Bulb Scales Research Articles

In Vitro Propagation of Lilium longiflorum Bulbs Using NAA and BAP Plant Growth Regulator Treatment

Lily (Lilium longiflorum) is a perennial herbaceous plant with white trumpet-shaped flowers, fragrant and bulbous. In vitro culture through bulbs is one of way propagation of lily plants, but it requires a long time and only produces limited plants. In vitro propagation is a very promising technique for plant propagation because it can produce a lot of plant seeds in a short time. Bulbs are one of the fastest explants for growing shoots in lilies, but it is not known for certain which cuts of explants from bulb scales are best for multiplying in vitro. This study aims to determine the effect of lily bulb explants and the concentration of NAA and BAP growth regulators on the growth of lily bulb explants. The best results were obtained on the base and middle cuts explant of bulb scales compared to the tip cuts explant ones. The best results of the growing percentage, the number of shoots and the best growing time are shown in the combination treatment of growth regulator 1 mg L−1 NAA and 1 mg L−1 BAP. The optimum results on the number of micro bulbs were found in the treatment of growth regulators 0.5 mg L−1 NAA and 1 mg L−1 BAP. The best results of the average time formed micro bulb was in the treatment of 1 mg L−1 NAA and BAP with middle explant cuts, and treatment concentrations of 0.5 mg L−1 NAA and BAP in the base explant section. The base and middle bulb explants are able to regenerate or grow higher shoots. This is caused by the presence of endogenous natural auxin and the spread of auxin in plant parts not in the same amount. Therefore when added to the exogenous growth regulator such as auxin or cytokines to culture media will further trigger the formation of micro tubers more quickly,. It can increase the concentration of endogenous growth regulators in cells, help growing process and developing tissue.
 
 Keywords: Bulb, lily, micro bulbs, in vitro, shoots

Transplantation of native species to existing vegetated slopes: Importance of optimal habitat and initial nutrient reserves

Although ecosystem degradation is often caused by existing slopes vegetated with commercial species, limited data are available for implementing practical approaches for nature restoration by transplanting native species to those vegetated slopes. Existing vegetated slopes are characterized by spatial heterogeneity in the habitat environment and competition with existing vegetation, which affect the behaviors of transplanted native species. In this study, we examined the factors affecting the survival, growth, verticillation, and flowering of Lilium pensylvanicum, a native coastal-meadow species transplanted to a vegetated road slope in Hokkaido, Japan. In the fourth year after transplantation, the number of surviving plantlets was 364 (57% of 640 transplanted plantlets) and the number of plantlets that shifted to verticillate leaves was 181 (50% of the survivors). The flowering plantlets accounted for 9% (n = 34) of the survivors. The generalized linear model showed that plantlet survival is strongly promoted by a larger bulb scale, that is, nutrient reserves. Furthermore, the survival ratio was greatly improved in the drought-prone, stressful habitats with low vegetation coverage, even for plantlets with relatively smaller bulb scales. However, in the moist habitat with high coverage, the survival ratio was greatly decreased regardless of the bulb scale size. Growth, verticillation, and flowering were facilitated by higher soil nutrient availability, such as a lower C/N ratio and thicker soil layer. Therefore, transplanting of native species to vegetated slopes requires the selection of stress-tolerant species with large nutrient reserves and identification of optimal habitats for transplanting, namely stressful environments with reduced vegetation coverage.

Callus mediated organogenesis in Lilium polyphyllum D. Don: A critically endangered Astavarga plant from North Western Indian Himalaya

Lilium polyphyllum D. Don (Liliaceae) is a critically endangered herbaceous perennial, commonly known as snow lily or Ksheerkakoli. Bulbs of the plant are of immense medicinal use and have astringent and anti-inflammatory properties. Over exploitation of the species from wild and degradation of habitats are posing threats to its existence. Therefore, an attempt has been made to develop a propagation protocol and promote mass multiplication from bulb scales of L. polyphyllum. Maximum callus was induced from the bulb scales in basal MS medium containing 2,4-D (0.5, 1, 1.5, 2 and 3 mg/l), BAP (0.5, 1, 1.5 and 2 mg/l) and NAA + BAP (0.5+2 mg/l). Maximum shooting (98.75%) was achieved in the callus when shifted to MS medium supplemented with BAP (1 and 2 mg/l) and NAA (0.5 mg/l), with an average of 1-2 cm shoot length per culture. The well developed in vitro regenerated shoots were transferred to the rooting medium and 100% rooting was achieved in half-strength MS basal medium supplemented with IBA and IAA (1 and 2 mg/l). The in vitro regenerated plantlets were shifted to a glasshouse in the sterilized soil and vermiculite mixture for acclimatization. After survival, plantlets were finally transferred to the open environment with 90% survival success.

