WPM Medium Research Articles

In vitro adventitious shoot regeneration from leaf explants of some apricot cultivars

ABSTRACT Apricot is one of the most valuable commercial fruits. In vitro propagation of apricot is very important for rapid multiplication of cultivars with desirable traits and production of cleaning up and virus-free plants. Low frequency of multiplication is the main limiting factor for traditional propagation methods. In this regard, the objective of our investigation was to study the morphogenetic capacity of apricot leaf explants of the promising cultivars ‘Iskorka Tavridy’, ‘Magister’ and ‘Bergeron’ for regeneration system development and solving some breeding questions. The source of explants was in vitro plants regenerated and cultured on QL medium. Leaves were maintained in the dark at 24±1 °C in thermostat for three-four weeks. Morphogenic callus and structures were mainly formed at the central and proximal parts of leaves on MS, QL and WPM media with 1.5 or 2.0 mg L-1 BAP and 1.5 or 2.0 mg L-1 IAA in different combinations, or TDZ (0.6 and 1.3 mg L-1). Callus with adventive buds was transferred to regeneration medium and placed into a growth chamber at 24±1 °C and 16-hour photoperiod with a light intensity of 37.5 μmol m-2 s-1. The best results were obtained when adaxial leaf surface was in contact with the culture medium. Frequency of leaf callus formation on MS medium with 1.5 mg L-1 BAP and 1.5 mg L-1 IAA was higher in the explants of ‘Iskorka Tavridy’ (80.0%) than in - ‘Bergeron’ (50.0%) and ‘Magister’ (36.7%). The best results of callogenesis for ‘Magister’ was obtained on MS medium with 1.3 mg L-1 TDZ (53.3%). Active microshoot regeneration in ‘Iskorka Tavridy’ cultivar was shown on MS medium with BAP and IAA and in ‘Magister’ cultivar - on MS medium with TDZ. Rhizogenesis was obtained on half strength MS medium with 2.0 mg L-1 IBA.

Studies on micropropagation of Rosa setigera michx. through multiple shoot induction

The present paper describes the studies carried out to determine the effect of nutrition on micropropagation of Rosa setigera. The nodal explants harvested from 4th to 8th node of juvenile shoots were inoculated over the media composed of salts and vitamins of MS, WPM, SH and B5 medium. These media contained 2–4% of sucrose or glucose or maltose as the carbon source. The explants harvested in rainy season were inoculated over the medium fortified with 2.2μM BAP, while those harvested in winter season were inoculated over the medium containing 0.5μM NAA and 8.8μM BAP. It was observed that the regeneration efficiency of explants harvested in rainy season was best with SH salts and vitamins, 3% glucose and 2.2μM BAP. Similarly, the explants harvested in winter season displayed their regeneration potential to maximum over the medium composed of WPM salts and vitamins, 3% glucose, 0.5μM NAA and 8.8μM BAP. The maximum elongation of microshoots was obtained with the medium containing 0.6% agar and rooting over the ½ strength MS medium containing 9.8μM IBA.

IN VITRO CALLUS INDUCTION OF ‘SHIRAZ’ GRAPE (Vitis vinifera L.) USING DIFFERENT MEDIUM AND GROWTH REGULATOR COMBINATION

One of grape cultivars widely used as raw material for wine production is ‘Shiraz’ cultivar. Propagation of this cultivar is necessary for the provision of grape as a wine making material in Bali. In vitro culture is an alternative propagation technique than can be employed to produce planting materials in a shorter time. This research aims to determine the most suitable medium and growth regulator combination in inducing grape cv. ‘Shiraz’ callus in vitro. The study was conducted from November 2017 until February 2018 at Plant Tissue Culture Laboratory, Biology Study Program, Udayana University. The explants used were young stem of grape cv. ‘Shiraz’ and the experiment was conducted using factorial design with two factors. The first factor was basal medium used (MS and WPM) and the second factor was IBA concentration (0; 0.5 and 1 mgL-1) and BAP (0, 1 and 2 mgL-1). The results showed that the highest percentage of callus induction (60%) was obtained on WPM medium without growth regulator combination (control). However, the fastest time of callus appear was on MS medium + 2 mgL-1 BAP without IBA, which was 17 days after planting. The texture and color of callus resulted on this research were friable with white, greenish white, greenish yellow and green in color.

