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Somatic embryogenesis in the endemic black iris
Abstract
Somatic embryogenesis was achieved from callus, cell suspension and protoplast culture systems in the endemic black iris (Iris nigricans). Subculture of friable callus fragments on embryogenesis induction medium (EIM) containing 4.5 μM 2,4-dichlorophenoxyacetic
acid (2,4-D), 0.5 μM kinetin, 4.5 μM 1-naphthaleneacetic acid (NAA) and 300 mg l-1 proline in the dark was necessary before transfer to regeneration medium (RM). Regeneration was studied by transferring friable
callus fragments from EIM to RM containing (0.0, 4.5, 9.0, 13.5 μM) of either 6-benzyladenine (BA), 2-isopentenyladenine (2iP),
zeatin or thidiazuron (TDZ) in combination with 0.49 μM indole-3-butyric acid (IBA), 0.45 μM 2,4-D. Maximum embryogenesis
was obtained at 4.5 μM BA while zeatin and TDZ were not effective and embryogenesis did not occur with these treatments. Sucrose
at 0.2 M was more effective for embryogenesis when compared to glucose or fructose. Growing cells in suspension culture on
EIM containing 4.5 μM 2,4-D in combination with 0.2 M sucrose for four weeks and transferring cells to RM (containing 4.5
μM BA) gave significant embryogenesis with maximum number of embryos (3568 embryos/g cells). Using 4.5 μM 2,4-D in protoplast
culture was necessary for the best protoplast division and colony formation. In all experiments, embryos developed on RM were
transferred to hormone-free medium (HFM) and 90% converted to rooted plantlets. Produced plantlets gave 95% survival ex vitro. Plantlets developed to whole plants in the greenhouse and flowered.
... The highest embryogenesis was achieved in medium culture containing 4.5 µM BA. It should be mentioned that zeatin or thidiazuron had no effect on embryogenesis (Shibli and Ajlouni 2000). Determining the cultivation protocol under in vitro conditions, with the aim of preserving wild plant species, provides the possibility of creating a source of information for future studies (Zhou and Wu 2006). ...
... In this study, the leafbase explant succeeded in producing callus. The success of propagation of different species of iris plants in the conditions of in vitro culture using leafbase explants has been confirmed in other studies (Shibli and Ajlouni 2000;Khawar et al. 2005;Jevremović et al. 2013;Boŝnjak Mihovilović et al. 2019;Kereša et al. 2009;Cerasela et al. 2014). Various factors, such as the type and concentration of plant growth regulators, play a role in the formation and type of callus produced from explants (Ikeuchi et al. 2013;Smith 2013;Abbasi et al. 2007). ...
... It has been reported that the use of proline in the medium culture encourages the formation of embryogenic calli (Jéhan et al. 1994). Proline has been used in the medium culture to induce callus in other studies (Jevremović et al. 2009;Shibli and Ajlouni., 2000). In this study, subculture the callus into medium culture containing different concentrations of BA and 1.07 µM NAA caused the production of shoots from callus. ...
Choosing the best method of plant propagation is one of the basic principles in preserving wild plants as a genetic source. Iris ferdowsii Joharchi & Memariani, sp. is a newly introduced plant and is in danger of extinction. In order to induce callus, leafbase explants of seedlings grown from in vitro seed embryos were used. Leafbase explants obtained from 21-day-old seedlings. Callus induction was performed in MS basal culture medium containing different concentrations of BA and 2,4-D. The results showed that the use of 4.52 µM 2,4-D + 10.92 µM BA was the best combination to induce callus from the explant (51.11%). High quality calluses were obtained in this combination. By increasing the concentration of BA and 2,4-D in the culture medium, the quality of callus decreased and also the amount of callus production decreased. After transferring calli to MS culture medium containing different concentrations of BA and NAA, regeneration was observed in them. The best callus regeneration compound was 7.28 µM BA + 1.07 µM NAA. Callus regeneration in these treatments was more than 80%. Rooting was performed in ½ MS medium without plant growth regulator. Also, the use of perlite substrates and 17 °C is the best condition for the acclimation of seedlings produced under in vitro culture. The result were shown that callus induction by leafbase is the effective way to propagate Iris ferdowsii in outside the natural plant habitat.
... Leaf base of Iris, or also known as shoot base (Jevremović et al., 2013), was used as explant for establishing in vitro regeneration process in several Iris species (Gozu et al., 1993;Jehan et al., 1994;Shibli and Ajlouni, 2000;Jevremović et al., 2013;Stanisǐć et al., 2015). To accomplish plantlets from leaf base explants, plant growth regulators (PGRs) play vital roles and interfere with the success rate. ...
... Gavinlertvatana and Prutpongse (1991) suggested that herbaceous plants are highly responsive to BA, and most cultured herbaceous species produce robust, well-formed shoots after treating with BA. Therefore, BA was widely used during in vitro propagation of several Iris species for directly generating new shoots from explants (Boltenkov and Zarembo, 2005;Wang et al., 2018;Tikhomirova, 2020) or callus (Yabuya et al., 1991;Leifert et al., 1992;Jehan et al., 1994;Shibli and Ajlouni, 2000;Nasırcılar et al., 2011;Bae et al., 2012Bae et al., , 2013Stanisǐć et al., 2015). Moreover, BA showed higher shoot regeneration efficiency than thidiazuron (TDZ) as reported by Stanisǐć et al. (2015). ...
... TDZ sometimes failed to induce shoots from explants (Violeta et al., 2013). In addition, BA had a lower antagonistic effect on root formation process of I. nigricans than 2ip (Shibli and Ajlouni, 2000). With this information, BA was used in this study. ...
Iris collettii Hook.f. and I. domestica (L.) Goldblatt & Mabb. are perennial plants that have a high potential as new economic ornamental plants due to their beautiful flowers. However, these plants are typically propagated through seeds or bulbs which cannot achieve large volumes of plants to the floriculture industry. Thus, to prevent the problem of smuggling these plants from their natural habitat, an in vitro shoot multiplication method for both Iris species was developed. In vitro plantlets at the height of 5-6 cm were used as starting materials. Leaf bases at 1.5 cm in length were excised from starting materials and used as explants. At eighth weeks of culture on Murashige and Skoog (MS) agar medium augmented with 0 (control), 1, 2, and 4 mg L-1 N6-benzyladenine (BA), leaf bases of I. collettii cultured on MS medium supplemented with 1 mg L-1 BA showed the greatest shoot formation (3.13 shoots explant-1 and 2.31 cm). Whereas I. domestica provided maximum new shoot numbers explant-1 (5.60) from leaf bases culturing on MS medium augmented with 2 mg L-1 BA. This study also found that the media containing BA resulted in shorter regenerated shoots of I. domestica when compared to the control. Root formation was not found in any treatments except I. domestica cultured on BA-free medium. These results could be used as basic information for optimizing complete in vitro propagation protocol and germplasm conservation method of both Iris species in further study.
... (Gozu et al., 1993), I. nigricans Dinsm. (Shibli and Ajlouni, 2000), I. pumila L., I. reichenbachii Heuff. (Jevremović et al., 2006) and I. adriatica Trin. ...
... Explants with induced calli were then subcultured on the B medium (as CIM with 3.5% sucrose and without L-proline) supplemented with 0.45 M 2,4-D, 4.44 M benzyladenine (BA) and 0.49 M indole-3-butyric acid (IBA) for further development of somatic embryos, as previously described by Shibli and Ajlouni (2000) and Kereša et al. (2009), and maintained under low light intensity (25 E*m -2 *s -1 ) and 16/8 h photoperiod for 5 weeks. Embryogenic calli were subcultured on hormone-free medium containing MS salts and vitamins, 0.25% Phytagel, 2% sucrose and 200 mg*L -1 casein hydrolysate (C medium). ...
... There were substantial differences among treatments (CIM1-3) in efficiency of callus induction (Tables 2 and 4). Treatment CIM1 is identical to the hormone constitution used in studies of Jéhan et al. (1994), Shibli and Ajlouni (2000), Al-Gabbiesh et al. (2006) and Kereša et al. (2009) for callus induction of different Iris species, and mostly resulted in good callogenesis either from leaf bases or flower explants. CIM2 medium was used by Jéhan et al. (1994) and Laublin and Cappadocia (1992), who have observed the best callogenesis of ovary explants on this medium. ...
The present study was undertaken to evaluate efficiency of callogenesis and regeneration by somatic embryogenesis of the subendemic Iris species Iris illyrica from Croatia and to select highly regenerative donor plants/genotypes. Leaf base segments were used as explants. Callogenesis and somatic embryogenesis were induced on MS media supplemented with: (1) 4.52 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 4.83 M 1-naphthaleneacetic acid (NAA) + 0.46 M kinetin (Kin); (2) 4.52 M 2,4-D + 4.6 M Kin; (3) 13.4 M NAA + 2.3 M Kin. Transfer of embryogenic calli onto hormone-free medium enabled the development of mature somatic embryos. Frequency of callogenesis was influenced by the donor plant. Among 15 donor plants tested, 3 of them exhibited high regeneration capability and produced 87 regenerants. Seven morphological traits were observed in order to assess phenotypic variability of flowering regenerants. Regenerants with higher values of fall and standard width and length show potential for further breeding of new varieties of Illyrian iris. Flowering and non-flowering regenerants had the same ploidy level as donor plants. Also, the nuclear DNA content (2C = 12.936 ± 0.038 pg) of this species was estimated for the first time using flow cytometry.
... Plant tissue culture in Jordan had witnessed a significant advance in the past decades (Shibli, 1995 Shibli and Ajlouni, 2000). ...
... Micropropagation of bulb plants as an alternative to the conventional methods for vegetative propagation attracts much attention, because of its advantages (Shibli and Ajlouni, 2000). It increases many times the multiplication level and enables to obtain materials free from disease (Chang et al., 2000). ...
... Cell suspension cultures are widely used in cell culture engineering due to the favorable flow ability and ease of operation as well as higher mass transduction and absorption rates (Zhigang et al., 2005). Liquid suspension cultures consist of mixtures of cell aggregates, cell clusters and single cells (Elfahmi, 2006;Shibli and Ajlouni, 2000;Ogita, 2005). The growth rates of such cultures are generally much higher than on agar. ...
... Iris species can be propagated from seeds, bulbs, or rhizomes with a low propagation speed like other geophytes (Jevremovic and Radojevic, 2002;Boltenkov et al., 2007). Therefore, in recent years, a number of studies have reported in vitro propagation of Iris species such as I. ensata (Yabuya et al., 1991;Boltenkov et al., 2007), I. germanica (Shimizu et al., 1997;Wang et al., 1999aWang et al., , 1999b, I. nigricans (Shibli and Ajlouni, 2000), I. pumila (Jevremovic and Radojevic, 2002), I. ensata, I. setosa, I. sanguinea (Boltenkov and Zarebno, 2005), I. hollandica (Fidalgo et al., 2005), I. atrofusca, I. petrana, I. vartanii (Al-Gabbiesh et al., 2006, and I. adriatica (Keresa et al., 2009) from a range of explants. ...
