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In vitro Propagation of Endangered Iris Species
Abstract
Calli from Iris petrana and I. atrofusca were produced from flower bases, leaf bases and anthers. The flower bases responded positively to the culture conditions and gave the best results. Callus induction medium was Murashige and Skoog (MS) supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 μM Kinetin and 4.5 μM 1-naphthaleneacetic acid (NAA). Formation of green structures was noticed on calli of both species after transferring calli to callus culture medium (MS supplemented with 4.5 μM N<sup>6</sup>-benzyladenine (BA), 0.45 μM 2,4-D and 4.5 μM NAA). Embryo-like structures were appeared after transferring calli to embryogenesis induction medium (MS supplemented with 4.5 μM 2,4-D, 0.5 μM Kinetin, 4.5 μM NAA and 300 mg L<sup>-1</sup> proline). Both green and albino plantlets were observed after calli were transferred to regeneration medium {MS supplemented with 4.5 μM BA, 0.45 μM 2,4-D and 0.49 μM indol-3-butyric acid (IBA)}.
... (Boŝnjak Mihovilović et al. 2019), (Kereša et al. 2009), Iris odaesanensis Y. N. Lee (Bae et al. 2013), Iris germanica (Xu et al. 2015;Cerasela et al. 2014), Iris reichenbachii (Jevremović et al. 2015) and etc. In the reproduction of Iris genus in vitro condition, various explants were used to induce callus, including: rhizome (Bae et al. 2012(Bae et al. , 2013, leafbase (Bae et al. 2013;Jevremović et al. 2013;Stanišić et al. 2015;Boŝnjak Mihovilović et al. 2019;Cerasela et al. 2014;Al-Gabbiesh et al. 2006), zygotic embryos (Jevremović et al. 2009(Jevremović et al. , 2013(Jevremović et al. , 2015Boltenkov et al. 2004), root cutting (Bae et al. 2012(Bae et al. , 2013, Leaf (Bae et al. 2012) and shoot apex (Xu et al. 2015). In Iris sibirica, the use of TDZ (4.54 µM) in the MS medium culture only led to the induction organogenic callus from the leafbase explant; While the produced calluses in this medium culture with 2,4-D (4.52 µM) were divided into three groups: embryogenic calluses (4.5%), organogenic calluses (12.4%) and calluses without regeneration (77.3%) (Stanišić et al. 2015). ...
... The positive effect of callus induction in MS medium culture containing 4.5 µM 2,4-D, 0.5 µM Kin and 4.5 µM NAA has been confirmed in iris plant. Also, the simultaneous use of 4.5 µM BA, 0.45 µM 2,4-D and 0.49 µM IBA caused shoot regeneration from callus (Al-Gabbiesh et al. 2006). Cultivation of Iris ensata Thunb. ...
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.
... 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.
... Although tissue culture propagation of Irises has been accomplished with various explants, continuous availability of the source of explant and its aseptic nature, and ability to respond to induction and propagation methods are, however, crucial for the successful establishment of a regeneration protocol 6,10,12 . Disinfection of the source mate- rial prior to inoculation or isolation of explant is essential as microorganisms that reside on the explant surface may find the in vitro environment optimal for their growth and affect the overall success of tissue culture 6,10,23 . Direct contact of the explants with disinfectants, in order to fully eliminate the contaminants, can severely affect their regeneration potency 10 . ...
Abstract Iris sanguinea is a perennial flowering plant that is typically cultivated through seeds or bulbs. However, due to limitations in conventional propagation, an alternate regeneration system using seeds was developed. The protocol included optimization of sterilization, stratification and scarification methods as iris seeds exhibit physiological dormancy. In addition to chlorine-based disinfection, alkaline or heat treatment was used to break seed dormancy and reduce contamination. When seeds were soaked in water at 80 °C overnight, and sterilized with 75% EtOH for 30 s and 4% NaOCl solution for 20 minutes, contamination was reduced to 10% and a 73.3% germination was achieved. The germinated seedlings with 2-3 leaves and radicle were used as explants to induce adventitious buds. The optimal MS medium with 0.5 mg L−1 6-benzylaminopurine, 0.2 mg L−1 NAA, and 1.0 mg L−1 kinetin resulted in 93.3% shoot induction and a proliferation coefficient of 5.30. Medium with 0.5 mg L−1 NAA achieved 96.4% rooting of the adventitious shoots. The survival rate was more than 90% after 30 days growth in the cultivated matrix. In conclusion, a successful regeneration system for propagation of I. sanguinea was developed using seeds, which could be utilized for large-scale propagation of irises of ecological and horticultural importance.
