Bulblet Regeneration Research Articles

In vitro propagation of native Ornithogalum species in West Mediterrenean region of Turkey

Ornithogalum is a popular genus especially, as an ornamental plant. In this study, eight native Ornithogalumspecies grown in West Mediterrenean Region of Turkey were cultured on MS media supplemented with combinations of BAP (1.0, 2.0, 4.0 mg L-1) and NAA (0.25, 0.50 mg L-1). The results indicated that the highest rate of proliferation was induced on Murashige and Skoog (MS) to which 4 mg L-1 BAP + 0.5 mg L-1 NAA (4.97 bulblets/explant) while the lowest rate of bulblet proliferation was obtained in 2 mg L-1 BAP + 0.25 mg L-1 NAA (2.27 bulblets/explant). Ornithogalum umbellatum showed the highest bulblet regeneration within the species. The regenerated bulblets were transferred into plastic viols containing mixture of peat moss and perlite (1:1) after 5 months. Key words: Ornithogalum spp, bulb scale explant, bulblet regeneration, in vitro propagation.

In vitro bulblet regeneration from immature embryos of endangered and endemic Muscari azureum

A high frequency of bulblet regeneration was achieved for the endemic and endangered ornamental plant Muscari azureum using immature embryos. Immature embryos of M. azureum were cultured on a callus induction medium consisting of N6 mineral salts and vitamins, 400 gL-1 casein + 40 gL-1 sucrose + 2 mgL-1 L-proline, 2 mgL-1 2,4-D and 2 gL-1 Gelrite. Then the embryogenic callus clusters were transferred to a bulblet induction medium consisting of MS mineral salts and vitamins containing different concentrations and combinations of BAP, KIN, TDZ, Zeatin, IAA, NAA, 30 gL-1 sucrose and 7 gL-1 agar. Prolific bulblet multiplication (over 13 bulblets/embryo) was achieved from immature embryos after 5-6 months of culture initiation. Well-developed bulblets were excised and individually rooted on ? strength MS medium supplemented with 1 mgL-1 IBA, 0.5 gL-1activated charcoal, 20 gL-1sucrose and 6 gL-1agar and acclimatized. <br><br><font color="red"><b> This article has been retracted. Link to the retraction <u><a href="http://dx.doi.org/10.2298/ABS150608072E">10.2298/ABS150608072E</a><u></b></font>

Bulblet Regeneration from Tepal Explant in Lily Cultivars

Protocol for bulblet regeneration from tepal segments was developed in Asiatic hybrid lily cultivars. The explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of ∞-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The days for bulblet initiation decreased with the addition of NAA and/or BA in the medium. The bulblets were initiated much earlier (26.9 d) with 1 mg l−1 NAA and 0.5 mg l−1 BA. About 75% of the cultures with 1.2 bulblets per explants were observed in growth regulator-free medium. However, the regeneration response increased to 80.82% with 1.5 mg l−1 NAA and 0.5 mg l−l BA. The number of bulblets per explant and average fresh weight was highest with 1.5 and 1 mg l−1 NAA in combination with 0.5 mg l−1 BA. Among the cultivars, maximum number of bulblets was recorded for ‘Alaska’, whereas heaviest bulblets were observed in ‘Pollyana’. The bulblets attaining the size of 14–16 cm flowered in two and a half years without any phenotypic variations. ...

Stimulating effects of different basal media and cytokinine types on regeneration of endemic and endangered Muscari aucheri

In this study a simple protocol has been developed for the endemic and endangered Muscari aucheri plant. 2-4 bulb scale explants of M. aucheri were cultured in different basal media (Nitsch mineral salts and vitamins, Orchimax Medium mineral salts and vitamins) supplemented with 2 mgL-1 2,4-D, 20 mgL-1 mannitol, 20 mgL-1 sucrose, 0.5 mgL-1 NAA and different concentrations of BAP, KIN, 2iP and TDZ. The medium was solidified with 2 mgL-1 Gelrite. The Orchimax and Nitsch & Nitsch medium promoted shoot and bulblet regeneration. BAP, KIN and 2-iP were suitable for induction of the bulblet for 2-4 bulb scales of Maucheri. The highest number of bulblets per explant for 2 and 4 scales was achieved on the Orchimax medium supplemented with 1 mgL-1 KIN and 2 mgL-1 KIN, respectively. Whereas, the maximum number of bulblets per explant for 4 scales was recorded on the Nitsch medium supplemented with 2 mgL-1 BAP. However, the Nitsch medium containing 10 mgL-1 2-iP gave the best number of bulblets per explant for 2 scales. Bulblets acclimatized very well under greenhouse and garden conditions. .