... The MS medium with 0.5 mg/L TDZ plus 0.5 mg/L NAA and 1 mg/L TDZ plus 0.5 mg/L NAA produced the largest number of regenerated shoots per explant. Although TDZ has been proven to be effective for in vitro regeneration of various plants (Çöçü et al., 2004;Erişen et al., 2011;Nasırcılar et al., 2011), Shibli and Ajlouni (2000) reported that embryogenesis did not occur with TDZ treatment in Iris nigricans. ...
... In treatments including BAP, shoot regeneration response changed with species and I. schachtii was found to be better than I. sari. The promotive effect of BAP alone or in combination with NAA-inducing callus and adventitious bulblet regeneration was previously reported in other Iris species (Kawase et al., 1995;Suzhen et al., 1999;Shibli and Ajlouni, 2000;Boltenkov and Zarembo, 2005;Boltenkov et al., 2007). ...
Plant tissue culture is an efficient technique for conserving endemic plant species. A reproducible in vitro regeneration protocol was developed for the endemic Iris sari and Iris schachtii in the present study. The highest number of shoots per explant was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L thidiazuron (TDZ) plus 0.5 mg/L alpha-naphthaleneacetic acid (NAA) and 1 mg/L TDZ plus 0.5 mg/L NAA, whereby 96.88% and 100% shoot induction with 9.55 and 11.34 shoots per explant of Iris sari and Iris schachtii were recorded, respectively. Regenerated shoots were successfully rooted on MS medium with either 1 mg/L indole-3-butyric acid (IBA) or 1 mg/L IBA plus 0.2 mg/L NAA. Rooted shoots were transferred to pots containing either a peat-soil-sand (1:1:1) mixture or a hydroponic culture containing Hoagland solution to acclimatize the regenerated plants to the greenhouse chamber. Approximately 90% of plants were transferred ex vitro successfully.
... Subsequently, to ensure the continuation of such species, emphasis should be directed at cultivating them by tissue culture and optimizing the conditions for their production. In previous studies, in vitro cultivation of different Iris species was achieved by somatic embryogenesis using apex callus of rhizomes, flowers and leaves, cell suspension and protoplast culture systems (Reuther 1977;Gozu et al. 1993;Jéhan et al. 1994;Shimuzu et al. 1996Shimuzu et al. , 1997Shibli and Ajlouni 2000). ...
... The three threatened Iris species under investigation were collected from northern and southern Jordanian provinces and were identified by plant taxonomist. For the various stages of the in vitro cultivation, Murashige and Skoog (1962) (MS) medium, with necessary growth regulators, were used as reported by Shibli and Ajlouni (2000) for I. nigricans. The media used for the cultivation of the Iris species in vitro and the components and their concentrations in the MS medium are described in Tables 1 and 2, respectively. ...
... The in vitro conditions used in this study were adapted from Shibli and Ajlouni (2000). The investigators described the successful use of a combination of growth regulators with MS media in callus formation from I. nigricans (leaf base) (Shibli and Ajlouni 2000). ...
The genus Iris is the largest and most complex genus of the family Iridaceae. Several Iris species occur naturally in Jordan. Of these, Iris atrofusca, Iris petrana and Iris vartanii are threatened species. Callus from the former two species was successfully propagated using juvenile flower bases. Irone-type essential oil was first determined by TLC and confirmed using GC-MS.
... Successful regeneration of iris species from leaf-base explants was reported by several authors (Gozu et al., 1993;Shimizu et al., 1997;Shibli and Ajlouni, 2000;Jevremović et al., 2013). In this study, leaf bases proved to be suitable explants for I. sibirica regeneration as organogenic and embryogenic calli developed in most of the explants. ...
... The 2,4-D is used more often in combination with other plant growth regulators (Gozu et al., 1993;Jéhan et al., 1994;Radojević and Subotić., 1992;Shimizu et al., 1997;Shibli and Ajlouni., 2000;Fidalgo et al., 2005) than sole (Laublin and Cappadocia, 1992;Fidalgo et al., 2005;Jevremović et al., 2008) for morphogenesis induction in Iris sp. ...
... Up to now, there have been no literature data about the effect of TDZ on I. sibirica morphogenesis and generally it has been rarely used for the regeneration of other iris species (Ascough et al., 2009). An unsuccessful attempt of I. nigricans somatic embryo regeneration was reported by Shibli and Ajlouni (2000), while organogenic calli and adventitious shoots were successfully induced in zygotic embryos culture of Iris pseudopallida (Jevremović et al., 2008). Leaf-base explants of I. sibirica were twice as responsive to the same concentration of TDZ (1.0 mg/l). ...
Efficient protocols, safe from somaclonal variation, were developed for regeneration of Iris sibirica plants via organogenesis and somatic embryogenesis from leaf-base explants cultivated on Murashige and Skoog media supplemented with thidiazuron (TDZ, 1.0 mg/l) or 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/l). The morphogenic response and callus formation efficiency differed significantly between 2,4-D (80.9%) and TDZ (67%) morphogenesis induction treatments. TDZ induced only organogenic calli, while calli obtained with 2,4-D were composed of three types differing in color and consistency: white, friable — embryogenic calli (4.5%, 3.8 mg/explant), green, compact — organogenic calli (12.4%, 48.4 mg/explants) and yellow — non-regenerative calli (77.3%, 254.4 mg/explant). The cultivation of embryogenic calli on medium with 2,4-D and Kinetin resulted in further development of somatic embryos (54 embryos/g of calli) which germinated with a frequency of 62% after being transferred to a medium without plant growth regulators. Stable shoot cultures were established by transferring organogenic calli with shoot primordia to media with 0.1 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l 6-benzylaminopurine (BA), while further cultivation on media of the same composition (TDZ or 2,4-D) resulted in the reduced growth and rhizogenesis, respectively. The TDZ induction treatment resulted in higher number of shoots per explant (7.9) than the 2,4-D treatment (4.3). After successful rooting and ex vitro acclimatization, plants were grown in the field and flowered to seed production. Flow cytometry, chromosome counting and random amplification of polymorphic DNA (RAPD) analysis indicated no evidence of genetic variation in plants regenerated via somatic embryogenesis or organogenesis. The results suggest that established protocols are safe for use in genetic transformation procedures or large-scale production of true-to-type I. sibirica plants.
... At the same time, similar to vegetative reproduction, as long as 4-5 years are required to obtain sufficient quantities of planting stock. Plant tissue culture is a powerful alternative technique for conservation and propagation of plants, especially for those that are rare and difficult to propagate by conventional methods (Shimazu et al. 1997;Wang et al. 1999a;Shibli and Ajlouni 2000) and improves the quality of valuable planting stock (Baruch and Quak 1966;Mielke and Anderson 1989). ...
... The developed techniques for clonal reproduction are used as an alternative way to conserve rare iris species (Radojevic and Subotic 1992;Shibli and Ajlouni 2000). ...
... Numerous studies demonstrated that the hormonal composition of the medium is the most important factor for in vitro regeneration of irises (Radojevic et al. 1987;Laublin and Cappadocia 1992;Radojevic and Subotic 1992;Gozu et al. 1993;Jehan et al. 1994;Shimizu et al. 1996;Wang et al. 1999b). Various iris species have been propagated through organogenesis or somatic embryogenesis, using explants from the leaf base (Gozu et al. 1993;Shibli and Ajlouni 2000), mature zygotic embryos (Radojević and Subotić 1992; Boltenkov et al. 2004), ovary sections and root sections (Laublin and Cappadocia 1992), but, so far, no studies have been reported on callus derived organogenesis in I. odaesanensis. ...
Iris odaesanensis Y. N. Lee. is an important endangered and native plant belonging to the family Iridaceae in Korea. This study describes a method for rapid micropropagation of this species via from leaf, rhizome and root explants derived calli. Leaf, rhizome and root explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) for callus induction. Rhizome explants yielded calli at a frequency of 72% when cultured at 1.0 mg/l 2,4-D. Calli were maintained at 1.0 mg/l 2,4-D. These calli were transferred to MS medium supplemented with 0, 0.5, 1.0, and 2.0 mg/l 2,4-D in combination with 0, 0.5, 1.0, and 3.0 mg/l BA for adventitious shoot induction. The highest number of adventitious shoot (228.9 per petri-dish) were formed at 1.0 mg/l 2,4-D and 1.0 mg/l BA. WPM medium was the best to convert calli into plantlets, where up to 98.2% of calli were regenerated into plantlets. This in vitro propagation protocol should be useful for conservation of this endangered plant.
... Furthermore, similar to vegetative reproduction, the time required to obtain sufficient quantities of planting stock is 4-5 years. On the other hand, the cell and tissue culture method has become popular in iris reproduction, since it considerably increases plant multiplication factors (Shimazu et al. 1997;Wang et al. 1999a;Shibli and Ajlouni 2000) and improves the quality of valuable planting stock (Baruch and Quak 1966;Mielke and Anderson 1989). The developed clonal reproduction techniques are used as an alternative method of conserving rare iris species (Radojevic and Subotic. ...
... The developed clonal reproduction techniques are used as an alternative method of conserving rare iris species (Radojevic and Subotic. 1992;Shibli and Ajlouni 2000). ...
... As is common in irises, propagation by splitting rhizomes is slow (Hussey 1975;Jehan et al. 1994); thus, micropropagation in vitro might be the method of choice. Various iris species have been propagated through organogenesis or somatic embryogenesis, using explants from the leaf base (Gozu et al. 1993;Shibli and Ajlouni 2000), mature zygotic embryos (Radojević and Subotić 1992; Boltenkov et al. 2004), ovary sections (Laublin and Cappadocia 1992), and root sections (Laublin et al. 1991). ...
Iris dichotoma Pall. is an important endangered plant belonging to the family Iridaceae. A method was developed for the rapid micropropagation of I. dichotoma through plant regeneration from leaf, rhizome, and root explant-derived calli. Leaf, rhizome, and root segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; ) for callus induction. Callus production was highest at 2,4-D, where 73.8% and 45.5% of cultured rhizome and root cuttings, respectively, produced calli. The viable calli were maintained at an induced concentration of 2,4-D (). They were then transferred to MS medium supplemented with various concentrations of 2,4-D () in combination with 6-benzyladenine (BA: 0, 1.0 and ) for adventitious shoot regeneration. The addition of a low concentration of 2,4-D into BA-containing medium significantly increased the frequency of shoot regeneration in leaf, rhizome, and root-derived calli. The highest number of adventitious shoots (26.4 per callus) formed at 2,4-D and 1.0 mg/l BA. For rooting of the shoots, half- strength MS medium supplemented with different concentrations of indole 3-butyric acid (IBA) was tested. The optimal results were observed using half-strength MS medium supplemented with IBA, on which 98% of the regenerated shoots developed roots with an average of 3.5 roots per shoot within 45 days. The plantlets raised in vitro were acclimatized and transferred to soil with 95% success. This in vitro propagation protocol will be useful for conservation and mass propagation of this endangered plant.