alışmaları sonucunda elde edilen sürgünler ile köklendirme çalışmaları yapılmıştır. Çalışmalar sonucunda, en iyi çoğaltım oranı eksplant başına 10,3 adet sürgün ile 0,5 mg l-1 NAA + 1,0 mg l-1 BAP içeren MS ortamında gözlenmiştir. Çoğaltılan sürgünlerin köklendirilmesinde, en yüksek köklenmenin sağlandığı ortam, bitkicik başına 16,5 adet kök ile 1,0 mg l-1 IBA+2,0 mg l-1 JA içeren MS ortamında bulunmuştur. Bitkiciklerin bulb çapları incelendiğinde ise en iyi bulb gelişiminin 4,19 mm bulb çapı artış ortalamasıyla 1,0 mg l-1 IBA+0,2 mg l-1 NAA+2,0 mg l-1 JA ve 1,0 mg l-1 IBA içeren MS ortamlarından elde edildiği gözlenmiştir. ABSTRACT: Tissue culture techniques, one of the biotechnological applications for the protection of plant biodiversity, are often used especially in the protection of endemic plants. Iris sari Schott ex Baker, an endemic species of the family Iridaceae, has natural spreading areas in our country. The aim of our study is to investigate the vegetative multiplication and effective rooting possibilities of Iris sari Schott ex Baker, one of the geophytes of Turkey, with in vitro techniques. For this purpose, immature embryos belonging to the Iris sari Schott ex Baker, plant have been used as initial explant. Rooting studies were done with shoots obtained as a result of in vitro proliferation studies. As a result of the studies, the best rate of reproduction was observed in MS medium containing 0.5 mg l-1 NAA + 1.0 mg l-1 BAP with 10.3 shoots per explant. In rooting of proliferated plantlets, the medium in which the highest rooting is achieved was found in MS medium containing 1.0 mg l-1 IBA+2.0 mg l-1 JA with 16.5 roots per plant. When the bulb diameters of plantlets were examined, it was observed that the best bulb development was achieved from MS medium containing 1.0 mg l-1 IBA+0.2 mg l-1 NAA+2.0 mg l-1 JA and 1.0 mg l-1 IBA with a bulb diameter increase average of 4.19 mm.
Iris severe mosaic virus (ISMV, Potyviridae) can threaten the sustainability of iris production and the marketability of the plants. Effective intervention and control strategies require rapid and early detection of viral infections. The wide range of viral symptoms, from asymptomatic to severe chlorosis of the leaves, renders diagnosis solely based on visual indicators ineffective. A nested PCR-based diagnostic assay was developed for the reliable detection of ISMV in iris leaves and in rhizomes. Considering the genetic variability of ISMV, two primer pairs were designed to detect the highly conserved 3' untranslated region (UTR) of the viral genomic RNA. The specificity of the primer pairs was confirmed against four other potyviruses. The sensitivity of detection was enhanced by one order of magnitude using diluted cDNA and a nested approach. Nested PCR facilitated detecting ISMV on field-grown samples beyond the capabilities of a currently available immunological test, and in iris rhizome, which would facilitate ensuring clean stock is planted. This approach dramatically improves the detection threshold of ISMV on potentially low virus titer samples. The study provides a practical, accurate, and sensitive tool for the early detection of a deleterious virus that infects a popular ornamental and landscape plant.
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%.