Rooting of <i>Vaccinium corymbosum</i> L. microshoots cv. «Blue-Berry» in culture <i>in vitro</i> and <i>ex vitro</i>

This paper discusses a method of micropropagation of Vaccinium corymbosum L. cv. Blue-Berry. The results showed that WPM supplemented with 1,0 mg/l of zeatin in combination with 0,1 mg/l of indolyl-3-butyric acid was more effective for the multiplication of blueberry axillary shoots. The maximum increase in the number of healthy axillary shoots was observed in the fourth subculture, whereas the phenomenon of hyperhydration (vitrification) began to appear in the fifth subculture. In addition, it was established that the presence of indolyl-3-butyric acid and 1,0 g/l of activated charcoal in the nutrient medium lead to the development of good root system of the Vaccinium corymbosum cv. Blue-Berry. The highest mean number of roots formed per explant was obtained on WPM medium, supplemented with 0,5 mg/l indolyl-3-butyric acid after 10 weeks. Acclimatization of in vitro regenerated plantlets of Vaccinium corymbosum with a developed root system in ex-vitro conditions (pH 3,54) showed a 100% survival rate.

Induction of callus and adventitious shoots on epicotyl and hypocotyl segments of cumaru (Dipteryx odorata)

Somatic embryogenesis from callus induced in epicotyl and hypocotyl segments can be viable native species in order to better -benefit ratio costs, and rates of clonal multiplication. In this sense, two trials were established to induce callus and adventitious buds on hypocotyl and epicotyl segments of cumaru bean seedlings germinated in vitro in different concentrations and combinations of growth regulators. At first, we used the MS medium supplementwith ANA (0.0, 1.5 mg.L-1) and TDZ (0.0, 4.0 and 8.0 mg.L-1) distributed in factorial 2 x 3 x 2 (x auxin cytokinin x explant) with eight replications. In the second, it was used the WPM medium supplemented with BAP (2.0 mg L-1) and plus 2,4-D (2.0 and 4.0 mg L-1) in a factorial 2 x 2 (auxin x explant) with 15 repetitions each. They were evaluating callus formation and the average number of adventitious shoots during the period of 90 days. The results indicated that the highest average for callus formation was observed when the explants were subjected to concentrations of 8.0 mg L-1 TDZ combined with 1.5 mg L-1 ANA in MS medium. For the formation of buds, the WPM medium plus 2.0 mg L-1 2,4-D in the second experiment, induced higher number of shoots, being significant the use of auxin, and its interaction with the type of explant.

In vitro establishment and anatomical analysis of Bauhinia holophylla (Bong.)

Bauhinia holophylla (Bong.) Steud. is an endemic legume of the Brazilian Savanna that has economic and medicinal importance. The natural and conventional propagation occurs through seeds, whose production occurs during a few months per year. Currently, the Brazilian Savanna agricultural expansion aggravates the propagation and conservation of B. holophylla species. Considering the few studies about the environmental factors effects on germination and growth of B. holophylla, micropropagation is a promising alternative for seedlings production and species conservation. The aim of this work was to establish a protocol for in vitro seed germination, shoots induction and elongation of B. holophylla explants. To obtain in vitro seedlings, seeds were germinated on culture medium WPM 50 and 100%, MS 50 and 100% of salts concentration or water + agar. After 30 days, nodal segments of B. holophylla from seedlings were isolated and inoculated into WPM culture medium supplemented with the 6-benzylaminopurine (BAP) at concentrations of 0, 0.48, 8, 16 μM. After induction of shoots, the explants were inoculated in WPM culture medium containing different concentrations of gibberellic acid (GA3): 0, 5, 10 and 20 μM. In the experiments the following parameters were evaluated: germination percentage, oxidation, stem length, leaf number and number of nodes. At the end of the multiplication experiment, leaves were collected for histological analysis. For in vitro seeds germination, the water + agar substrate showed higher percentages of germination (91%), not differing from the culture media MS 50% and WPM 100%. However, for growth parameters, the best culture medium was WPM 100%. For nodal segments multiplication, there was a significant increase on shoot number related with BAP concentration. For shoots elongation, there was reduction on stem size with the increase of GA3 concentration. In the anatomical analyses, the development of palisade and spongy parenchyma and vascular bundles was observed in B. holophylla in vitro leaves.