... IRIS ADRIATICA TRINAJSTIĆ EX MITIĆ propagated through organogenesis or somatic embryogenesis, using explants from the leaf base (Gozu et al., 1993;Shibli and Ajlouni, 2000;Jevremović and Radojević, 2006), mature zygotic embryos (Radojević and Subotić, 1992;Boltenkov et al., 2007), ovary sections (Laublin and Cappadocia, 1992) and root sections (Laublin et al., 1991). Propagation of I. adriatica via in vitro plant regeneration has not been reported. ...
... Although a comparative study evaluating the regeneration capacity of several monocotyledonous ornamental species ranked Iridaceae last (Hussey, 1975), several authors have reported sufficient or efficient regeneration in various Iris species, using embryogenesis or organogenesis after callus induction on solid agar medium (Radojević and Subotić, 1992;Gozu et al., 1993;Shibli and Ajlouni, 2000;Jevremović and Radojević, 2006) or in suspension culture (Shimizu et al., 1997;Wang et al., 1999;Shibli and Ajlouni, 2000;Jevremović and Radojević, 2002). ...
... Although a comparative study evaluating the regeneration capacity of several monocotyledonous ornamental species ranked Iridaceae last (Hussey, 1975), several authors have reported sufficient or efficient regeneration in various Iris species, using embryogenesis or organogenesis after callus induction on solid agar medium (Radojević and Subotić, 1992;Gozu et al., 1993;Shibli and Ajlouni, 2000;Jevremović and Radojević, 2006) or in suspension culture (Shimizu et al., 1997;Wang et al., 1999;Shibli and Ajlouni, 2000;Jevremović and Radojević, 2002). ...
Plant regeneration via somatic embryogenesis and organogenesis was achieved in leaf base and ovary culture of the Croatian endemic Iris adriatica Trinajstić ex Mitić. Callus induction from leaf base explants occurred in the dark on three media with MS mineral solution containing 4.52 μM dichlorophenoxyacetic acid (2,4-D), 4.83 μM naphthaleneacetic acid (NAA), 0.46 μM kinetin (Kin), 5% sucrose and 200 mg L-1 casein hydrolysate. The media differed only in vitamin and/or proline content. Calli from ovary culture were achieved on MS medium containing 45.25 μM 2,4-D. The mean percentage of callus induction from leaf base explants was 18.9%, with no significant differences between media, and 27.3% from ovary sections. All embryogenic calli were formed on MS media containing 0.45 μM 2,4-D, 4.44 μM benzyladenine (BA) and 0.49 μM indole-3-butyric acid (IBA) under low light intensity (25 μE m-2s-1). Transfer of embryogenic calli to hormone-free medium enabled the development of mature somatic embryos on the surface of 6.0% of induced calli produced from leaf base explants and 4.0%
of those from ovary sections. Genotype had the main effect on plant regeneration efficiency in Iris adriatica.
... In vitro propagation, which includes the use of somatic embryogenesis, is one of the most effective methods for propagation and multiplication of medicinal plants (Mostafa et al. 2010;Al-Qudah et al. 2011;Shatnawi et al. 2011). Somatic embryogenesis allows the production of large numbers of genetically uniform plants which can have the same growth characteristics as seedderived plants (Shibli and Ajlouni 2000;Shibli et al. 2001;Duquenne et al. 2006;Abdul Karim and Ahmed 2010). In this study, a protocol for plant regeneration via somatic embryogenesis was developed because A. palaestinum plant establishment from seeds has been difficult in vitro. ...
... Among the regeneration medium with 2iP, the medium with 4.9 μM 2iP resulted in the highest number of embryos (524.9 embryos/g callus FW) and shoots (201.8 shoots/g callus FW; Table 1). In "Nabali" olive (Olea europea L), the highest rate of embryogenesis (1,680 embryos/g callus FW) was obtained using a medium containing 9.8 μM 2iP (Shibli et al. 2001), whereas in Iris nigricans, a medium containing 4.9 μM 2iP resulted in 2,686 embryos/g callus FW (Shibli and Ajlouni 2000). Somatic embryos of Zantedeschia hybrids developed into plantlets on BM supplemented with 4.9 μM 2iP (Duquenne et al. 2006). ...
... Among the carbohydrate sources (sucrose, fructose, and glucose) and concentrations in embryogenesis induction media tested here, 785.2 embryos/g callus developed after transfer of callus to an embryogenesis induction medium containing 3 %w/v sucrose (Table 2). A similar result was seen in I. nigricans, in which the maximum number of embryos developed on BM containing 3.4 % sucrose (Shibli and Ajlouni 2000). In O. europea, increasing the sucrose, glucose, or fructose concentrations in the BM increased embryogenesis, and maximum embryogenesis was obtained with 6.8 % sucrose (Shibli et al. 2001). ...
Somatic embryogenesis was obtained from callus of wild arum (Arum palaestinum). Callus was induced from sterilized corm bud sprouts cultured on basal medium containing 4.4 μM 6-benzyladenine and 5.4 μM 1-naphthaleneacetic acid. Callus was maintained under dark conditions using basal medium with 4.4 or 8.8 μM 6-benzyladenine and 5.4 or 10.8 μM 1-naphthaleneacetic acid. The highest callus weight and most desirable callus phenotype were achieved using basal medium containing 8.8 μM 6-benzyladenine and 5.4 μM 1-naphthaleneacetic acid. Friable calli were cultured in the dark on basal medium containing 4.5 μM 2, 4-dichlorophenoxyacetic acid, 0.46 μM 6-furfurylaminopurine, 5.4 μM 1-naphthaleneacetic acid, and 1.7 mM proline to induce embryogenesis before transfer to regeneration medium. Embryos that developed on regeneration medium were transferred to medium minus plant growth regulators for germination. Ninety percent of the germinating embryos developed into rooted plantlets. Rooted plants were grown in the greenhouse and acclimatized successfully with a 95 % survival rate. This is the first report of successful somatic embryogenesis and plant regeneration in A. palaestinum.
... Ascough et al. (2009'un bildirdiğine göre Iris türlerinde yapılan ilk çalışma 1945 yılında Randolph tarafından yürütülen embriyo kültürüdür. I. ensata (Yabuya et al. 1991;Boltenkov et al. 2007), I. germanica (Shimizu et al. 1997;Wang et al. 1999aWang et al. ,1999b, I. nigricans (Shibli and Ajlouni 2000), I. pumila (Jevremovic and Radojevic 2002), I. ensata, I. setosa, I. sanguinea (Boltenkov and Zarebno 2005), I. hollandica (Fidalgo et al. 2005), I. atrofusca, I. petrana, I. vartanii (Al-Gabbiesh et al. 2006, I. adriatica (Keresa et al. 2009), I. sari ve I. schachtii (Uzun et al. 2014) gibi birçok Iris türünde kök, yaprak, çiçek organları, soğan segmentleri, olgunlaşmamış embriyo ve zigotik embriyo gibi birçok eksplant kullanılarak rejenerasyon çalışmaları yapılmıştır. Ancak Iris galatica ile ilgili herhangi bir çalışmaya rastlanmamıştır. ...
... Değişik araştırıcılar tarafından da belirtildiği gibi İris türlerinin çoğunun, tohum ekiminden çiçek açacak zamana ulaşması ortalama 4-5 yıl süre alması ve vejetatif çoğaltım hızının da düşük olması nedeniyle bir takım hızlı çoğaltma tekniklerinin bu bitkiler üzerinde geliştirilmesi gerekmektedir (Boltenkov et al. 2007;Shibli and Ajlouni 2000;Wang et al. 1999a;Wang et al. 1999b). Bu çalışmada da endemik ve az tehdit altında bulunan Iris galatica türünün doku kültürü teknikleri ile in vitro çoğaltımı amaçlanmıştır. ...
... There are scarce reports about the use of thidiazuron (TDZ), a very potent cytokinin for the multiplication of irises. (Shibli and Ajlouni 2000) used TDZ to regenerate shoots from calli derived in leaf base culture of I. nigricans, endemic iris from Jordan, but with no success. Formed calli are cultured on relatively high concentrations of TDZ (1-3 mgL -1 ) and only several plantlets but mainly roots were produced. ...
... TDZ is a substitute phenylurea and acts like a strong cytokinin. According to previous work with (1-3 mgL -1 ) TDZ, there is no significant effect on plant regeneration from callus in leaf base culture of black iris (Shibli and Ajlouni 2000). In our experiments, TDZ induced the expansion of shoot meristems in zygotic embryo cultures. ...
... Somatic embryogenesis is the fastest system of plant regeneration and generally considered to be prerequisite for genetic transformation (Jeknic et al., 1999;Karami et al., 2006). Therefore, several protocols for iris regeneration via somatic embryogenesis from a variety of explant types have been developed (Jevremovi c and Radojevi c, 2006;Kim et al., 2009;Laublin et al., 1991;Radojevi c et al., 1987;Radojevi c and Suboti c, 1992;Shibli and Ajlouni, 2000), including I. germanica regenerated from leaf bases, apices, sepals, petals, and ovaries (Jehan et al., 1994). Jehan et al. (1994) found that the optimal medium for obtaining embryogenic callus was MS medium supplemented with 2.9 g/L proline. ...
... Sucrose, as one of the most commonly used carbohydrate sources in plant tissue culture, has been known to play a key role in the formation and maturation of SEs (Fuentes et al., 2000). Sucrose at 0.2 M (6%, w/v) was found to produce the maximum number of embryos of I. nigricans (Shibli and Ajlouni, 2000). Meanwhile, Kim et al. (2009) found that 0.22 M sucrose (6.6%, w/v) induced the highest frequency of SEs of I. pseudacorus. ...
An improved three-stage protocol for plant regeneration via somatic embryogenesis of the horticulturally important plant Iris germanica L. was developed using shoot apex segments as explants. At the first stage of the experiment, 60% of callus was obtained from shoot apex segments of I. germanica on Murashige and Skoog’s (MS) medium supplemented with 4.52 μM 2,4-dichloropheoxyacetic acid (2,4-D) and 0.44 μM 6-benzyladenine (6-BA). When nonembryogenic calli were subcultured on MS medium with 11.31 μM 2,4-D and 0.44 μM 6-BA, maximum frequency of embryogenic callus (66.0%) was obtained. At the second stage, the treatment of 9% (w/v) sucrose resulted in the optimum somatic embryo (SE) formation (70.0%). More than 90.0% of SEs germinated with bipolar structure and regenerated into plantlets on plant growth regulator-(PGR)free MS medium during the third stage. Regenerated plantlets were successfully acclimatized in greenhouse environment with little somaclonal variation. Histological study showed that somatic embryogenesis stages were asynchronous and SEs developed from the surface and inner tissue of embryogenic calli.