In vitro bulb formation of direct and indirect regeneration of Lilium orientalis cv. \u201cStarfighter\u201d plants

BackgroundLily is one of the most important commercial cut flower species and one of the three major bulb crops in the commercial market. The aim was to assess and establish a suitable protocol for increasing the number of Lilium bulblets and enlarge their size.MethodologyDifferent plant growth regulators (TDZ, 2,4-D, 2ip, and picloram) at different concentrations for direct and indirect micropropagation were used. Also, the effect of various concentrations of sucrose alone or with growth inhibitor on bulblet formation during three repeated subcultures was examined with anatomical study.ResultsFor direct shoot organogenesis, culturing leaf explant on MS culture medium containing TDZ (0.5 mg/l) + 2,4-D (10 mg/l) led to the highest number of shootlets after 1 month. For indirect micropropagation, MS was supplemented with 2ip (0.5 mg/l) + picloram (5 mg/l)-induced callus tissues which were differentiated to embryos then weak shoots after 3 months. For more proliferation, the highest numbers of shootlets, leaves, and bulb scales were observed on MS culture medium supplemented with 1.0 mg/l of BA plus 0.2 mg/l NAA. Using 60 g/l of sucrose plus 3 or 6 mg/l of paclobutrazol led to the highest number of bulblets/explant for the third subculture. The highest bulblet diameters were recorded with 120 g/l sucrose plus 6 or 3 mg/l paclobutrazol.ConclusionThis study had reached to optimize a suitable protocol for direct and indirect micropropagation of Lilium orientalis cv. “Starfighter” bulbs and increase the number and size of bulbs that can be beneficial for increasing the production and reduce their price. This study will help the producers for commercial purposes.

Metabolite and expression profiling of steroidal alkaloids in wild tissues compared to bulb derived in vitro cultures of Fritillaria roylei – High value critically endangered Himalayan medicinal herb

Bulbous Fritillaria roylei (known as Jangli lahsan) is an important critically endangered medicinal herb of Astavarga group and widely used in traditional medicinal system. Being a medicinal potential but endangered herb, biotechnological interventions are urgently needed for its restoration and production of bioactive compounds. In the present study, in vitro cultures were established from bulb scales as explant by using optimized sterilization protocol (79.67% survival). From different combinations and concentrations of plant growth regulator tested; high efficiency callus induction (88.89%) and in vitro plantlets regeneration (77.78%) response was obtained in Murashige and Skoog medium supplemented with 0.5 mg/L thidiazuron +2.0 mg/L picloram and 1.0 mg/L kinetin +0.5 mg/L naphthaleneacetic acid, respectively. For the quantification of steroidal alkaloids i.e. sipeimine and peimine; selective, sensitive, and rapid liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analytical method was developed and validated. Quantification of sipeimine and peimine was done for the first time in established in vitro cultures and naturally grown tissues of F. roylei i.e. bulbs, leaves, stem and flower bud. Among in vitro raised cultures, higher sipeimine content (1.98 μg/g) was detected in callus. Whereas, maximum sipeimine content (599.72 μg/g) was observed in floral bud compared to other wild tissues. Peimine content was detected in bulb (1.13 μg/g), leaf (0.78 μg/g) and stem (0.67 μg/g). Qualitative and quantitative phytochemical screening revealed potentiality of this important medicinal herb for commercial utilization in drug formulation to the industries. Maximum antioxidant activity (IC50) was exhibited in floral buds (0.11 mg/mL), while among in vitro raised cultures; regenerated plantlets (0.80 mg/mL) shown higher scavenging activity than callus (2.71 mg/mL). Higher total phenol content (42.86 μg gallic acid/mg) was observed in bulb whereas, flavonoids content is higher in leaves (68.36 μg quercetin/mg) followed by callus (58.22 μg quercetin /mg). Putative steroidal alkaloids gene expression studies using RT-qPCR showed positive correlation with metabolite content. For the first time, present investigation concluded that floral buds, leaves and stem could also be used as an alternative potential source for steroidal alkaloids and callus culture as sustainable approach for metabolite production to meet industrial standards and demands.