Sterilization and Callus Formation of Rubber Meristem (Hevea brasiliensis Muell. Arg.)

Rubber plant (Hevea brasiliensis Muell Arg) is one of the important plantation commodities in Indonesia because of its role as a source of income. It stimulates the economic growth around the rubber plantation area. The propagation of rubber is still using conventional methods like grafting. The technique of tissue culture through callus induction is one of the alternatives of mass propagation of rubber seedling with quick and efficient time. The sterilization method is very important to determine the success of tissue culture techniques. Therefore, the aim of this research is to know the best method of sterilization and callus formation in rubber explants. The basic media used were WPM and MS, with BAP of 2 ppm and NAA 0.1 ppm. The best result of sterilization is by soaking 5% fungicide solution for 5 minutes, 5% Clorox solution for 15 minutes, betadin 10% solution for 5 minutes, and finally it rinsed with sterile water three times. The best medium uses WPM medium for callus induction, with 0.5 cm callus length and embryonic callus. In contrast, the MS medium has 0.4 cm callus length and non embryonic callus.

In vitro establishment of shoot meristems of Ilex paraguariensis and identification of endophytic bacteria

Yerba mate (Ilex paraguariensis A. St.-Hil.) is a species of great economic, social and environmental importance for the southern regions of Brazil, Uruguay and Argentina. Currently the most diverse products are obtained from mate leaves, including mate tea. The objective of this study was to establish shoot meristem cultures (meristematic dome and a few primordia) of elite clones and identify the endophytic bacteria present in the explants. We tested the effect of clones (F1, F2, A03 and A07), culture media (MS, 1/2MS, 1/4MS and WPM), cytokinins (kinetin, BA and 2iP), activated charcoal (1, 2 and 3 g L−1), and disinfecting agent (sodium hypochlorite and mercuric chloride) on in vitro establishment. F1 and F2 clones were the most responsive for shoot meristem in vitro culture. WPM medium supplemented with 8.8 µM 2iP, 0.2 µM NAA and 3 g L−1 activated charcoal was the most suitable for the in vitro establishment of the F1 clone. No phytotoxic effect of the disinfectant was observed and some meristems sprouted. The isolated endophytic bacterium was identified for the first time in yerba mate as Agrobacterium larrymoorei. To conclude, we were able to establish in vitro culture of yerba mate using meristems as explants but the tissues were not free of endophytic microorganisms which could interfere with explant response.

Establishment of optimal conditions for micropropagation by node culture and multiple shoots formation from sucker explants of thornless Blackberry (Rubus fruticosus L. cv. BB21)

This study was conducted to develop a simple, rapid, and reliable method for in vitro propagation of disease-free and true-to-type clones from sucker explants of thornless blackberry (Rubus fruticosus L. × R. parvifolius L.). To induce multiple shoots, the sucker explants were sterilized in 1% NaOCl solution, and then were aseptically cultured on the full and 1/2 MS solid medium supplemented with BAP (0.1, 0.5, 1.0, 2.0 mg/L). After six weeks of culture, the highest frequency (85.4%) of shoot formation from sucker explants was obtained on the full-strength MS medium with 1.0 mg/L BAP. Node explants obtained from multiple shoots were cultured on the various media of full- or half-strength of AD, B5, MS, SH, QL, WPM media, respectively. After 30 days of culture, plant growth was good on the half-AD, half-QL medium. After 90 days of culture, plant growth was good on the full MS and full SH medium. The survival rate of the plantlets after transfer to plastic pots containing soil mixture (sand: soil: vermiculite was 1:1:1, vol.) in the greenhouse was 98%. The results indicate that a multiple-shoot procedure can be applied for an efficient mass propagation of Rubus fruticosus L. × R. parvifolius L.