... In contrast, some researchers report that both induction and regeneration of somatic embryos occurs in the presence of an auxin (Bieniek et al. 1995, Büyükalaca and Mavituna 1995, Shimizu et al. 1997, Anbazhagan and Ganapathi 1999, Shibli and Ajlouni 2000, Barry-Etienne et al. 2002, Kosky et al. 2002. In the case of Panax ginseng, induction of embryogenic callus occurred on an auxin enriched medium while regeneration of somatic embryos took place on a cytokinin enriched medium (Kevers et al. 2000). ...
... In some species, the requirement for induction of embryogenic cells is absolute, while in others, cytokinins act to enhance the process by increasing the rate at which embryos regenerate. For the induction of embryogenic callus, iP has been used for coffee (Barry-Etienne et al. 2002), and BA for Hevea brasiliensis (Martre et al. 2001), black iris (Shibli and Ajlouni 2000) and dune reed (Wang et al. 2001). For embryo development and formation, kinetin has been used for cotton (Gawel and Robacker 1990), asparagus (Reuther 1996) and ginseng (Kevers et al. 2000(Kevers et al. , 2002. ...
Plant liquid culture offers many benefits over solidified media. Growth and multiplication rate of shoots, roots, bulblets and somatic embryos is enhanced in liquid culture, as a consequence of better availability of water and nutrients resulting from a lower resistance to diffusion and closer contact between the explant and the medium. Morphogenic development, such as meristemoid production and somatic embryogenesis, is influenced by the physical environment provided by the liquid culture, and benefits from reduced gradients within the medium and dilution of exuded toxins. Chemical growth regulating factors, including plant hormones, growth retardants and other bioregulators are also discussed in terms of their influence on growth and development. Automation and scaling up for bioreactor production of propagules requires optimisation before becoming commercially viable. Considerable potential exists for future research into the molecular aspects regulating developmental pathways such as somatic embryogenesis, storage organ formation and meristemoid induction.
... Since then, researchers have cultured callus of many species of Iris, using explant sources such as young inflorescence, floral parts, mature embryo (Yabuya et al., 1991) [20] , shoot apex (Radojevic and Subotic, 1992) [14] , leaf base and rhizome apex (Jehan et al., 1994) [6] . Various Iris species have been propagated through organogenesis or somatic embryogenesis, using explants from the leaf base (Shibli and Ajlouni, 2000 [15] , mature zygotic embryos (Boltenkov et al., 2004) [2] , ovary sections (Laublin and Cappadocia, 1992 [8] , and root sections (Laublin et al., 1991) [9] . However, there is no such research report available about the ideal hormonal combination in successful in vitro regeneration of iris sp. ...
Leaf base explants of Iris ensata were cultured in MS basal medium containing different plant growth hormones. MS supplemented with 2,4-D (1.0 mg/l) and TDZ 0.1 mg/l) showed earlier callus initiation (25.30 days) in greater percentage of explants [96.67% (79.69%)] and produced higher callus weight at both 15 (0.373g) and 30 (2.961g) days after culture. Regenerated calli when cultured in MS + GA3 (2.0 mg/l) + BAP (1.0 mg/l) recorded earlier regeneration (29.20 days) with greater percentage of explants [99.70% (86.97%)], greater micro shoot length (8.64 cm) and leaf length (7.00 cm); while MS+GA3 (2.0 mg/l) + BAP (2.0 mg/l) produced maximum number of micro shoots per culture (16.30) with broadest leaf (1.32 cm). MS supplemented with 2.0 mg/l BAP yielded maximum number of leaves per micro shoot (11.70). In vitro regenerated micro shoots when cultured in MS medium containing IBA (1.0 mg/l) + NAA (1.0 mg/l) reported earlier root initiation (26.07 days) with higher number of roots per plant (9.07) having greater length (9.32 cm). Percent of rooted micro shoots were found highest [99.62% (86.51%)] when tried with MS + IBA (1.5 mg/l) + NAA (1 mg/l). Diameter (1.019 mm) of regenerated in vitro roots was found higher with MS + IBA (2.0 mg/l) + NAA (1.0 mg/l). A mixture containing equal proportion of vermiculite and vermicompost (v /v) resulted greater survivability [86.85% (68.69%)] in shorter duration (12.43 days) during subsequent acclimatization of in vitro regenerated plantlets of Japanese Iris. Culture of explants in hormone free MS basal medium showed least response in all growth and development aspects in vitro. Least percent of acclimatized plantlets [55.42% (48.15%)] was obtained from the hardening medium containing equal proportion of garden soil and vermicompost.
... In other studies, the growth regulator kinetin has been used to induce protocorm formation in Dactylorhiza majalis [51,52]. The effect of these kinds of PGRs on embryogenesis has been reported on black iris [53], Phalaenopsis [54], and Rhynchostylis retusa [55]. Effective PGR treatments were 3 mg L −1 TDZ on protocorm and 1 mg L −1 NAA in nodal explant of E. veratifolia and 3 mg L −1 TDZ and 2 mg L −1 Kin on protocorm explant of D. umberosa ( Table 1). ...
Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others.
... In other studies, the growth regulator kinetin has been used to induce protocorm formation in Dactylorhiza majalis [51,52]. The effect of these kinds of PGRs on embryogenesis has been reported on black iris [53], Phalaenopsis [54], and Rhynchostylis retusa [55]. Effective PGR treatments were 3 mg L −1 TDZ on protocorm and 1 mg L −1 NAA in nodal explant of E. veratifolia and 3 mg L −1 TDZ and 2 mg L −1 Kin on protocorm explant of D. umberosa ( Table 1). ...
Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others.
... In other studies, the growth regulator kinetin has been used to induce protocorm formation in Dactylorhiza majalis [51,52]. The effect of these kinds of PGRs on embryogenesis has been reported on black iris [53], Phalaenopsis [54], and Rhynchostylis retusa [55]. Effective PGR treatments were 3 mg L −1 TDZ on protocorm and 1 mg L −1 NAA in nodal explant of E. veratifolia and 3 mg L −1 TDZ and 2 mg L −1 Kin on protocorm explant of D. umberosa ( Table 1). ...
Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums.
In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others.
... The I. bismarckiana species propagates naturally through rhizomes where their seed production is extremely low. In addition, most bulbous and rhizomatous Iris species including I. bismarckiana do not produce more than 5 to 10 daughters annually [48,49]. Somatic embryogenesis techniques that were implemented in embryo culture labs were successful for I. bismarckiana [20] and other iris species [48] regeneration, where acclimatization and reintroduction protocols support such achievement [48]. ...
Conservation of endangered plant species in their indigenous regions is of crucial importance, especially for those grown in semi-arid regions. The objectives of this study were to explore the Nazareth iris’s (Iris bismarckiana) natural habitat and identify new suitable sites to initiate a reintroduction program of this endangered plant species in a semi-arid environment. The study was conducted in Dibbeen Forest Reserve, Jordan, where six zones inside the reserve [A–F] were assessed in addition to zone G outside the reserve borders that represents the area where I. bismarckiana still exists. Habitat selection variables (topography, soil physical and chemical properties, climatic data, and potential risks and benefits) from all zones within the reserve were cross matched with that of zone G. The results showed that climatic data of all selected sites were suitable for reintroduction; all sites are open to direct sunlight most of the day. The minimum soil depth was greater than 40 cm in all zones, while soil respiration level revealed that zone A (a recreation site) was below the recommended thresholds. The percentage of stone volume (>2 mm) in the soil profile was high in zones D and F. Zones E, C, and F were extremely steep (>40 degrees), which undermined their potential to be suitable habitats. All sites are susceptible to high human disturbance risk except zone B, which is protected and under continuous surveillance by the Reserve Botanist. Considering all measured suitability indicators, including slope degree suitability (<25), soil respiration (57–77 mg kg−1), soil stone percentage value (8.3%), tree canopy cover (open area), and human disturbance potential (low risk), zone B holds promise as a suitable site for a I. bismarckiana reintroduction program. Therefore, the initiation of long-term reintroduction programs within this site with timely surveillance is urgently needed to conserve and support such valuable species self-regeneration.
... Since then, researchers have cultured callus of many species of Iris, using explant sources such as young inflorescence, floral parts, mature embryo (Yabuya et al., 1991) [20] , shoot apex (Radojevic and Subotic, 1992) [14] , leaf base and rhizome apex (Jehan et al., 1994) [6] . Various Iris species have been propagated through organogenesis or somatic embryogenesis, using explants from the leaf base (Shibli and Ajlouni, 2000 [15] , mature zygotic embryos (Boltenkov et al., 2004) [2] , ovary sections (Laublin and Cappadocia, 1992 [8] , and root sections (Laublin et al., 1991) [9] . However, there is no such research report available about the ideal hormonal combination in successful in vitro regeneration of iris sp. ...
Leaf base explants of Iris ensata were cultured in MS basal medium containing different plant growth hormones. MS supplemented with 2,4-D (1.0 mg/l) and TDZ 0.1 mg/l) showed earlier callus initiation (25.30 days) in greater percentage of explants [96.67% (79.69%)] and produced higher callus weight at both 15 (0.373g) and 30 (2.961g) days after culture. Regenerated calli when cultured in MS + GA3 (2.0 mg/l) + BAP (1.0 mg/l) recorded earlier regeneration (29.20 days) with greater percentage of explants [99.70% (86.97%)], greater micro shoot length (8.64 cm) and leaf length (7.00 cm); while MS+GA3 (2.0 mg/l) + BAP (2.0 mg/l) produced maximum number of micro shoots per culture (16.30) with broadest leaf (1.32 cm). MS supplemented with 2.0 mg/l BAP yielded maximum number of leaves per micro shoot (11.70). In vitro regenerated micro shoots when cultured in MS medium containing IBA (1.0 mg/l) + NAA (1.0 mg/l) reported earlier root initiation (26.07 days) with higher number of roots per plant (9.07) having greater length (9.32 cm). Percent of rooted micro shoots were found highest [99.62% (86.51%)] when tried with MS + IBA (1.5 mg/l) + NAA (1 mg/l). Diameter (1.019 mm) of regenerated in vitro roots was found higher with MS + IBA (2.0 mg/l) + NAA (1.0 mg/l). A mixture containing equal proportion of vermiculite and vermicompost (v /v) resulted greater survivability [86.85% (68.69%)] in shorter duration (12.43 days) during subsequent acclimatization of in vitro regenerated plantlets of Japanese Iris. Culture of explants in hormone free MS basal medium showed least response in all growth and development aspects in vitro. Least percent of acclimatized plantlets [55.42% (48.15%)] was obtained from the hardening medium containing equal proportion of garden soil and vermicompost.
... Therefore, traditionally, irises are propagated by bulbs or rhizomes vegetatively which is slow and ine cient (Hussey, 1975;Jehan et al., 1994). In a year, through vegetative propagation, iris produce only three to ve daughter bulbs per mother bulb (Shibli and Ajlouni, 2000) and the new bulbs produced are most likely susceptible to the fungal, bacterial, and iris borer. Therefore, to meet the demand of quality planting material of this commercially important cultivar, others e cient methods of propagation are required. ...