In vitro culture and micropropagation of the Baetic-Moroccan endemic plant Lapiedra martinezii Lag. (Amaryllidaceae)

Lapiedra martinezii Lag. is a potential medicinal and ornamental plant facing conservation challenges. Thus, this study was focused on determining the conditions for culture initiation and propagation using in vitro techniques. The optimal sterilization procedure combined thermotherapy at 54°C for 60 min and immersion in 7% (w/v) Ca(ClO)2 solution for 20 min. The most suitable medium to initiate bulb scales cultures was Gamborg B5 medium containing 500 mg L−1 casein, 2 mg L−1 adenine, 10 mg L−1 glutathione and 10 g L−1 sucrose. The most productive multiplication medium tested was Murashige and Skoog medium containing 30 g L−1 sucrose, 4.0 mg L−1 6-benzylaminopurine, and 0.12 mg L−1 1-naphtaleneacetic acid. Most plants developed in vitro rooted spontaneously in the multiplication phase. The vast majority of the plants (89%) were successfully transferred to ex vitro conditions, and 100% survived over 1 yr of cultivation outdoors. Sucrose at a concentration of 60 g L−1 was the most effective treatment to increase the biomass of bulblets. High auxin/cytokinin ratios produced the highest callus induction efficiency. The vast majority of callus developed in dark conditions, but none regenerated in the combinations of growth regulators previously tested. The plants obtained by micropropagation did not show significant differences in morphometric traits compared with the wild specimens, which supported the stability of the materials produced in vitro. This is the first report on cell cultures and micropropagation of L. martinezii, and the results can be applied to other Amaryllidaceae for industrial or conservation purposes.

Storage and growth temperatures affect growth, flower quality, and bulb quality of Hippeastrum

The effects of storage and growth temperatures on growth, flower quality, and bulb quality of Hippeastrum sp. were studied. Bulbs of Hippeastrum sp. ‘Apple Blossom’ with circumferences in the range of 22–24 cm were selected and stored for 12 weeks at one of three different temperatures (i.e., room temperature [RT: 30 ± 2 °C], 13 ± 2 °C, and 15 ± 2 °C). After storage, the bulbs were grown in a growth chamber at one of three growth temperatures—that is, RT (30 ± 2 °C), 15 ± 2 °C, and 25 ± 2 °C. The results showed that storing bulbs at RT for 12 weeks reduced their quality in terms of fresh weight, diameter, and firmness, whereas storing bulbs at 13 °C or 15 °C retained their quality in terms of fresh weight and firmness. Electron scanning imaging of the starch granules in the bulb scales showed that prior to storage, the leaf base cells were filled with large starch granules of either lenticular shape or spherical shape. After storing for 12 weeks at RT, the starch granules in the bulb scales were sharply reduced in number and were predominantly of the small, spherical form. A growing temperature of 25 °C could stimulate better growth with respect to plant height, flower diameter, bulb diameter, bulb fresh weight, and bulb dry weight, compared with RT (30 °C). A significant interaction between storage and growing temperatures was found for all parameters except for stalk length. A bulb storage temperature of 15 ± 2 °C for 12 weeks combined with a growing temperature of 25 ± 2 °C produced the best results for both flower and bulb production.