Tissue culture techniques, one of the biotechnological applications for the protection of plant
biodiversity, are often used especially in the protection of endemic plants. Iris sari Schott ex Baker, an endemic species of the
family Iridaceae, has natural spreading areas in our country. The aim of our study is to investigate the vegetative
multiplication and effective rooting possibilities of Iris sari Schott ex Baker, one of the geophytes of Turkey, with in vitro
techniques. For this purpose, immature embryos belonging to the Iris sari Schott ex Baker, plant have been used as initial
explant. Rooting studies were done with shoots obtained as a result of in vitro proliferation studies. As a result of the studies,
the best rate of reproduction was observed in MS medium containing 0.5 mg l-1 NAA + 1.0 mg l-1 BAP with 10.3 shoots per
explant. In rooting of proliferated plantlets, the medium in which the highest rooting is achieved was found in MS medium
containing 1.0 mg l-1 IBA+2.0 mg l-1
JA with 16.5 roots per plant. When the bulb diameters of plantlets were examined, it was
observed that the best bulb development was achieved from MS medium containing 1.0 mg l-1IBA+0.2 mg l-1NAA+2.0 mg
l
-1JA and 1.0 mg l-1IBA with a bulb diameter increase average of 4.19 mm
The article gives the results of research work in morphogenesis and regenerative capacity of specimen in vitro culture of floral axis and perianth of some cultivars of I. sibirica L. It is stressed that morphogenesis proceeds in accordance to hemmogenesis and rhizogenesis, omitting callusogenesis. The spears formed de novo are solely endogenous. The spears close to the perianth carried floral elements at prophyllum primordium, and had typical monocotyledon constitution.
Never before in history has the issue of preserving biodiversity been of such significance than today, to the extent that it has become an important global issue. Nowadays, humans have a massive impact on biodiversity whether in rural, urban, or wilderness settings. Besides, biodiversity today is also being significantly constrained by climate change and the introduction of new varieties of plants. Plant tissue culture is a method that can be useful for collecting, storing, and multiplicating of plant germplasms. Nowadays, many valuable germplasms are in danger of extinction, especially in Iran. Thus, there is a necessity to preserve valuable Iranian ornamental geophytes such as Oxalis articulata, Eminium jaegeri, Muscari kurdicum, Leopoldia tijtijensis, Gagea calcicola, Tulipa faribae, Gagea alexii, and Allium. In this study, plant tissue culture as well as in vitro conservation techniques as means for medium- and long-term conservation of Iranian ornamental geophytes is reviewed. There are only few studies on plant tissue culture of Iranian ornamental geophytes despite their great importance in Iran. To our knowledge, there is no report on cryopreservation of the abovementioned plants. In conclusion, the methods presented in this review can be utilized for conserving these valuable germplasms for future generations. © 2018, Institute of Bioorganic Chemistry. All rights reserved.
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.
Amentoflavone has been characterised from leaves of Patersonia glabrata. This is the first report of a biflavone in the Monocotyledoneae. The quinone plumbagin, a characteristic constituent of three dicotyledonous families, is now found to be a useful marker for the genus Aristea; it also occurs in two Sisyrinchium species and in Sparaxis tricolor. Mangiferin, a C-glucosylxanthone known previously in the Iridaceae only from Crocus, Iris and Gynandriris has now been found in Eleutherine, Rigidella, Gelasine and Tigridia. results is discussed.
Suspension cultures of Vitis vinifera were cultured in different media in order to establish a model system for promoting high levels of phenolic substances identical with those found in wine. These media were: a low sucrose maintenance medium (MM) and four high sucrose media (differing mainly in sucrose and mineral contents) which were shown to induce secondary metabolism. In MM medium, polyphenol accumulation in the cells was low, and concentrations of 0.1 mg/gfw for condensed tannins and 0.3 mg/gfw for anthocyanins were reached within two weeks of cultivation. Values of 1.4 and 6.4 mg/gfw, respectively, were obtained with a low nitrate and high sucrose medium (HM1), but cell proliferation was reduced. To obtain a maximal production of polyphenols, we investigated the most effective conditions for cell growth and polyphenol production (a high mineral and high sucrose medium, IM1; inoculum dilution of 1.25:10). Under these conditions, the cells produced mainly anthocyanins (1100 mg/l), proanthocyanidins (300 mg/l) and catechins (25 mg/l).