Callus Induction and Plant Regeneration from Leaf Segments of Unique Tropical Woody Plant Parasponia andersonii Planch

The purpose of the present study was to develop effective methods for callus induction, shoot regeneration, and rooting for Parasponia andersonii. Leaf explants of P. andersonii were placed on Lloyd and McCown’s (WPM) medium supplemented with various concentrations of TDZ and NAA for callus induction. Callus induction was observed on media containing 0.1 - 0.2 mg/l TDZ with 0.05 mg/l NAA. Maximum shoot regeneration was observed when the calluses were cultured on MS supplemented with TDZ and IBA. Shoots cultured on WPM medium supplemented with 0.5 mg/l IBA had the maximum rooting percentage (100) in 3 weeks. Rooted plants were transplanted to a potting mixture containing vermiculite (50%) and peat (50%) (v/v). After 2 months, more than 20% of plants survived and were transferred to the greenhouse. Thus, a new effective method has been developed for P. andersonii micropropagation that can be used in studies of plant-Rhizobium symbiosis and for the generation of transgenic Parasponia plants.Plant Tissue Cult. & Biotech. 28(1): 45-55, 2018 (June)

Olive tree in vitro establishment under different culture media and explant collection periods

In the species Olea europaea L. the tissue culture techniques have not been sufficiently studied by the lack of efficient establishment of protocols for various cultivars. The objective of this work was to evaluate the effect of different culture media and sampling times of explants on in vitro establishment of six cultivars of olive (Ascolano 315, Leccino, Maria da Fé, Coratina, Arbequina and Frantoio). The work was divided into two experiments conducted in the Fruit Tree Propagation Laboratory, Crop Science Department, Faculty of Agronomy Eliseu Maciel, Federal University of Pelotas, Brazil. Stock plants of the olive cultivars maintained in semi- hydroponic system were used. In the first experiment, nodal segments were used to establish in vitro culture medium MO and WPM, consisting of salts and vitamins. In the second experiment four seasons to obtain the explants were evaluate (autumn, winter, spring and summer). The WPM medium promoted greater establishment of olive explants. There is a higher phenolic oxidation in olive explants collected in winter. Spring is indicated for collecting explants, therefore favors the in vitro cultivar Maria da Fé, while Ascolano 315 and Arbequina cultivars have a higher rate of in vitro establishment by collecting explants over the year

PRODUTIVIDADE DE MICROCEPAS DE GRÁPIA (<i>Apuleia leiocarpa</i>) MANTIDAS<i> IN VITRO</i>

O objetivo deste trabalho foi avaliar a produtividade de microcepas de grápia (Apuleia leiocarpa) mantidas em diferentes condições de cultivo in vitro, visando maximizar a taxa de multiplicação. Microcepas oriundas da poda drástica de plantas assépticas foram cultivadas em meio de cultura WPM acrescida de 0; 2,2; 4,4; 6,6 e 8,8 µM de 6-benzilaminopurina (BAP) e subcultivadas em meio de cultura WPM sem citocinina. Microcepas de grápia também foram cultivadas em meio de cultura WPM, MS ou ½ MS, acrescido ou não de 1,5 g L-1 de carvão ativado e submetidas a três coletas de brotos aos 30, 60 e 90 dias de cultivo. A manutenção das microcepas em meio de cultura WPM ou ½ MS suplementado com 8,8 µM de BAP, seguido do subcultivo em meio de cultura sem citocinina aumenta o número e o crescimento de brotações. O cultivo de microcepas de grápia em meio de cultura com carvão ativado aumenta a produção de microestacas por microcepas, enquanto a ausência de carvão ativado favorece a formação de calos e brotos por organogênese indireta. A manutenção in vitro de microcepas de grápia maximiza a taxa de multiplicação, constituindo-se de uma técnica promissora para a produção de plantas de grápia.

Efficient Technique for Long-Term <i>in Vitro</i> Storage of Transgenic Aspen Genotypes

In vitro culture of isolated cells from tissues and organs is sometimes used to preserve and reproduce unique genotypes of woody plants. The technique, however, requires regular subculturing which raises storage costs and creates risks for contamination and accumulation of somaclonal variations. We examined the effects of sugar composition of culture medium, the length of photoperiod, light intensity, and ambient temperature on the survival of plant material in vitro. The study was performed on 49 genotypes of Populus tremula (46 transgenic genotypes carrying GFP-, Xeg- and Gus-genes, and 3 control (wild-type) genotypes). It was shown that effective storage of plants was achieved through optimization of the combined effects of all storage parameters under study. Based on the experimental data, we developed a protocol for long-term in vitro storage of desirable genotypes without subculture and with a survival rate of up to 98%. The best results were obtained when the plant material was pre-cultured on a WPM medium containing 15 g/L sucrose, 7.5 g/L sorbitol and 7.5 g/L mannitol, and then stored at +4°C under a 24-hour light day cycle with only 8 hours of light per day and maximum light intensity of 2000 lux. Post-storage recovery was done by culturing on a medium containing 1 mg/L gibberellic acid. The developed method can be used for effective in vitro storage of the studied genotypes for up to 24 months without subculture.