The effects of different concentrations of nano silver on elimination of bacterial contaminations and stimulation of morphogenesis of Sorbonne lily in vitro culture

Nano silver (NS) have been known as a novel material which cannot only destroy the structure of cell microorganism but also have positive effects on in vitro plant morphology. In this research, NS was used to sterilize lily samples and stimulate lily morphogenesis in vitro. Separated bulb scales were treated in solutions of NS with four different concentrations (100, 200, 300 and 400 ppm) in five different times (60, 90, 120, 150 and 180 min) that were compared with controls being soaked in sodium hypochlorite solution. Besides, four different concentrations (2, 4, 6, and 8 ppm) of NS were also added to culturing medium that aimed to test effects of NS on lily plant growth parameters in vitro. The result illustrates that NS with 300 ppm concentration was the most useful solution for sterilizing the lily samples in 120 min., making up 87.78% sample survival and clean. Moreover, NS enhanced bulblet formation from bulb scales samples. On the medium supplemented with 4 ppm NS, 100% bulb scales were formed new bulblets with a coefficient, 2.98 after six weeks culturing; and on the rooting medium, 6 ppm NS made up the highest percentage of bulblets samples rooting, 87.33%, with the highest root number per bulblet, 3.67 and average 2 cm in length, each.

Seed propagation and micropropagation of Lilium fargesii

Green lily (Lilium fargesii) is an endemic rare species in China, which seldom distributed in Mts. Qinling and Bashan, with narrow distribution and scarce population. The seed germination rate is 13.3%, which is not affected by storage temperature significantly. Meanwhile, the plug germination rate is 2.7%, which is probably limited to the maturity of seeds themself. The differentiation rate of bulb scales from different populations has a huge range on the same medium. The orthogonal test shows that the MS and sucrose concentration were the main factors that affect bulblet expansion, and the optimal medium is MS + 60 g L‑1 sucrose. There is no significant effect with PP(333). The germination rate of in vitro bulbet is only 14.0% and the survival rate is 13% after one winter in Beijing outdoors.

Plant regeneration by organogenesis from bulbous explants in Fritillaria imperialis L., a wild rare ornamental species at the risk of extinction

Fritillaria imperialis L. (Liliaceae) is a rare and endangered ornamental plant grown in mountain regions and Zagros altitudes, Ilam province, Iran. This species is in danger of extinction due to invasive collection. Plant regeneration was done by organogenesis from bulb scales as explants cultured on Murashige and Skoog (MS) media fortified with different concentrations of kinetin (KIN, 0.00, 0.50, 1.00 and 2.00 mg l-1) and α_naphthaleneacetic acid (NAA, 0.00, 0.50, 1.00 and 2.00 mg l-1), either individually or in combination. The largest number of leaf (3.80), root (5.86) and callus (8.16) per explant was regenerated on the medium containing 0.50 mg l-1 KIN and 1.00 mg l-1 NAA. Maximum viability percentage (96.66%) was obtained in medium supplemented with 1.00 mg l-1 KIN. In vitro regenerated plantlets were cultivated in plastic pots containing peat moss and perlite (1:1). The plantlets were successfully acclimatized in an adaptation greenhouse with a survival rate of 95% exhibiting normal developmental patterns.

Influences of Storage Conditions, Cultivation and Culinary Practices, On the Antioxidant Capacity of Red Bulbs of Some Onion Varieties Grown in Burkina Faso

Red onion bulb is a vegetable containing micronutrients with antioxidant properties. However, certain factors can have an impact on the content of these compounds. The present research evaluate the effects of parameters such as cultural practices, storage conditions and culinary practices, on the antioxidants content of red onion bulb of some varieties grown in Burkina Faso. The identification of the factors was carried out through field surveys, and their effects on the antioxidants content were assessed and appreciated through especially specific related bibliographic and laboratory test data. The results showed that84.78% of farmers use chemical fertilizers and all of them use chemical pesticides on onion crop. They don’t use premises or equipment suitable for storage. About dishes,7.61% of cooks, peel, cut and wash onion bulb before steam cooking, boiling water cooking or oil frying them at high temperature during 30 minutes to 3 hours. Some cooks use to braise onion bulb scales as ingredients for different dishes such as barbecue. Only 02,72% of cooks grind the onion bulbs with a pestle, before using the crushed as such on barbecue, or slow cooking it in different sauces. Research results show that these culinary practices contribute declining significantly the final intake of antioxidant for the consumer's body.