Higher plants are not only the most important producers of natural products including foods, wood, fibers and oils, but also the richest sources of medicinal substances. In recent years, however, there have been increasing difficulties in securing an ample supply of medicinal plants because of a drastic decrease in plant resources due to human disturbance of the natural environment, ruthless exploitation, increasing labor cost, and technical and/or economic difficulties in cultivating wild plants. We hope to bypass some of these difficulties by introducing cell culture systems for medicinal compound production.
The first and, to date, the most extensive practical application of tissue culture techniques to horticultural crops involves the multiplication of ornarmental plant species. The early work by Morel (4) with orchids, the development of the Murashige and Skoog medium (5), and the efforts of Murashige (6, 7) to enhance the practical side of tissue culture science stimulated the development and widespread use of tissue culture as a means of micropropagating ornamentals. Herbaceous ornamentals have adapted to tissue culture techniques with relative ease; more attention and efforts have been required to successfully apply in vitro culture techniques to woody plant species.
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.
17 species of monocotyledons, other than orchids (treated in a separate paper) from Jordan are discussed. Some species are recorded for the first time; others are rare and recorded from new localities. The taxa belong to the families Araceae, Gramineae and Iridaceae. Maps showing their distribution in Jordan are provided, as well as remarks on their habitat, especially for the genus Crocus.
Zusammenfassung Die verschiedenen Differenzierungsmuster im Kallus der Gattungen Iris und Asparagus wurden histologisch untersucht. Es konnte im Kallus aus explantierten Sproßscheiteln von Iris-Sektionshybriden auf einem Medium nach Linsmaier/skoog (L/S) mit 2,4-D-Zusatz die Entstehung einer großen Zahl nichtzygotischer Embryoide nachgewiesen werden. Die bipolare Struktur entwickelte sich verhältnismäßig spät aus kleinzelligen, rundlichen oder länglichen Arealen, die sich mit einem Protoderm allmählich gegen den übrigen Kallus abgrenzten. Nach Abschluß der embryoiden Organisation wiesen die kallusbürtigen Embryonen das typische Strukturmuster eines monokotylen Embryos auf und keimten wie zygotische Embryonen ohne Zusatz von Wachstums-regulatoren auf nährstoffarmen Medien. Aus Sproßsegmenten von xx-, xy- und yy-Stämmen von Asparagus officinalis wurde auf L/S durch Zusatz von NES und Kinetin ein üppiger Kallus induziert. Auf IES - und BAP-haltigen Medien im Verhältnis 1: 1 oder 10: 1 haben sich neben Sprossen und Wurzeln auch embryoide Formen entwickelt. Die Sproßanlagen entstanden einzeln oder in Dreiergruppen am Kallusrand, die Wurzelanlagen wurden im Innern des Kallusgewebes aufgefunden. Der embryogene Charakter globulärer Formen, bestehend aus plasmareichen Zellen im peripheren Kallusbereich, wurde beschrieben. Bipolare Gebilde mit Sproß-und Wurzelpol, Prokambiumstrang und Kotyledoanlage wichen mehr oder weniger stark vom Bau zygotischer Embryonen ab. In der Weiterkultur auf wuchsstoffarmen Medien keimten diese Embryoide wie zygotische Embryonen. Die Mehrzahl der in Büscheln aus älteren Kalluskulturen herauswachsenden 3 bis 4 mm großen und äußerlich von Embryoiden nicht zu unterscheidenden weißen Gebilde sind hypertrophierte Phyllocladienhomologe. Sie gehören entweder zu einem breiten Scheitelmeristem und stellen somit einen aberranten Vegetationskegel dar oder sie verwachsen unter Aussparung eines zentralen Hohlraumes. In dieser Höhle mit einer basalen Öffnung befinden sich ein bis drei normal strukturierte Sproßknospen.
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.
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.