In vitro propagation of avocado (Persea americana Mill. cv. Hass) through morphogenesis

Teniendo en cuenta la importancia y el potencial de desarrollo del cultivo de aguacate (Persea americana Mill.) en Colombia, y los problemas que se presentan para el cultivo eficiente en campo, principalmente por la variabilidad genética del material de propagación y por condiciones fitosanitarias adversas, el presente trabajo tuvo como finalidad desarrollar una metodología para la propagación clonal in vitro del cultivar “Hass”. La introducción in vitro de explantes apicales y segmentos nodales se realizó a partir de plantas madres injertadas. El efecto del pretratamiento de etiolación sobre el número de brotes desarrollados y el porcentaje de fenolización in vitro, se evaluó sometiendo las plantas madre y los explantes introducidos in vitro a condiciones de fotoperiodo (12/12h) y oscuridad; esta última condición es la que redujo significativamente la fenolización de los explantes, y estos a su vez formaron un mayor número de brotes nuevos con mayor altura y en menor tiempo. En la etapa de multiplicación en el medio de cultivo WPM se obtuvo un efectivo control del proceso de fenolización. Durante los subcultivos hubo disminución de elongación de los explantes apicales, y a partir de los segmentos nodales, se logró una inducción importante de nuevos brotes; sin embargo, solo algunos alcanzaron la altura requerida para mantener la tasa de multiplicación a través de los subcultivos. Finalmente durante el endurecimiento, se obtuvo un 82 % de supervivencia de las vitroplantas.

Effect of growth factors on the production of biomass of the hairy root of Urena lobata

Urena lobata, one of the traditional medicine, is used to treat inflammation, arthritis or improve lipid profiles in liver and blood. There also has been a lot of studies in the world about its potential biological activities. Interestingly, root extract usually has higher biological activities than the whole plant or other parts. Therefore, applying several modern biotechnology techniques could help to initially obtain this potential resource. In this research, we demonstrated the application of some growth factors which can increase the biomass of hairy root such as: inoculum density, light condition, medium and proportion of marco-nutrient. The results showed that when hairy roots were cultured at the inoculum density of 10 g/L FW, under illumination at 3000 lux, WPM medium supplemented 4 % sucrose were more suitable for the growth of hairy root. At the same time, when all these factors were put fogther the haing root grouthr was improved. The fresh weight (FW) and dry weight (DW) increased 2.01-fold and 1.94-fold, respectively.

Micropropagation of mature Quercus ilex L. trees by axillary budding

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 to 100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044 µM BA plus 0.46 µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L−1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L−1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8 µM or 24.6 µM indole-3-butyric acid and 0.54 µM α-naphthalene acetic acid.

Inisiasi Tunas Kokoleceran (Vatica bantamensis) pada Berbagai Jenis Media Tanam dan Konsentrasi BAP (Benzyl Amino Purine) Secara In Vitro

Kokoleceran (Vatica bantamensis) is an endemic plant of Banten which is only in Ujung Kulon, and has been designated as identity of Banten province. Now the existence of Kokoleceran has been endangered. Based on data from the IUCN, since 1998 there has been no research. Kokoleceran initiation needs technology for preventing from the extinction. One of the mass propagation is through the technique culture in vitro. This research aimed to get the precise medium and BAP concentration for Kokoleceran, and knowing the response of Kokoleceran growth in in vitro. This research was done in November 2015 until March 2016 at the Laboratory of Biotechnology, Department of Agroecotechnology, Faculty of Agriculture, University of Sultan Ageng Tirtayasa, Serang, Banten. This research used Completely Randomized Design (CRD) two factors. The first factor was medium that were MS and WPM media. The second factor was BAP concentration which consisted of four levels namely 0 mg L-1, 1 mg L-1, 2 mg L-1, and 3 mg L-1. The results showed that the use of different media and BAP concentrations had no impact on the time appear of shoot, shoot number, and root number. There was effect from both treatments on medium color, and growing of callus on the explants.