In vitro propagation from axenic explants of Lilium oxypetalum (D. Don) Baker, an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.

Bulblet production from node explant grown in vitro in hybrid lilies

An attempt was made to regenerate bulblets from ex vitro node explants of hybrid lilies in the year 2007. Node sections (3-4 mm) isolated from the middle part of the stem in hybrid lilies were cultured on Murashige and Skoog (MS) medium supplemented with several combinations of NAA and BA. Growth regulator-free medium was not effective in inducing bulblet regeneration. A significant increase in the percent of explant producing bulblets and number of bulblets per explant was observed when 2 mg/l NAA was used in combination with 1.5 or 2 mg/l BA. The heaviest bulblets were obtained with 2 mg/l NAA in combination with 1.5 mg/l BA after 90 days of culture. The cultivar Apeldoorn produced greater number of bulblets whereas more weighty bulblets were produced in the cultivar Beartix. 1 or 2 mg/l IBA was most effective in producing roots. The rooted bulblets were hardened with 80-82% survival success after 30 days of transfer in the pots.

Bulblet regeneration from ex vitro root explant in lily hybrids

The influence of growth regulators on &lt;i&gt;in vitro&lt;/i&gt; bulblet formation from &lt;i&gt;ex vitro&lt;/i&gt; roots was studied in asiatic and oriental hybrids of &lt;i&gt;Lilium&lt;/i&gt; The root segments (3–4 mm) isolated from the middle zone of 2–3 cm &lt;i&gt;ex vitro&lt;/i&gt; root were cultured on Murashige and Skoog (MS) medium containing 1 or 1.5 mg/dm&lt;sup&gt;3&lt;/sup&gt; naphthalene acetic acid (NAA) and/or benzyladenine (BA). Bulblets were not produced in the presence of NAA and BA alone. A significant increase in the per cent explants producing bulblets was observed with 1.5 mg/dm&lt;sup&gt;3&lt;/sup&gt; NAA and 1 mg/dm&lt;sup&gt;3&lt;/sup&gt; BA. Maximum number of bulblets and average fresh weight per bulblet was observed with 2 mg/dm&lt;sup&gt;3&lt;/sup&gt; NAA and 1.5 mg/dm&lt;sup&gt;3&lt;/sup&gt; BA after 90 days of culture. No differences were found among cultivars in bulblet regeneration of explant or bulblet number although more weighty bulblets occurred in cv. Apeldoorn. About 82% bulblet survival was recorded in coco peat after 30 days of transfer to pots.

In Vitro Regeneration of Red Squill Urginea Maritima (L.) Baker. Using Thidiazuron

ABSTRACTThe study reports an efficient in vitro micropropagation of Red squill (Urginea maritima (L.) Baker), an important medicinal plant of Mediterranean region, from single and double scale explants. The explants were cultured on MS medium containing different concentrations of Thidiazuron with initial culturing in darkfor eight weeks followed by transfer to 16 h light photoperiod or culturing them in 16 h light photoperiod only. The results showed that initial growth of explants in dark for eight weeks was inhibitory and produced negligibly low number of bulbs on both explants. Contrarily, 16 h light photoperiod resulted in varying frequency of bulblet regeneration on single and double scale explants. Double scale explants were more potent for bulblet regeneration compared to single scale explants. Single scales produced only axillary bulblets with a frequency of 0.90–1.33 bulblets per expalnt. Contrarily, both adventitious and axillary bulblets were recorded on double scales. These explants produced maximum number of 1.17 adventitious bulblets and 3.58 axillary bulblets per explant on MS medium containing 0.55 mg/l TDZ. The frequency of axillary bulblets regeneration remained higher compared to the frequency of adventitious bulblet regeneration on all concentrations of TDZ. All axillary bulblets rooted on the mother regeneration media. However, the adventitious bulblets could only be rooted on MS medium supplemented with 1 mg/l IBA. All bulblets established in the growth chamber were transferred to the field for further observations and flowering.