Dutch Iris is a commercially important bulbous ornamental crop. Its high demand in global floriculture market necessitates the production of high-quality planting material. In the present investigation, the significance of meta-topolin (mT), a novel aromatic cytokinin, was studied on in vitro morphogenesis of Iris × hollandica Tub. cv. Professor Blaauw (Dutch Iris). Cytokinin mT (1.0 mg L − 1 ) as a better substitute to BAP (6-Benzylaminopurine) and Kn (Kinetin), has resulted in maximum shoot induction response (86.11 per cent) and increased emergence of micro shoots (4.95 shoots/explant with average shoot length of 5.65 cm) from twin scale explants. Photoperiod also affected the shoot induction, wherein initial 1-week (w) dark incubation significantly increased shoot induction response (94.44 per cent). Both meta-topolin and initial 1w dark incubation resulted in improved quality of regenerated shoots by reducing the incidence of necrosis and preventing vitrification. The collective consequence of cytokinin-auxin (1.0 mg L − 1 mT + 0.25 mg L − 1 NAA) showed considerably higher number of shoots (17.53) with mean shoot length (6.90 cm) and maximum number of bulbets (2.74). Positive effect of increased sucrose concentration (90 g L ¹ ) and paclobutrazol (5 mg L ¹ ) on in vitro bulblet formation and bulblet size was also observed. The superiority of mT over BAP was also found during in vitro rhizogenesis. Shoots derived from mT medium were healthy and long enough, thus showed better rooting response (63.3 %) on ½ MS medium + 0.5 mg L − 1 IAA after 4 weeks. The mT raised plantlets were acclimatized in a greenhouse and showed 89.6 % survival under ex vitro conditions The clonal fidelity of the in vitro regenerated plants was evaluated using Start Codon Targeted (SCoT) markers. Out of 36 primers, 13 primers showed clearly scorable monomorphic bands, thus displaying genetic uniformity among in vitro regenerated plantlets. This meta-topolin mediated protocol can be routinely used for the rapid large scale production of this valuable floriculture crop.
... The propagation of the iris through seedlings is difficult due to the poor rate of seed set and germination (Simonet 1932). Therefore, traditionally, irises are propagated by bulbs or rhizomes vegetatively which is very slow and inefficient, and can produce only three to five daughter bulbs per mother bulb per year (Hussey 1975;Jehan et al. 1994;Shibli and Ajlouni 2000). Moreover, the new bulbs produced vegetatively are most likely susceptible to the fungal, bacterial, and iris borer. ...
Dutch iris is a commercially important bulbous ornamental crop. Its high demand in global floriculture market necessitates the production of its high-quality planting material. In the present investigation, an efficient in vitro propagation system has been developed for Iris × hollandica Tub. cv. Professor Blaauw (Dutch iris) using meta-Topolin (mT) for the first time. Effect of various concentrations of BAP, Kn, and mT (0, 0.5, 1.0, 1.5 and 2 mg L⁻¹) along with varying photoperiods (16 h light and dark incubation for 1, 2, 3 and 4 weeks) on in vitro shoot induction from the twin scale explants was studied. Of the cytokinins tested, different doses of mT has resulted in better shoot induction response from twin scale explants than BAP and Kn. Photoperiod duration has also affected shoot induction response significantly. Along with dark incubation for 1 week, cytokinin mT at 1.0 mg L⁻¹ in MS medium has resulted in maximum shoot induction response (91.63%) with increased emergence of micro shoots (4.83 shoots/explant with average shoot length of 5.02 cm). Efficacy of BAP and mT alone or in combination with auxins for in vitro shoot multiplication was also compared. The synergistic effect of cytokinin-auxin in multiplication medium comprising MS + 1.0 mg L⁻¹ mT + 0.25 mg L⁻¹ NAA resulted in considerably higher number of shoots (17.53) with mean shoot length (7.06 cm) and maximum number of bulblets (2.74). Positive effect of increased sucrose concentration (90 g L⁻¹) alone or with paclobutrazol (5 mg L⁻¹) on in vitro bulblet formation and bulblet size was observed respectively. The superiority of mT over BAP was also found during in vitro rhizogenesis. Shoots raised on the mT medium were healthy and long enough, thus showed better rooting response (63.83%) on ½ MS medium + 0.5 mg L⁻¹ IAA after 4 weeks of incubation. About 89.16% survival rate was recorded for in vitro raised plantlets under ex vitro conditions. Analysis of clonal fidelity of thirteen in vitro regenerated plants was done using SCoT markers. Out of 36 primers, 13 primers showed clearly scorable monomorphic bands, thus displaying genetic uniformity among in vitro regenerated plantlets. This mT mediated protocol can be routinely used for the rapid large scale production of this valuable floriculture crop.
... For example, wild pear (Pyrus syriaca) was preserved at ultra-low temperature by Tahtamouni and Shibli in 1999 where cryopreservation technique was newly introduced to Jordan. Also, micropropagation was used for plant propagation and conservation of the national flower of Jordan's black iris (Iris nigricans) via somatic embryogenesis (Shibli and Ajlouni, 2000). ...
Plant biodiversity expresses one of the principal natural resources for all nations as it provides food, medicine, and shelter for all living besides its important role in balancing ecosystems and mitigating climate change. Jordan has paid great attention to its unique heritage of plant biodiversity since the foundation of the country, especially in terms of conservation. Notable conservation efforts have been made by Jordanian governments during the first hundred years of the country's foundation. These efforts were highly fruitful thanks to the unlimited governmental support to all projects related to the conservation of Jordan Flora. So, on the occasion of the first centenary of the country’s foundation, this review article was prepared to highlight some of the efforts made by Jordan during the past hundred years to conserve this national wealth of plant biodiversity.
... Colchicum chalcedonicum is known as a calcitrant species. Because of its advantages, micro-propagation of corm plants could be an alternative to the conventional techniques for vegetative propagation, increasing many times the multiplication level, enabling the plant materials to be freed from diseases especially for medicinal plants (Shibli & Ajlouni 2000, Chang et al. 2000. ...
Colchicum calcedonicum Azn is one of the endemic species distributed in Turkey, where many endemic plant species occur. It has long-oval shaped corm under the soil, and usually 3-4 leaves on it. In vitro production of endemic species using callus culture has become promising study for conservation. The aim of this study is to generate an efficient callus protocol for in vitro production of C. chalcedonicum. To sterilize the explants, 0.25% (w/v) mercuric chloride (HgCl2) was used for 20 min. In addition to mercuric chloride, surface sterilization was conducted by using 6.5% NaCl with Tween 80 for 30 min. We used 19 different mediums and the primary callus formation was obtained in Murashige & Skoog's basal medium (MS) supplemented with 2,4-D (2 mg L-1), 2IP (0.5 mg L-1), 3% sucrose and 0.05% active carbon. Our study demonstrated the active carbon usage was effective for the primary callus formation. This study is the first report for primary callus formation of C. chalcedonicum. However, our work is a pioneering study to improve callus formation protocol system for in vitro conservation of endemic species C. chalcedonicum.
... The seed oil of Iris Pseudocorous, Iris Sibrica, and Iris missouriansis has been found to contain appreciable amounts of phenols, quinines and resorcinols, substituted with homologous alkyl or alkenyl side chains [14] . Triterpenoids with an unusual spiro -bicyclic structure [15] and irisolidone, irigenin and iridin [16] have been isolated and characterized from rhizomes of Iris germanica. The most abundant protein isolated from iris bulbs has been identified as 1-ribosomal inactivating protein [17] . ...
The present study reports the antibacterial activity of different extracts of five different Iris plant species growing in Kashmir Himalayas. Water,
methanol and hexane extracts of the rhizome of Iris croceae, Iris ensata, Iris germanica, Iris hookeriana and Iris kashmiriana were prepared and screened
for phytochemical studies and antibacterial activities against five bacterial strains including both Gram positive and Gram negative. The different extracts
of these species showed broad spectrum antibacterial activity with methanol extract showing highest zone of inhibition followed by hexane and aqueous
extracts. The phytochemical analysis of the different extracts of these five species revealed the presence of flavonoids, isoflavonoids, glycosides and
tannins, while as Alkaloids were absent in these plant species.
... Improving solanine production in in vitro cultures of Solanum nigrum L. using different chemical and physical factors reported in various medicinal plants by exposing the cultured explants to different growth conditions in vitro such as, using different types of culturing media, growth regulators, sugars and manipulating some physical environmental factors, such as, temperature, moisture and light (Robbins et al., 1996;Shibli and Ajlouni, 2000;Chawla, 2002;Jasmin et al., 2011). ...
Solanum nigrum L. is a medicinal plant of solanaceae family with distinguished therapeutic properties. Traditionally, S. nigrum. had been used as an anti-tumorgenic, antioxidant, hepatoprotective, diuretic, and antipyretic agent. The most important alkaloid member in this plant is solanine. Therefore, this study was conducted to utilize tissue culture techniques for the enhancement of solanine production in the in vitro grown cultures of this promising neglected plant. For callus growth and development experimental part, the highest callus growth parameters (callus diameter (21.4 mm) and callus fresh weight (2202.4 mg) were obtained in callus grown on Murashige & Skoog MS media supplemented with 2,4-Dichlorophenoxyacetic acid (2.0 mg·L-1) plus 1.5 mg·L-1 Thidiazuron. Similar trend was also obtained in cell suspension culture experiment, as maximum growth was recorded at similar hormone combination. Moreover, High-performance liquid chromatography analysis revealed that, solanine was affected by growth regulator type and concentration. The highest solanine levels were obtained when the explants were treated with 6-benzylaminopurine at level of 2.0 mg·L-1, as solanine content reached up to (2.61, 1.53 mg.g-1) for callus and cell suspension, respectively, while, microshoot contained the highest solanine (4.52 mg.g-1 DW) at 6-benzylaminopurine level of 1.6 mg.L-1. Additionally, carbon source had positively affected solanine level, where 0.2 M sucrose resulted in production of the highest amounts (3.13, 2.03 and 1.20 mg.g-1 DW) of solanine in microshoots, callus and cell suspension, respectively. Also, exposing microshoots and callus to light intensity of (100 µmol.m -2 s-1) yielded the highest solanine content (4.03 and 1.26 mg.g-1 DW, respectively),while the lowest solanine levels (1.50 and 0.48 mg.g-1 DW) were observed in plant material exposed to the lowest light intensity treatment (25 µmol.m -2 s-1) .
... Meanwhile, defining the proper type and concentration of the growth regulators for each plant species is crucial for a successful callus growth and development 13 . Moreover, sugar type plays a significant role in growth of in vitro grown plants, and the impact of many carbohydrates on organs growth in addition to secondary metabolites production in these organs were extensively researched 14,15 . ...
... Meanwhile, defining the proper type and concentration of the growth regulators for each plant species is crucial for a successful callus growth and development 13 . Moreover, sugar type plays a significant role in growth of in vitro grown plants, and the impact of many carbohydrates on organs growth in addition to secondary metabolites production in these organs were extensively researched 14,15 . ...