Endemik Astragalus trojanus Stev’in Nodal Kültür ile Mikroçoğaltımı

The clonal propagation with nodal cultures of the medicinal endemic plant species Astragalus trojanus Stev. was aimed in this study. Seeds were germinated in Murashige and Skoog (MS) medium, and the nodal segments obtained from seedlings were used as an explant. Firstly, the effects of MS and (Woody Plant Medium) WPM nutrient media on shoot multiplication were investigated. The explants were cultured in MS or WPM nutrient media containing at different concentrations (0.2, 0.5, 1.0, 2.0 mg L -1 ) 6-benzyl amino purine (BAP). After 4 weeks from culture, the highest number of shoots (2.80 number/explant) was obtained in WPM medium containing 0.5 mg L -1 BAP. Nodal segments were cultured in WPM medium containing different concentrations of BAP, thidiazuron (TDZ), kinetin (KIN) (0.2, 0.5, 1.0, 2.0 mg L -1 ) to determine the effect of cytokinin source and concentrations on shoot multiplication. At the end of 4 weeks, it was determined that TDZ and KIN had similar effect on number of shoots. The highest number of shoots (2.09 number/explant) was obtained from WPM media containing BAP. Regenerated shoots were transferred to WPM media containing 1 or 3 mg L -1 indole-3-butyric acid (IBA), naphtalene acetic acid (NAA) or indole-3-acetic acid (IAA) for rooting. After 4 weeks from culture, the most root number (4.33 number/explant) were obtained from the WPM medium containing 3 mg L -1 IAA, while the highest rooting percentage was obtained from WPM medium containing 3 mg L -1 IBA with 80%. Rooted seedlings were transferred to plastic cups containing peat and perlite in a ratio of 1:1 and successfully acclimatized. Plant culture was completed at 15 weeks. The effective clonal propagation method was described for the endemic species Astragalus trojanus Stev.

In Vitro Biomass Production of Astragalus gilvus

Biomass production of medicinal plants is crucial to take advantage of their biological activities. In vitro biomass production is a beneficial, controllable and ecofriendly way of this process. In this study, an efficient protocol for biomass production of in vitro grown Astragalus gilvus Boiss shoots was specified. Shoots were exposed to different concentrations of 6-benzyladenine, indole-3-butyric acid, indole-3-acetic acid and doubled concentrations of vitamins (nicotinic acid, pyridoxine and thiamine). To identify the effects of growth regulators, growth parameters such as shoot elongation, shoot formation per explant, single and multiple shoot formation percentages were examined and biomass accumulations such as fresh (FW) and dry (DW) weights and relative dry weight percentages were also specified. Maximum biomass production rates (FW = 0.46 g/month/explant and DW = 0.031 g/month/explant) were obtained in WPM medium supplemented with doubled vitamin concentration and 1 mg/L BA amongst the media used. In this medium, a total of 168 shoots obtained and this was the 19.33% of all shoots regenerated. In respect to data obtained, a three-step process is offered for high quality biomass production.

Transformation of pomegranate (Punica granatum l.) a difficult-to-transform tree

Abstract In this study, an Agrobacterium-mediated transformation method was developed for pomegranate, a difficult-to-transform plant. We first optimized callus induction and shoot regeneration efficiency. Induction of calluses was best achieved from the intermodal stem sections on WPM medium supplemented with 12 μM BA and 8 μM NAA. The highest frequency of shoot regeneration (69.33%) and number of shoots (7.16) per piece of callus were obtained when calluses were incubated on WPM medium supplemented with 12 μM BA and 2 μM NAA. Callus pieces were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBin19 carrying the neomycin phosphotransferase (nptII) gene as a selectable marker and green fluorescent protein (GFP) gene as a reporter. After 4 weeks in WPM selection medium (the best callus induction medium supplemented with 50 mg L−1 kanamycin), putative transgenic calluses were obtained. From such calluses transformed shoots began to appear approximately 3 week after the kanamycin resistance transgenic callus pieces had been placed on the best shoot regeneration medium containing 50 mg L−1 kanamycin. GFP fluorescence was observed in putatively transformed calluses and shoots. Molecular analysis confirmed the integration of the nptII transgene in transformed shoots. The transgenic shoots were rooted and successfully acclimatized.