Efficient in vitro multiplication in Ornithogalum ulophyllum Hand.-Mazz. from twin scale explants

Ornithogalum ulophyllum Hand.-Mazz. with beautiful white flowers is an important medicinal and ornamental plant of the Middle Eastern countries and need exploitation for commercial propagation. The study reports in vitro mass proliferation of bulblets achieved from twin scales and “ in vitro regenerated bulblet” explants on MS medium supplemented with various concentrations of BAP–NAA. The best regeneration on twin scales and “ in vitro regenerated bulblets” was obtained on MS medium containing 2 mg l −1 BAP–0.5 mg l −1 NAA and 2 mg l −1BAP–1 mg l −1 NAA, respectively. However, bulb scales seemed to be more potent for bulblet regeneration. A large number of the developing bulblets rooted on the regeneration medium. Remaining non-rooting bulblets were rooted on MS medium containing 1 mg l −1 NAA. All plants were acclimatized in the environmental chamber for 4 weeks and were transferred to the greenhouse for flowering. Regenerated bulblets developed into morphologically normal plants.

Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of Fritillaria imperialis L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l−1 NAA + 0.4 mg l−1 IAA) with higher level of cytokinin (1 mg l−1 BAP). In contrast, auxins (0.6 mg l−1 NAA + 0.4 mg l−1 IAA) with lower level of cytokinin (0.1 mg l−1 BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.

히아신스(cv. Pink Pearl)의 인편 기내 배양시 효과적인 자구의 재생과 생장

The regeneration and growth of bulblets from the bulb scale segments of Hyacinthus orientalis L. cv. Pink Pearl were more efficient in IBA than IAA at the same concentrations (1.0 and 3.0 ㎎/l).. The normal (base-down) orientation of explants was more effective for bulblet regeneration and root growth than the inverted (base-up) orientation. The growth of bulblets and roots was increased higher in the perlite than the agar medium. These results suggested that the alternate culture system, first cultured in the agar medium for bulblet regeneration, and then in the perlite medium for bulblet growth, may be more useful for efficient in vitro culture of hyacinth (H. orientalils) cv. Pink Pearl.

In vitro Propagation of Lilium longiflorum Var. Ceb-Dazzle Through Direct Somatic Embryogenesis

Experiments were carried out to investigate the effects of various concentrations of Picloram (0, 1, 2, 3, 6 and 9 mg L(-1)), TDZ (0, 0.5, 1, 1.5 and 2 mg L(-1)), NAA (1.5 mg L(-1)) in combination with TDZ (0.08, 0.2 and 0.4 mg L(-1)), 2,4-D (2.5, 5 and 10 mg L(-1)) combined with BAP (0.25 mg L(-1)) and different types of explants (basal, central and distal part of the bulb scale) on direct somatic embryogenesis induction of Lilium longiflorum var. Ceb-Dazzle. The explants were surface sterilized and cultured on MS medium supplemented with 3% sucrose, 0.3% Phytagel and various concentrations of mentioned growth regulators. It was found that Picloram at a concentration of 2 mg L-' was the most effective treatment for induction of direct somatic embryogenesis and gave the highest number of embryos (18.6) on each explant. The explants from basal part of the bulb scale showed the best responses (19.9 embryos/explant). TDZ alone or combined with NAA in various concentrations was not able to induce somatic embryogenesis, but gave direct bulblet regeneration. Similar results were obtained for 2, 4-D and BAP combination treatments. Induced somatic embryos were transferred to MS medium without growth regulators for maturation and matured plantlets were successfully acclimatized and transferred to in vivo conditions.

(79) Direct Bulblet Regeneration from Mature Embryo: A Rapid, Efficient and Genotype-independent In Vitro Morphogenesis Pathway for Preservation of Endangered Wild Populations of Fritillaria imperialis and Fritillaria persica