The proof is in the bulb: glycerol influences key stages of lily development.

A bulb is a whole plant condensed into an underground organ. A geophyte's bulb comprises both food reserves and important developmental history that may affect its whole growth. In Easter lily (Lilium longiflorum), bulb size is associated with the plant's flowering pathway - vernalization or photoperiod - and also affects sprouting, flower quality and abortion rate. The aim of this study was to investigate the reasons for the major physiological differences between large and small bulbs. Lily bulbs start their development from secondary meristems along the stem, with large bulbs being heavier and bear more scales than small ones. Peeling the outer scales of a large bulb converts its physiological responses into those of a small bulb, implying that the physiological discrepancies in plants developing from large or small bulbs are mediated by factors inherent to the bulb. We therefore performed broad analyses of the metabolite composition in the scales of bulbs subjected to temperature regimes affecting further plant development. We found a striking association between the level of glycerol, a primary metabolite mostly synthesized in the outer scales, and a delay in sprouting and flowering time, and reduction in abortion rate. Exogenous glycerol application to the bulbs before planting corroborated these results. Moreover, transcriptome analyses showed that flowering-promoting gene expression was downregulated in the bulb after glycerol treatment, while potential flowering inhibitor as well as a dormancy-related gene expressions were upregulated. Based on these studies, we postulate that glycerol is a major factor influencing both vegetative and reproductive development in lily.

In vitro propagation of a cut flower variety Muscari armeniacum Leichtlin ex Baker through direct bulblet proliferation pathways

Muscari armeniacum is one of the important ornamental cut flowers in floriculture industry, which is native to Southern Europe, North Africa, Western Asia and Asia Minor. In this study, bulb explants (basal plate of bulb having meristems), bulb scales, and leaf segments from in vitro derived bulblets were cultured in Murashige and Skoog (MS) medium with different plant growth regulator concentrations and combinations to assess their effects on different bulblet organogenesis pathways. The results demonstrated that cytokinin in combination with auxin is required for both axillary and adventitious bulblet regeneration. Benzyladenine (BA) combined with α-naphthalene acetic acid (NAA) showed significant effects compared to other growth regulator combinations tested. The 4.0 µM BA + 2.0 µM NAA was found to be the best for axillary bulblet formation from bulb explants. Likewise, bulb and leaf segments showed the best response in adventitious bulblet organogenesis when they were cultured in BA-NAA combinations. Out of several concentrations of BA with NAA, 4.0 µM BA + 1.0 µM NAA was optimum for adventitious bulblet regeneration. Bulblets, properly isolated from both axillary and adventitious proliferation systems, showed maximum percentage of rooting on half strength MS medium containing 2.0 µM indole-3-butyric acid (IBA). However, all the auxins at more than 2.0 µM concentration showed reduced root formation through either callus formation at the base of shoots or malformation of roots. All the in vitro regenerated plantlets were successfully acclimatized under ex vitro environment with 60% survival rate using garden soil.

A procedure for indirect shoot organogenesis of Polianthes tuberosa L. and analysis of genetic stability using ISSR markers in regenerated plants

Efficient in vitro regeneration system was well-established in Polianthes tuberosa L., an attractive ornamental and aromatic plant of horticultural importance. Various explants (Crown explants, leaves, petals, flowering stems and bulb scales) and different plant growth regulators (PGRs) were evaluated for both callus induction and shoot regeneration. Results showed that the maximum callus formation was obtained in crown explants using Murashige and Skoog (MS) medium supplemented by 1.95 μM 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T). The highest rate of shoot regeneration frequency was obtained on MS medium enriched with 2.26 μM thidiazuron (TDZ) + 250 mg l−1 proline from crown callus. The regenerated shoots were elongated on MS medium supplemented with 2.22 μM 6-benzylaminopurine (BAP). All shoots were successfully rooted in MS medium containing 9.89 μM indole-3-butyric acid (IBA) within a 15-day under a 16/8 h photoperiod. Plantlets were successfully hardened and adapted in the greenhouse with a survival rate of 100% and demonstrated normal growth. Flow cytometry and Inter Simple Sequence Repeats (ISSR) markers analysis indicated that there was no variation between mother and regenerated plants. The established regeneration protocol would be very useful for genetic engineering of P. tuberosa L. leading to the production of new flower colour and shape, pest and disease resistance and fragrance modification.