Peganum harmala L. (harmal) is a medicinal plant which has been used for ages in folk medicine for the treatment of many human diseases, including lumbago, asthma, colic, cancer, depression and malaria. Unfortunately, this valuable herb is facing the danger of extinction and decline due to climate change and human activities. So, introducing harmal to tissue culture systems might contribute to efforts of propagation, conservation and production of elite secondary metabolites needed for medicine. For this aim, this study was conducted to experiment the possibility of callus establishment in harmal from leaf discs to be used later on as a row material for in vitro micropropagation. Also the antibacterial activity of callus and wild plant extracts against two bacteria strains was assessed. Callus establishment was experimented by culturing young leaf discs on MS medium supplemented with various concentrations of 2,4-D (0.0, 0.2, 0.6, 1.2 and 2.0 mg/l), where full rate of callusing was obtained at level of 2.00 mg/ L. Moreover, best callus diameter (3.98 cm), fresh weight (3.79 mg) and dry weight (0.096 mg) were obtained in the MS medium plus 1.5 mg/L TDZ in combination with (2.00 mg/ L) 2,4- D. Meanwhile, the highest callus growth parameters were recorded in media supplemented with 0.1 M sucrose, while the lowest growth values were obtained in mannitol experiment at all levels. Moreover, the obtained results for antibacterial activity of callus cultures and wild plant extracts indicated that, both extracts were able to inhibit growth in both bacterial strains, but the wild type extract showed a stronger inhibitory effects on Salmonella sp. and Bacillus subtilis (inhibition zone: 1.6 and 1.8 cm, respectively) than those obtained from callus extract. © 2017, World Food Ltd. and WFL Publishers. All rights reserved.
... Monosacharides also increased somatic embryogenesis and plant regeneration: glucose in Trticum aestivum (Chu et al. 1990), fructose in Coffea cane phora (Fuentes et al. 2000) and mannose in Panax ginseng (Tang 2000). On the contrary, both types of sugars were less effective compared to sucrose in the case of Iris nigricans (Shibli and Ajlouni 2000). The autoclaved fructose was completely ineffective in rooting induction in Quercus suber (Romano et al. 1995). ...
Context: Carbon plays a vital role in plant cell growth and regeneration in artificial media but the source of carbon deserves scientific investigation to analysis their comparative performance. Objectives: To analyze the comparative performance of different carbon sources (glucose, sucrose and sorbitol) in cell growth and regeneration efficiency of banana (Musa spp) cultivars. Materials and Methods: Male flowers of banana cultivars cv. Sabri, Gine and Ranginsagar were used in this experiment. Male flowers were cut into small pieces and they were transferred in petri dishes containing Murashige and Skoog media supplemented with 2 mg/l 2,4-D + 1mg/l NAA + 1mg/l IAA + 1mg/l Biotin + 1mg/l glutamine and 3% (w/v) different sugars: sucrose, glucose, and sorbitol singly or in combinations autoclaved in 121ºC temperature for 20 min. The pH of the medium was adjusted to 5.8. Results: Glucose showed the highest performance in callus induction and cell growth and 3% glucose proved as the optimal dose in media formulation for callus induction and cell growth. Sucrose and sorbitol behaves differently in embryo formation and they produced the highest and lowest number of embryos respectively in regeneration medium. In respect of overall performance the highest percentages of shoot and root formation was obtained in the media containing 3% sucrose. Conclusion: Glucose proved to be the best carbon source in callus induction and cell growth media.
... In this study, 30 g sucrose was found to be the best for embryogenic callus induction. These results were similar to those reported by Ganesan et al. (2007) in okra and in black iris by Shibli and Ajlouni (2000). For successful regeneration of synthetic seeds from the somatic embryos, good quality embryogenic callus is essential (Ganesan and Jayabalan 2004). ...
Synthetic seeds of cauliflower cv. Chillout were developed by encapsulating mature somatic embryos in neutral gel media. Somatic embryos were obtained by optimizing callus and cell suspension cultures of cauliflower. Friable, yellowish embryogenic calli were obtained on MS supplemented with 2 mg/l 2,4‐D and 0.5 mg/l BAP using hypocotyl as explants, while calli were regenerated in media consisting of 5 mg/l BAP, 2 mg/l Kn and 6 mg/l GA3. Somatic embryo‐ genesis was induced in cell suspension culture where auxins were removed in successive steps triggering conversion of globular cells into the heart, torpedo stage (71%) and finally into cotyledonary/somatic embryos (28%). The mature somatic embryos were encapsulated by mixing mature cell suspension with sodium alginate and calcium chloride mixture (1 : 4). Developed synthetic seeds germinated into complete plantlets when placed in neutral gel media. Germination efficiency of synthetic seeds decreased to about 50 per cent after 12 weeks of storage at 4ºC followed by a rapid decrease to zero per cent after 16 weeks. It was also observed that cauliflower plantlets from synthetic seeds survived successfully when transferred to soil demonstrating that cauliflower synthetic seeds is a promising step towards their in vivo direct use.
... In this study, the addition of 2,4-D in the nutrient medium with KIN stimulated the formation of somatic embryos, which reached a maximum at a concentration of 1 mg/ L. Previous studies [44,45] have shown that somatic embryos do not form in different tissue cultures of Narcissus pseudonarcissus without 2,4-D in the nutrient medium. This auxin and cytokinin combination proved to be adequate for the induction of somatic embryogenesis in many plants with rhizomes as well as in bulb-like Astromeria [46], Iris [47], and tulip [48]. Bulbs were maximally regenerated on a medium with an auxin and cytokinin concentration of 1 mg/L each, but their numbers were lower than in a medium enriched with TDZ. ...
Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4°C or on a medium with an increased concentration of sucrose (4.5%) for 30days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1mg/L), and the number and activity of isoforms were followed every 7days for 4weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process.
... The seed oil of Iris Pseudocorous, Iris Sibrica, and Iris missouriansis has been found to contain appreciable amounts of phenols, quinines and resorcinols, substituted with homologous alkyl or alkenyl side chains [14] . Triterpenoids with an unusual spiro -bicyclic structure [15] and irisolidone, irigenin and iridin [16] have been isolated and characterized from rhizomes of Iris germanica. The most abundant protein isolated from iris bulbs has been identified as 1-ribosomal inactivating protein [17] . ...
The present study reports the antibacterial activity of different extracts of five different Iris plant species growing in Kashmir Himalayas. Water, methanol and hexane extracts of the rhizome of Iris croceae, Iris ensata, Iris germanica, Iris hookeriana and Iris kashmiriana were prepared and screened for phytochemical studies and antibacterial activities against five bacterial strains including both Gram positive and Gram negative. The different extracts of these species showed broad spectrum antibacterial activity with methanol extract showing highest zone of inhibition followed by hexane and aqueous extracts.
The phytochemical analysis of the different extracts of these five species revealed the presence of flavonoids, isoflavonoids, glycosides and tannins, while as Alkaloids were absent in these plant species.
... Finally we confirmed that 30 g dm -3 of sucrose was an efficient carbon source for the embryogenic callus induction (Fig. 2). Sucrosemediated high frequency embryogenic callus induction was also observed in black iris (Shibli and Ajlouni 2000). With the other concentrations and forms of carbon sources tested, variations in colour, texture and a delayed response for callus induction was observed. ...
A simple and reliable protocol for regeneration of okra through somatic embryogenesis from suspension cultures has been developed. Embryogenic callus was obtained from hypocotyl explants cultured on media with Murashige and Skoog (MS) salts, Gamborg (B5) vitamins, 2.0 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg dm-3 naphthaleneacetic acid (NAA), 25 mg dm-3 polyvinylpyrrolidone and 30 g dm-3 sucrose. More number and high frequency of healthy embryoids appeared individually in suspension culture containing MS salts, B5 vitamins, 2.0 mg dm-3 2,4-D and 1.0 mg dm-3 kinetin. Formation of cell clusters from the single cells was clearly noticed during ontogeny. Matured embryos at the cotyledonary stage were transferred to agar solidified medium for germination. The best conversion of embrya into plantlets (67.3 %) was recorded on media with half strength MS salts, B5 vitamins, 0.2 mg dm-3 benzylaminopurine (BAP) and 0.2 mg dm-3 gibberellic acid (GA3). The plantlets were transferred to soil and hardened in the plastic pots. After proper acclimatization, the plantlets regenerated through somatic embryogenesis were compared to seed grown plants to observe any variation.
... In this study, 30 g sucrose was found to be the best for embryogenic callus induction. These results were similar to those reported by Ganesan et al. (2007) in okra and in black iris by Shibli and Ajlouni (2000). For successful regeneration of synthetic seeds from the somatic embryos, good quality embryogenic callus is essential (Ganesan and Jayabalan 2004). ...
Synthetic seeds of cauliflower cv. Chillout were developed by encapsulating mature somatic embryos in neutral gel media. Somatic embryos were obtained by optimizing callus and cell suspension cultures of cauliflower. Friable, yellowish embryogenic calli were obtained on MS supplemented with 2 mg/l 2,4-D and 0.5 mg/l BAP using hypocotyl as explants, while calli were regenerated in media consisting of 5 mg/l BAP, 2 mg/l Kn and 6 mg/l GA3. Somatic embryogenesis was induced in cell suspension culture where auxins were removed in successive steps triggering conversion of globular cells into the heart, torpedo stage (71%) and finally into cotyledonary/somatic embryos (28%). The mature somatic embryos were encapsulated by mixing mature cell suspension with sodium alginate and calcium chloride mixture (1: 4). Developed synthetic seeds germinated into complete plantlets when placed in neutral gel media. Germination efficiency of synthetic seeds decreased to about 50 per cent after 12 weeks of storage at 4°C followed by a rapid decrease to zero per cent after 16 weeks. It was also observed that cauliflower plantlets from synthetic seeds survived successfully when transferred to soil demonstrating that cauliflower synthetic seeds is a promising step towards their in vivo direct use.
... Segments of flower-stalk internodes of Ocidium (sweet sugars) were able to directly from somatic embryos and shoot buds, and both embryo and shoot bud-derived regenerants developed into healthy plantlets when potted in sphagnum moss and acclimatized in the green house [13]. A review for the plant regeneration of garlic Allium sativum L. via somatic embryogenesis was given by Lee et al. [14]; somatic embryogenesis from whole mature Bamboo (Bambusa ventricosa) was successfully experimented by Cheah et al. [15], somatic embryogenesis of the endemic black Iris (Iris nigricans) was achieved from callus; cell suspension and protoplast culture by stems for black Iris in Jordan by Shibli et al. [16]. Plant regeneration of tall bearded Iris from suspension cultured cells was protocoled by Wang et al. [17]. ...
