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In vitro plant regeneration in zygotic embryo culture of Iris reichenbachii
Abstract
Plant regeneration from mature zygotic embryo culture of Iris reichenbachii was studied. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D, 0-10 mg L-1) on induction of morphogenesis in vitro is investigated. The best response for callus induction is observed on MS medium with 10 mg L -1 2,4-D. White embryogenic, yellow non-embryogenic and green organogenic calli are observed. The fresh weight, as well as, percentage portion of embryogenic and organogenic calli is 2,4-D dependent. Shoots are succesfuly multiplied by adventitive organogenesis from organogenic callus. Germination of somatic embryos and rooting of shoots were achieved on MS solid medium without hormones.
... Two iris species, I. pseudopalida and I. reichenbachii are strictly protected as endemic plants of the Balkan Peninsula (Šilić 1990). Plant regeneration of I. reichenbachii has been reported earlier (Jevremović and Radojević 2002;Jevremović et al. 2006a) but there are no published reports about in vitro plant regeneration of Iris pseudopallida. ...
... In vitro sprouted cormlets rooted after 8 weeks in MS medium without hormone (86.7 ± 6.67%) with an average of 3.33 ± 0.33 roots per cormlet (Fig. 1D). Many studies have reported the rooting process on hormone-free medium in many members of the Iridaceae family: Babiana spp (McAlister et al. 1998), Iris ensata (Boltenkov et al. 2005Boltenkov et al. , 2007), Dierama luteoalbidum (Madubanya et al. 2006), Iris reichenbachii (Jevremonic et al. 2006), Sisyrinchium laxum (Ascough et al. 2010), as well as many other species. The occurrence of root formation on hormone-free basal medium may be due to the presence of endogenous auxins in regenerated cormlets (Blakesley et al. 1991). ...
The genus Crocus comprises plants with a potential to be developed as a new ornamental crop but to date, there are not many reports on in vitro propagation of many members of this genus. The present study involves in vitro propagation of Crocus cancellatus with ornamental and horticultural value. Two different types of corm explants (apical and basal halves of corms) were cultivated onto Murashige and Skoog’s (MS) medium supplemented with different levels of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). One to five cormlets emerged from every responding explant through direct organogenesis. Apical halves of corms were more highly responsive than basal halves and produced a maximum multiplication rate with 3.45 ± 0.06 cormlets per explant in 95.33 ± 2.33% of the explants in MS medium supplemented with 3% sucrose and 2 mg L−1 NAA and 1 mg L−1 BAP. The effect of cold storage temperature on in vitro cormlets sprouting was studied. Cormlets stored at 4°C for 8 weeks had more statistically significant positive effects on cormlets sprouting from the controls. In vitro rooting of cormlets was induced on MS medium without plant hormones.
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%.
The explant type, its anatomical structure, and hormonal composition of culture media are crucial for selecting explants of Iris L. genus for direct shoot regeneration in tissue culture. In case of vegetative bud culture, antibiotics must be added to the agar medium with due regard to the antibiotic sensitivity of microflora. On MS medium (Murashige and Skoog 1962) supplemented with BA (6 µM) + NAA (5 µM) + L-glutamine and adenine sulfate (100 mg/l), the embryos of I. hybrida and I. sibirica developed into adventitious shoots and the multiplication coefficient of I. ensata increased in subsequent passages. In the budding phase, the rhachis and perianth tube maintained meristematic tissues and exhibited morphogenetic capacity. The fragments of generative organs of I. ensata, I. hybrid and I. sibirica formed the shoots on MS media containing BA (4–8 µM) and NAA (3–5 µM). The highest percentage of the shoots (100%) formed from the perianth tube explants had the structure similar to perianth lobes instead of the first leaf primordia. Further culture resulted in the formation of the shoots typical of these species of iris.
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.
У раду су приказани резултати анализе садржаја мангиферина, (1,3,6,7-тетрахидроксиксантон-C2-β-D-гликозид) у различитом биљном материјалу балканске ендемичнеперунике (Iris reichenbachii Heuffel). Помоћу методе течне хроматографије под великимпритиском (HPLC), садржај мангиферина анализиран је у биљном материјалу сакупљеном наприродним стаништима, током гајења у култури in vitro, као и у регенерисаним биљкама гајеним уусловима ex vitro (стакленик и башта института). Kод биљака сакупљених са природних стаништасадржај мангиферина у биљном ткиву зависио је од биљног органа, као и од места где су биљкесакупљене. Током индукције регенерације биљака у култури in vitro применом културе зрелихзиготских ембриона на хранљивим подлогама обогаћеним са 2,4-дихидрофенокси сирћетномкиселином продукција мангиферина у биљном ткиву зависила је од састава хранљиве подлоге каои од степена диференцијације ткива. Највећа продукција мангиферина током гајења у условима invitro постигнута je током индукције формирања изданака док је најмања продукција мангиферинадобијена у ембриогеним калусним културама где је забележена синтеза мангиферина само утраговима. После криопрезервације врхова изданака методoм витрификацијe у капљици добијен јеисти ниво синтезе мангиферина у изданцима, као и пре криопрезервације. Највећи садржајмангиферина уочен је у надземним деловима биљака регенерисаних у култири ткива иаклиматизованих на спољашње услове. Садржај мангиферина код биљака добијених применомове методе био је знатно већи него код биљака сакупљених из природних станишта. У овом радупредстављен је потенцијал техника културе ткива и криопрезервације у циљу ex situ заштите једнеендемичне биљне врсте као и потенцијал ових техника за производњу секундарних метаболитакао што је мангиферин. Применом ових техника може се произвести овај секундарни метаболит запотребе фармацеутске индустрије, без уништавања биљног материјала са природног станишта.
Here, we report an overview about in vitro regeneration and multiplication of several iris species that grow naturally in our country. Research includes two dwarf endemic species (Iris pumila, I. reichenbachii) and two tall bearded iris species (I.germanica, I. sibirica). Plant regeneration by culture in vitro can be achieved by organogenesis and/or somatic embryogenesis on solid, as well as liquid media in mature zygotic embryos or leaf base culture. The best morphogenetic potential was obtained with plant regeneration by somatic embryogenesis from cell suspensions when the highest number of plants can be produced.
A simple protocol has been developed for plant regeneration by somatic embryogenesis of Southern Adriatic iris (Iris pseudopallida Trinajstić), an endemic species of the Balkan Peninsula. Somatic embryogenesis was induced in zygotic embryo culture on media supplemented with 2,4-dichlorophenoxy acetic acid (2-10 mgL-1) as the sole plant growth regulator, where both embryogenic calli and somatic embryos were induced. Subsequent decrease of 2,4-D in the media promoted formation of somatic embryos. Developed somatic embryos germinated on medium without growth regulators. The regenerated plantlets had diploid chromosome number. Planted plantlets acclimatized very well under greenhouse and garden conditions.
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.
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.