An efficient protocol for in vitro regeneration and conservation of Shirui lily (Lilium mackliniae Sealy): a lab-to-land approach to save the rare endangered Asiatic lily species

An efficient protocol for direct and indirect shoot regeneration and proliferation from bulb scales of Shirui lily (Lilium mackliniae Sealy), an endangered Asiatic lily species endemic to the Shirui hill peak, Manipur, India, has been developed. Bulb scales were isolated from mature bulbs and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ). For direct shoot regeneration from bulb scale explants, 0.5 mg L−1 BAP yielded the highest shoot induction (3.5 shoots per scale; a 96.7% response). For indirect de novo organogenesis, optimum callus induction was achieved with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), and shoot organogenesis was higher (16.2) when subcultured onto 0.5 mg L−1 BAP medium. Multiple shoot regeneration and pseudo-bulb formation protocols were assessed; the highest shoot proliferation (10.1) occurred with 0.5 mg L−1 BAP and 1.0 mg L−1 gibberellic acid (GA3). Rooting response was 96% with 0.5 mg L−1 1-naphthalene acetic acid (NAA), with multiple roots per shootlet. Plantlet survival was increased to 92.5% during the hardening-off process by using hydroponics with Hoagland’s solution in a mist chamber. Clonal fidelity was assessed through random amplified polymorphic DNA (RAPD) analysis comparing the mother plant and regenerated plantlets. After confirming genetic uniformity, the pseudo-bulblets with four to six leaves and three to four roots were successfully established at the Shirui hills peak. This in vitro regeneration and ex vitro conservation approach could be helpful to save this rare endangered species in a sustainable way.

Differential response to heat stress in outer and inner onion bulb scales.

The formation of brown protective skin in onion bulbs can be induced by rapid post-harvest heat treatment. Onions that are peeled to different depths and are exposed to heat stress show that only the outer scales form the dry brown skin, whereas the inner scales maintain high water content and do not change color. Our study demonstrates that browning of the outer scale during heat treatment is due to an enzymatic process that is associated with high levels of oxidation components, such as peroxidase and quercetin glucoside. De novo transcriptome analysis revealed differential molecular responses of the outer and inner scales to heat stress. Genes involved in lipid metabolism, oxidation pathways, and cell-wall modification were highly expressed in the outer scale during heating. Defense response-related genes such as those encoding heat-shock proteins, antioxidative stress defense, or production of osmoprotectant metabolites were mostly induced in the inner scale in response to heat exposure. These transcriptomic data led to a conceptual model that suggests sequential processes for the development of browning and desiccation of the outer scale versus processes associated with defense response and heat tolerance in the inner scales.

Effect of year, location, compost, and mixed oilseed cake on bulb and scale characteristic, nutrients and organic compounds in bulb and leaf, and storage quality in organic bulb onion

ABSTRACTThis study aimed to evaluate the effect of years and different locations, beef cattle manure compost (BCMC) and mixed oilseed cake (MOC) application rates on bulb and scale characteristics, nutrient content and nutrient uptake in bulb and leaf, and storage quality of intermediate-day onions (Allium cepa L.) at two organic onion growers' fields during the 2012/2013 and 2013/2014 growing seasons. Treatments included BCMC applied at rates of 0, 30 ton·ha-1 and MOC applied at rates of 0, 3, 6, 9 t·ha-1. Average temperature at two experimental locations during the growing season in 2013/2014 was 1.2°C higher than in 2012/2013, while total rainfall was 206.6 mm higher in 2012/2013 than in 2013/2014. Onion bulb diameter, bulb fresh weight, scale thickness and fresh leaf weight were significantly greater in 2013 than in 2014, but bulb dry matter was greater in 2014 than in 2013. Nitrogen, potassium (K) and magnesium (Mg) contents and uptakes in bulb were significantly higher in 2013/2014 than in 2012/2013. BCMC application at 30 t·ha-1 rate increased K and Mg contents in bulb and leaf as compared with no BCMC. Total soluble solids (TSSs), pyruvic acid (PA), total phenolics (TPs) and total flavonoids (TFs) contents were higher in 2013/2014 than in 2012/2013. BCMC application at 3 t·ha-1 decreased TSSs and PA content compared with no BCMC. MOC application increased PA content. Rot and weight loss on 8 September in cold room storage were lower in 2013/2014 than in 2012/2013, but the loss on 14 October and 10 December were not significantly different between two years. In conclusion, high air temperature and low rainfall in Mar. and Apr. increased fresh bulb weight and bulb thickness, while high temperature, low rainfall and long sunshine duration increased TSSs, PA, TPs and TFs contents. Compost and oilseed cake application increased fresh bulb weight, scale thickness, while they decreased TSSs and did not affect organic compounds.