Wild populations of Fritillaria sp. have dramatically decreased in Iran because of pest overflow and continual grazing. Previous studies have shown that Fritillaria cannot rapidly and efficiently propagate by traditional methods. In vitro tissue culture techniques have shown high potential for micropropagation of endangered plants. The use of bulb-scale pieces for tissue culture can result in the destruction of the endangered parent plant. Fritillaria is a heterozygous plant in which the genetic content of each embryo is different from others, even on the same plant. In this study, mature embryos of F. imperialis and F. persica were used as explant for the first time. Embryos were extracted from seeds and cultured on B5 medium supplemented with various combinations of BAP (0, 0.1, 1 mg/L), NAA (0, 0.4, 4 mg/L), and IAA (0, 0.4, 4 mg/L). Embryo explant showed low genotype dependency between different heterogenous and heterozygote populations of both F. imperialis and F. persica. The best response of bulblet regeneration in both F. imperialis and F. persica was obtained from 1 mg/L BAP + 0.4 mg/L NAA+4 mg/L IAA and direct organogenesis pathway, with 15 bulblets per explant for F. imperialia and 20 for F. persica. Because of the large number of embryos in a plant and their different genetic contents, established in vitro propagation by using embryo explant in this study can provide broad genetic resources and variations. As explained above, in vitro protocols can play a major role in rescuing F. imperialis and F. persica from extinction.

Different Developmental Stages of Mother Plants Induct Different Morphogenesis Pathways in In Vitro Culture of Fritillaria imperialis and Fritillaria persica

Fritillaria imperialis and Fritillaria persica are important medicinal and ornamental plants that are native to Iran. Wild populations of Fritillaria are at risk of extinction. For the first time, capability of developmental stages in respect to induction of different morphogenesis pathways from petal tissue was studied in detail. The developmental stages were green unopened flowers, colored unopened flowers, and open flowers. Direct bulblet regeneration and direct somatic embryogenesis were observed from green unopened flowers in both F. imperialis and F. persica. More bulblet regeneration was produced in F. imperialis in contrast with F. persica in colored unopened flowers. Somatic embryogenesis via callus was established in green unopened flowers of F. persica with cold pretreatment. The effect of light on induction of different morphogenesis pathways was nonsignificant except for green unopened flowers of F. persica with cold treatment. Our results showed that the developmental stages of petal explants play a significant role in micropropagation of Fritillaria and induction of different morphogenesis pathways.

In vitro propagation of an endangered medicinal bulbous plant Sternbergia clusiana Ker-Gawler (Amaryllidaceae)

SummarySternbergia clusiana belongs to the family Amaryllidaceae. It is an endangered medicinal plant grown in Jordan and neighbouring countries. A protocol has been developed for efficient rapid vegetative propagation of S. clusiana through direct bulblet regeneration. Two types of explants have been used: (1) single-bulb scales, and (2) twin-bulb scale explants. Field-derived material was successfully sterilised using 70% ethanol for 1 min, and 6.5% NaOCl with Tween 80 (2–3 drops 100 ml–1) for 30 min before dissecting out the bulb scales. Combination treatments were conducted using two types of growth regulators: 0, 1, 5, 10 or 20 µM indole-3-butyric acid (IBA) and benzyladenine (BA). The highest rate of regeneration from single-bulb scales (73%) was observed in induction medium containing 20 µM IBA without added BA. In addition, 73% of twin-bulb scale explants were induced to form bulblets directly when cultured on a medium containing 10 µM IBA and 1.0 µM BA. Bulblet plants were rooted on similar medium without any growth regulators. A plant regeneration system from single-scale and twin-bulb scale explants of S. clusiana was thus established.

Competence for In Vitro Bulblet Regeneration among Eight Lilium Genotypes

Competence for in vitro bulblet regeneration was investigated for eight diverse Lilium genotypes (L. `Citronella', L. `Stargazer', L. `Stones', L. `Lovely Girl', L. pumilum, L. lancifolium, L. lancifolium `Orange Star', and L. speciosum var. rubrum). Explants established from bulb scales were cultured on lily bulblet regeneration medium [Murashige and Skoog (1962) modified by reducing the NH4NO3 to 0.825 g·L-1 and KH2PO4 to 0.170 g·L-1, and adding (per liter) 30 g sucrose, 0.1 g myoinositol, 0.4 mg thiamine·HCl, 80 mg adenine sulfate, 16 μm naphthaleneacetic acid, 2 mL·L-1 Plant Preservative Mixture (PPM), and 4.5 g·L-1 AgarGel at pH 5.7] for 8 weeks before transfer to sphagnum peat moss and 4 weeks refrigeration at 5 °C in darkness. All genotypes produced bulblets in vitro. However, the response rate varied among genotypes. Explants of Lilium `Lovely Girl', L. `Citronella', and L. speciosum var. rubrum were most responsive with ≈90% producing bulblets. The number of bulblets per responding explant ranged from 5.2 (`Stones') to 2.3 (L. lancifolium). When comparing in vitro and greenhouse bulblet production, about twice as many bulblets were produced by explants in vitro compared to halved scales incubated in the greenhouse. The percentage of responding explants ranged from 33% to 360% greater for six of the genotypes tested when propagated in vitro compared to bulb scales propagated in the greenhouse. Following refrigeration, bulblets were transferred to the greenhouse for sprouting. Over 80% of bulblets obtained from L. `Citronella', L. lancifolium `Orange Star', L. lancifolium, and L. `Stones' sprouted in the greenhouse. This study illustrates that a diverse range of lily species can be successfully propagated in vitro using a single medium formulation and is the first report of bulblet regeneration in vitro for L. `Citronella', L. `Stones', L. `Lovely Girl', and L. lancifolium `Orange Star'.