In an attempt to propagate and conserve the rare, showy bulbous plants of Iris bismarckiana, newly recorded to the flora of Jordan and to contribute to the conservation the wild Iris species in Jordan, a simple rapid, time consuming protocol has been developed using plant regeneration via somatic embryogenesis. Somatic embryogenesis was induced in zygotic embryo culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (8 mg/L) as the sole plant growth regulator, where both embryogenesis calli and somatic embryos were induced. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos successfully from embryo clusters and transferred onto fresh MS medium. Data obtained were analyzed as a complete random design with three replications. Calli fragments that were transferred to embryogenesis induction medium (EIM) produced white embryo-like globular structures within two weeks. Within three more weeks, clusters of structures at various stages of development could be found on the same callus. The applied technique is rewarding and encouraging for further research on the endangered wild species of Iris in Jordan.
... Monosacharides also increased somatic embryogenesis and plant regeneration: glucose in Trticum aestivum (Chu et al. 1990), fructose in Coffea cane phora (Fuentes et al. 2000) and mannose in Panax ginseng (Tang 2000). On the contrary, both types of sugars were less effective compared to sucrose in the case of Iris nigricans (Shibli and Ajlouni 2000). The autoclaved fructose was completely ineffective in rooting induction in Quercus suber (Romano et al. 1995). ...
Context: Carbon plays a vital role in plant cell growth and regeneration in artificial media but the source of carbon deserves scientific investigation to analysis their comparative performance. Objectives: To analyze the comparative performance of different carbon sources (glucose, sucrose and sorbitol) in cell growth and regeneration efficiency of banana (Musa spp) cultivars. Materials and Methods: Male flowers of banana cultivars cv. Sabri, Gine and Ranginsagar were used in this experiment. Male flowers were cut into small pieces and they were transferred in petri dishes containing Murashige and Skoog media supplemented with 2 mg/l 2,4-D + 1mg/l NAA + 1mg/l IAA + 1mg/l Biotin + 1mg/l glutamine and 3% (w/v) different sugars: sucrose, glucose, and sorbitol singly or in combinations autoclaved in 121ºC temperature for 20 min. The pH of the medium was adjusted to 5.8. Results: Glucose showed the highest performance in callus induction and cell growth and 3% glucose proved as the optimal dose in media formulation for callus induction and cell growth. Sucrose and sorbitol behaves differently in embryo formation and they produced the highest and lowest number of embryos respectively in regeneration medium. In respect of overall performance the highest percentages of shoot and root formation was obtained in the media containing 3% sucrose.
Iris is a perennial flowering plant, usually cultivated through seeds or bulbs. However, due to the limitations of traditional reproduction, the establishment of a callus regeneration system is particularly important, and callus induction and plant regeneration are the keys to transgenic technology. The callus regeneration system was studied using the I. sanguinea flower stem as explants. The experiment investigated the impact of different disinfectant solution concentrations, disinfection exposure times, and hormone concentrations on the explant growth status at various culture stages. The research analyzed the impact of the experimental variables on explant regeneration to determine the optimum callus regeneration system for I. sanguinea. In this research, it was determined that when I. sanguinea flower stems, that were used for explants, were treated with 75% alcohol for 30 s and 2% NaClO for 8 min for disinfection, there was a 54.48% callus induction rate. The callus induction medium was Murashige and Skoog medium (MS) + 6-benzylaminopurine (6-BA) 1.0 mg/L + 1-naphthylacetic acid (NAA) 0.2 mg/L + 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg/L for 40 d. The optimum medium for callus multiplication was MS + 6-BA 0.5 mg/L + 2,4-D 0.5 mg/L, while the optimum medium for callus adventitious bud induction was MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L + kinetin (KT) 0.5 mg/L, 60 d. The adventitious bud differentiation rate from flower stem calli was 46.03%.
In this study the effects of different sucrose concentrations, and the combinations of jasmonic acid (JA) with auxins (IAA or NAA) or with cytokinin (2iP) on the bulb induction and rooting of in vitro plantlets of Hyacinthus orientalis subsp. chionophyllus Wendelbo, which is endemic in Turkey, were investigated. The effect of four different sucrose concentrations (30, 45, 60 and 90 g L-1) on bulb formation in tissue culture was investigated. These plantlets were cultured on MS medium supplemented with several concentrations and combinations of JA (0.0, 1.0, 2.0 mg L-1) and 2iP (0.0, 0.25 and 0.50 mg L-1), IAA or NAA (0.5, 1.0 mg L-1). In JA-2iP treatment, the highest number of bulblets (13.7 number/explant) was obtained by the combinations of JA 1.0 mg L-1 + 2iP 0.25 mg L-1. Also, the largest bulblets with the mean diameter of 7.9 mm were found on MS medium supplemented with JA 2.0 mg L-1. In JA-Auxin treatment, the mean root number per bulblet was highest (17.9 number/explant) and root formation rate was maximum (81.14%) on MS medium supplemented with IAA 1.0 mg L-1 + JA 2.0 mg L-1 .
In this study the effects of different sucrose concentrations, and the combinations of jasmonic acid (JA) with auxins (IAA or NAA) or with cytokinin (2iP) on the bulb induction and rooting of in vitro plantlets of Hyacinthus orientalis subsp. chionophyllus Wendelbo, which is endemic in Turkey, were investigated. The effect of four different sucrose concentrations (30, 45, 60 and 90 g L-1) on bulb formation in tissue culture was investigated. These plantlets were cultured on MS medium supplemented with several concentrations and combinations of JA (0.0, 1.0, 2.0 mg L-1) and 2iP (0.0, 0.25 and 0.50 mg L-1), IAA or NAA (0.5, 1.0 mg L-1). In JA- 2iP treatment, the highest number of bulblets (13.7 number/explant) was obtained by the combinations of JA 1.0 mg L-1 + 2iP 0.25 mg L-1. Also, the largest bulblets with the mean diameter of 7.9 mm were found on MS medium supplemented with JA 2.0 mg L-1. In JA – Auxin treatment, the mean root number per bulblet was highest (17.9 number/explant) and root formation rate was maximum (81.14%) on MS medium supplemented with IAA 1.0 mg L-1 + JA 2.0 mg L-1.
APolygonatum stenophyllum Maxim. is an important endangered plant belonging to the family Liliaceae. A method was developed for the rapid micropropagation of P. stenophyllum through plant regeneration from rhizome (1-year, 3-years, and 5-years) explant-derived calli. The rhizome segments were cultured in Murashige and Skoog (MS) medium supplemented with varying concentrations of 2,4-D (0, 0.5, 1.0, 1.5 mg.L⁻¹) for callus induction. In media supplemented with 0.5 mg.L⁻¹ of 2,4-D, 87% of 3-years rhizome produced callus. Subsequently, the callus was transferred to 1/2MS medium supplemented with various concentrations of IAA, IBA, NAA, and 2,4-D (0, 0.1, 0.5 and 1.0 mg.L⁻¹) for adventitious shoot formation. The highest percentage of adventitious shoot induction (57%) was observed in 1/2MS medium containing 0.5 mg.L⁻¹ of NAA. Elongation of the adventitious shoot was achieved in 1/2MS medium supplemented with 0.1 mg.L⁻¹ of BA. Rooting was achieved in 1/2MS medium without any hormones. It is hypothesized that the stated in vitro propagation protocol will be useful for conservation and mass propagation of the endangered Polygonatum stenophyllum Maxim. for bioresources.
The initiation stage of Iris ensata Thunb. tissue cultures is optimized when flower fragments containing meristem tissue and with morphogenetic capacity are used. Direct regeneration of inflorescence axis (rachis) and perianth tube explants in tissue culture led to the formation of shoots typical of this species. Examination of the anatomical structures of the ovary, pistil, and filament did not show any zones of meristematic activity, as examined over a 30-d cultivation period using these organs. These results could explain the lack of regenerative capacity of these explants.
This experiment was conducted to establish the in vitro propagation of Dendrobium moniliforme through protocorm like body (PLB) induction from the culture of leaf, stem and root explants. Frequency of PLB formation from root explants was higher than those of leaf and stem explants. PLB's proliferation in MS medium including 1.0 mg/L NAA was better than 1.0 mg/L IBA. Frequency of PLB's proliferation on medium with various concentrations of BA, Kinetin, Tidiazuron (0. 1.0, 3.0 mg/L) and NAA (0, 1.0 mg/L) was tested. The maximun proliferation of PLB's was obtained on MS medium supplemented with 3.0 mg/L BA and 1.0 mg/L NAA after 6 weeks of culture. Addition of 200 mg/L activated charcoal (AC) and 30 g/L sucrose was effective on PLB proliferation for D. moniliforme.
The objective of this study was the improvement of in vitro plant regeneration of few German iris varieties (Blue Bird Wine, Tangerine Charm, Glazed Orange, Jane Philips) via somatic embryogenesis using leaf base. For callus and embryogenesis induction MS medium added with dichlorophenoxyacetic acid, α-nafthyl acetic acid, and kinetin was used. The frequency of explants with regeneration ability was up to 40% in all four varieties selected for this study. The number of regenerants per explant varied between 0 and 8. For micropropagation, the most crucial aspect is to maintain the genetic conformity, the regenerants have to be identical to the mother plant. For the assessment of in vitro stability/instability of the regenerated plants, we used RAPD molecular markers. For DNA extraction young leaves from 10 in vitro growing plants were sampled. The 10 RAPD primers were tested for detection of the genetic polymorphism among regenerated and mother plants. Only 5 produced reproducible fragments. RAPD analysis did not reveal any type of polymorphism between randomly selected in vitro plants and the mother plant, indicating the clonal nature of progenies. Our results showed an advantage of this in vitro propagation protocol because the risk of somaclonal variation is reduced.
A rapid and efficient system for papaya (Carica papaya L. cv. Co7) regeneration through indirect somatic embryogenesis has been established. Embryogenic calli were obtained from zygotic embryos (excised from 110-120 dayold fruits) on CIM [half-strength MS (Murashige and Skoog, 1962) basal salts and vitamins, 400 mg/L of glutamine, 6% sucrose, 4 g/L phytagel] supplemented with varying concentrations 0.5-12.0 mg/L of 2,4-D. The frequency of somatic embryo formation was as high as 54.35% when cultured on medium containing 2.0 mg/L of 2,4-D. Suspension cultures were superior to static cultures (solid medium) for the maturation of somatic embryos. Matured (cotyledonary stage) somatic embryos cultured on MSR medium supplemented with 0.4 mg/L BAP and 0.02 mg/L NAA showed regeneration efficiency up to 62.32%. The emerging shoots with 2-3 trilobed leaves were rooted on MSL medium containing 1.0 mg/L of IBA with efficiency of 60.07%. Plants with well-developed shoots and roots were subsequently hardened on a potting mixture enriched with arbuscular mycorrhiza. The plantlets from zygotic embryo explants were morphologically similar and normal. No phenotypic variations have been observed in field-established plants. This regeneration protocol assures a high frequency of somatic embryogenesis, maturation and regeneration into plantlets.
