting capacity. Regenerated shoots rooted readily (80-90%) in half strength MS medium containing 1 mg/l indole-butyric acid (IBA) and further established well in compost.