Enhanced bud and bulblet regeneration from bulbs of Nerine sarniensis cultured in vitro.

Nerine (Nerine sarniensis) cv. Salmon Supreme in vitro-grown bulblets, 7-9 mm in diameter, were cut in half longitudinally and used for adventitious bud initiation following dissection of the roots and two-thirds of the upper part of the bulblets. The terminal apex was injured with a hot, sterile microscope dissecting needle. The highest number of buds formed (seven to nine buds per halved bulblet) on a semi-solid Murashige and Skoog (MS) basal salts medium supplemented with 3% sucrose and either 1 microM 6-benzylaminopurine (BA) and 1 microM alpha-naphthaleneacetic acid (NAA) or 0.5 microM BA and 0.1 microM NAA. Bulblet halves were cultured in the dark for 11-13 weeks with one subculture after 6 weeks. Anatomical studies indicated that the initiation of adventitious buds on the abaxial side of the inner scales of the halved bulblet was adjacent to the basal plate and started from the leaf primordium and a meristematic bulge. Buds developed directly into small bulblets after they were transferred to semi-solid MS basal salts medium supplemented with 6% sucrose, 10 microM indole-3-butyric acid (IBA) and 0.25% activated charcoal. Small bulblets cultured in liquid MS medium supplemented with additional KH(2)PO(4 )(170 mg l(-1)), 6% sucrose and 0.1 microM NAA under a 16/8-h (light/dark) photoperiod for 8 weeks grew into larger bulbs faster than those cultured on semi-solid medium. The bulbs were rooted on a semi-solid medium after 4 weeks and then transferred to the soil. As many as 18 bulblets developed and rooted from one in vitro-grown bulb after 25-27 weeks.

Effects of light emitting diodes (LEDs) on the in vitro induction and growth of bulblets of Lilium oriental hybrid ‘Pesaro’

The effects of light generated by red, blue, red plus blue light emitting diodes (LEDs) on the in vitro regeneration of bulblets from scale segments of Lilium oriental hybrid ‘Pesaro’ were studied and responses were compared with explants grown under continuous dark and fluorescent light (16 h per day). Bulblet regeneration was observed under all the treatments, however, higher percentages of explants responded to fluorescent light and red plus blue LED and developed a higher number of bulblets per explant. Effect of red, blue, and red plus blue LEDs were also tested on the growth of in vitro cultured bulblets and compared with dark and fluorescent light grown bulblets. Red plus blue LED was suitable for bulblet growth, and bulblets grown under this condition were bigger in size and their fresh and dry weight, dry matter percentages were also high. This condition also induced the highest number of roots per bulblet and their fresh and dry weight and dry matter percentage were also high.

High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii

Fritillaria thunbergii Miq. bulb-scale sections were cultured using Murashige and Skoog (MS) medium supplemented with NAA (1.62 μM) and KN/2iP/BA (0.47–23.23 μM).

TEMPERATURE AND PHOTOPERIOD INFLUENCES ON IN VITRO BULB FORMATION OF HYACINTHOIDES PAIVAE

The aim of this study was to assess the interaction of cytokinin (BA), auxin (NAA) with light and temperature of in vitro bulblet production from explants of Hyacinthoides paivae S. Ortiz & Rodr.-Oubina. A combination BA (10 μM) and NAA (0.5 μM) gave a better response than other combinations. An incubation at 20 °C of temperature and 16 h photoperiod were the best for bulblet regeneration.