To establish the optimum conditions of in vitro plant regeneration, the leaf, rhizome, and root explants of Belamcanda chinensis were cultured on the MS medium supplemented with different concentration of 2,4-D and BA. The callus induction was more effective in the root explants than the leaf and rhizome explants, and was the best on MS medium containing 3.0 mg/L 2,4-D or 1.0 mg/L 2,4-D and 3.0 mg/L BA. The highest numbers of shoots were regenerated when callus were cultured on MS medium containing 3.0 mg/L 2,4-D for 4 weeks. However, the multiple shoots were induced on MS medium supplemented with the combination of 2,4-D and BA. The normal root formation from shoot was effective on the MS medium lacking any plant growth regulators. For acclimatization, the regenerated plantlets were cultured on MS medium without sucrose and plant growth regulators for 2 weeks, and then transferred to the pot.
Background: Bacterial Leaf Blight (BLB) is the most devastating disease of rice. This disease can reduce grain production by 20-50% and yield by 25%. The disease is widespread in Asia, United State, Latin America and Australia. In Pakistan, it is reported from all rice growing areas and it is increasing its area year by year. All famous Pakistan’s Basmati varieties are susceptible to BLB disease. The most economical way of controlling disease is to produce resistant varieties. Many different compounds are involved in resistance mechanism in host including phenolic compounds.
Methods: Plants inoculated with Xanthomonas oryzae pv. oryzae isolates i.e. 78.3 and 2.2 caused disease on all varieties were selected for investigation of phenolic compound. Randomized complete block design was used in field for inoculation and leaf collections for phenolic compounds determination. Data was phenolic compounds were analyzed through factorial ANOVA.
Results: The response of four varieties was different when inoculated with BLB isolate 2.2. Basmati 2000 variety of rice produced maximum amount of phenolic compounds after one week of inoculation of bacterial isolate 2.2 while the minimum total phenols were found in basmati 385 at 0 hrs. of inoculation (before inoculation). Xoo isolate 78.3 produced maximum phenolic compounds after 2 weeks in Basmati 2000.
Conclusion: Biochemical resistance due to high phenolic contents in Basmati-385 and Basmati-2000 is suggested. High phenol production may be due to loss of virulence in bacterial isolates. Both 78.3 and 2.2 Xoo isolates are significantly different in producing total phenols in all the varieties tested.
A protocol was developed for efficient plant regeneration of Iris germanica L. 'Skating Party' from suspension cultures. Suspension cultures were maintained in Murashige and Skoog (MS) basal medium (pH 5.9) supplemented with 290 mg·L-1 proline, 50 g·L-1 sucrose, 5.0 μM 2,4-D, and 0.5 μM Kin. Suspension-cultured cells were transferred to a shoot induction medium (MS basal medium supplemented with 10 mg·L-1 pantothenic acid, 4.5 mg·L-1 nicotinic acid, 1.9 mg·L-1 thiamine, 250 mg·L-1 casein hydrolysate, 250 mg·L-1 proline, 50 g·L-1 sucrose, 2.0 g·L-1 Phytagel, 0.5 μM NAA, and 12.5 μM Kin). Cell clusters that proliferated on this medium differentiated and developed shoots and plantlets in about 5 weeks. Regeneration apparently occurred via both somatic embryogenesis and shoot organogenesis. A series of experiments was conducted to optimize conditions during suspension culture to maximize subsequent plant regeneration. Parameters included 2,4-D and Kin concentrations, the subculture interval, and the size of cell clusters. The highest regeneration rate was achieved with cell clusters ≤280 μm in diameter, derived from suspension cultures grown for 6 weeks without subculturing in liquid medium containing 5 μM 2,4-D and 0.5 μM Kin. Up to 4000 plantlets with normal vegetative growth and morphology could be generated from 1 g of suspension-cultured cells in about 3-4 months. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); kinetin (Kin); 1-naphthaleneacetic acid (NAA).
Plantlet regeneration was achieved in culture of mature Iris setosa Pall. embryos via somatic embryogenesis and organogenesis, sequentially changing culture media. During short-term culture, embryogenic calli (6 %) were induced in MS mineral solution containing 5 % sucrose, 0.5 % agar and (in mg L-') 2,4-D 5, Kin 1, Pro 250 and CH 250. A reduced level of 2,4-D 1 mg L-1) in the above medium during the second subculture led to an increase of cultures forming embryogenic calli (39 %), organogenic calli (6 %) and non embryogenic calli (32 %. A thirty-day subculture of OC in MS with 2 % sucrose and (in mg L-1) AS 80, Tyr 100, GA3 1 and BAP 1 led to an increase in OC from 6 % to 60 % and adventitious bud regeneration. Shoot multiplication proceeded in the same medium with reduced GA3 (0.1 mgL-1. Somatic embryos developed into plantlets in MS containing 2,4-D (0.1 or 1 mg L-1) and Kin 1 mg L-1. Regenerated shoots were rooted in MS solid and liquid media, 1 % sucrose and (in mg L-') IAA 1, Kin 3 and GA31. Following transfer to soil, the plantlets with diploid chromosome number (2x = 2n = 36) developed normally.
Plantlets were regenerated from callus of Iris pallida, an important perfume plant. Only the leaf base attached to the rhizome had the ability to generate yellow-colored callus on LS medium supplemented with 1 mg/l 2,4-D and 0.1 mg/l KT in the dark. Yellow calli grew with partial differentiation into white tissue, probably embryogenic, during subculture on the same medium with a 16-h photoperiod. Only yellow-colored calli with the white tissue could differentiate into plantlets after transfer to kinetin- or gibberellin- supplemented LS medium. Regenerated plantlets which grew on the medium without growth regulators were transferred to the soil. After 2 years of cultivation in soil, the regenerated plants flowered and formed rhizomes. The components of the essential oil in the rhizome of regenerated plants were essentially the same as those in natural plants.
Irones are violet-scented ketonic compounds contained in the rhizome of certain species of iris. As cultivation of the iris tends to decrease, a selection program has been initiated to find the best performing clones in terms of growth and yield. Parallel to this selection, in vitro regeneration studies have been carried out in order to multiply interesting clones. A method of rapid multiplication by somatic embryogenesis associated with multibudding was developed. Callus was obtained from leaf bases, flower pieces or rhizome apices; the best explants were flower pieces. The induction media used to obtain embryogenic callus were Murashige & Skoog (1962) media. Assays with adding of proline in these media have showed that it could double the yield of embryogenic callus. The embryogenic expression medium was the Knudson's orchid agar (Knudson 1946) medium. Conformity of the plants obtained was checked by comparing their chemotypes with those of the mother plants.
The effects of various components of culture media and culture conditions on plantlet regeneration in cell suspension cultures of rice (Oryza sativa L.) were studied. It was found that plantlet regeneration was affected by the concentrations of medium components such as plant growth regulators, carbohydrates, inorganic nitrogen salts, and osmoticum in the N6 medium (Chu et al., 1975). Addition of 0.1 mg/L NAA in combination with 0.1 mg/L kinetin improved regeneration. A low level of sucrose (7g/L) promoted regeneration, and an increased sucrose level (above 20g/L) was highly inhibitory. A concentration of KNO3 twice as high as that of the N6 medium resulted in a doubling in the number of regenerated plantlets. Although sorbitol was necessary for plantlet regeneration, mannitol inhibited regeneration. This contradictory effect between common osmotica on regeneration suggests that sorbitol was physiologically rather than just osmotically active. In addition to the components of the regeneration medium, culture conditions such as medium volume and volume of culture vessel were found to be important factors affecting regeneration. Culture in 300 mL flasks containing 30 mL of medium resulted in a two-fold increase in the number of regenerated plantltets in comparison with culture in 100 mL flasks containing 20 mL of medium. Under optimum conditions, more than 200 plantlets per 20 mg of cell clusters regenerated in suspension cultures in 6 weeks.
In Iris germanica L., 'G1', 'Adorn' and 'Rococo', induction and proliferation of embryogenic calli were achieved by culture
of leaf-base explants on Murashige and Skoog (MS) medium supplemented with 1 mg l−1 2,4-D, 1 mg l−1 kinetin, 200 mg l−1 casein hydrolysate, 250 mg l−1 proline, 30 g l−1 sucrose and 2.5 g l−1 gellan gum. Among these cultivars, however, only in 'G1' could a suspension culture be established using a liquid N6 medium
with 1 mg l−1 2,4-D, 1 mg l−1 kinetin, 200 mg l−1 casein hydrolysate, 250 mg l−1 proline and 30 g l−1 sucrose. Murashige and Skoog medium with 1 mg l−1 gibberellic acid (GA3), 30 g l−1 sucrose and 2.5 g l−1 gellan gum was suitable for somatic embryo formation from suspension cells. When the somatic embryos were transferred to
solid, growth regulator-free MS medium and subcultured monthly, 36 shoots were obtained from 20 mg suspension cells.
A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m-2 s-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.
Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.
Pigment producing in vitro cells of Vaccinium pahalae (ohelo) were tested for their ability to survive cryopreservation and
retain pigment-production capacity after encapsulation-dehydration. Preculture of cells for 6 to 8 days in a medium containing
1.0 M sucrose was essential before dehydration. Reduction of bead water content before quenching in liquid nitrogen was highly
correlated (r = 0.94) with increased survival rate in cells after cryopreservation. Dehydration of beads for 4 h was satisfactory
for survival of cells. After quenching in liquid nitrogen, colored cells became pale, but pigment content was recovered once
cells resumed growth. After three subcultures, cells regained their maximum capacity for pigment accumulation. The percentage
of colored-to-total cell volume was not influenced by cryopreservation. Encapsulation-dehydration and cryopreservation did
not diminish the capacity of cells to produce anthocyanins and other flavonoids.
Protoplasts were isolated enzymatically from suspension cultures derived from embryogenic calli induced by leaf base culture of Iris germanica. In protoplast culture, the effects of glucose concentration, different sugars and combinations of 2,4-D and KIN on protoplast division and colony formation were examined. N6 medium supplemented with 0.1–1 mg/l 2,4-D, 1 mg/l KIN, 200mg/l casein hydrolysate, 250 mg/l proline, 0.2 M glucose and 20 g/l agarose was suitable for protoplast division and colony formation. When colonies formed were transferred onto hormone-free MS medium, many plantlets were regenerated through somatic embryogenesis. Thus, we could establish a plant regeneration system from protoplasts of I. germanica.
Somatic embryos of black iris (Iris nigricans) were cryopreserved using encapsulation-dehydration. Embryos size of 2-4 mm gave the highest survival after cryopreservation. Preculturing embryos on medium containing 0.75 M sucrose for 3 d at 22 degree C, then at 30 degree C for 1 d ensured maximum survival (60%). Viable embryos were brown in color after cryopreservation and during early recovery while dead ones where light brown or creamy. The first sign of regrowth was noted after 15 d. The final regrowth percentage of living embryo was 90% after 35 d. Only 10% of the embryos in all treatments showed secondary embryogenesis. Direct sowing in vivo of cryopreserved embryos was not successful in achieving germination.
Shoot-tip culture in the production of virus-indexed Dutch iris
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