Efficient protocol for in vitro propagation from bulb scale explants of Fritillaria ruthenica Wikstr. (Liliaceae), a rare ornamental species

An efficient protocol for in vitro propagation from bulb scale explants has been developed for Fritillaria ruthenica Wikstr., an endangered and rare species. The use of B5 medium supplemented with 5.0-µM 6-benzylaminopurine (BAP) and 2.0-µM α-naphthalene acetic acid (NAA) was found to be the most effective for adventitious bud induction and shoot multiplication (2.9 shoots per explant with 100% regeneration frequency). Adventitious regeneration occurred exclusively via direct organogenesis with the development of microshoots consisting of the rosette leaves, while bulb formation was not observed. The rooting microplants was carried out on a half-strength BDS hormone-free medium supplemented with activated charcoal at a concentration of 0.5 g/l resulted in 100% rhizogenesis with 5.2 roots per plantlet. Elevated sucrose concentrations (40.0 and 50.0 g/l) had no stimulating effect on bulblet differentiation at the in vitro rooting stage of F. ruthenica shoots. The regenerated plantlets were successfully transferred in a mixture of coconut fiber and sand (3:1) for acclimatization in greenhouse conditions with 72% of survival rate. The large bulbs developed only in ex vitro conditions at the end of the vegetation period.

Isolation and Characterization of Novel Pantoea stewartii subsp. indologenes Strains Exhibiting Center Rot in Onion.

Center rot of onion is an economically important disease caused by three Pantoea spp.: Pantoea ananatis, P. agglomerans, and P. allii. Symptoms caused by these three species are similar and include white streaking and necrosis of foliage; and, in some cases, the bacterium may enter the bulb, causing liquefaction and rot of bulb scales. Two bacterial strains were isolated from onion expressing symptoms indicative of center rot from two different outbreaks in Toombs County, GA in 2003 (PNA 03-3) and 2014 (PNA 14-12). These strains were initially identified as P. ananatis based on physiological and specific polymerase chain reaction (PCR) assays; however, further 16S ribosomal RNA (rRNA) and multilocus sequence analysis showed that the strains were more closely related to P. stewartii subsp. stewartii and P. stewartii subsp. indologenes. Further characterization using phylogenetic analysis, a P. stewartii subsp. indologenes-specific PCR assay, indole test, and pathogenicity on onion and pearl millet were conducted. Phylogenetic analyses (16S rRNA and atpD, gyB, infB, and rpoB genes) revealed that these strains formed a distinct cluster with the type strains of P. stewartii subsp. indologenes LMG 2632T and P. stewartii subsp. stewartii LMG 2715T separate from P. ananatis, P. agglomerans, and P. allii. Furthermore, onion strains were amplified with the P. stewartii subsp. indologenes-specific PCR assay. The pathogenicity assays with onion strains showed that they were pathogenic on onion and pearl millet, a known host of P. stewartii subsp. indologenes. However, the type strain of P. stewartii subsp. indologenes LMG 2632T was pathogenic only on pearl millet but not on onion. These results suggest that the onion strains PNA 03-3 and PNA 14-12 can potentially be novel P. stewartii subsp. indologenes strains capable of producing symptoms on onion. Hence, we recommend the inclusion of P. stewartii subsp. indologenes as the fourth member in the center rot complex of onion, along with P. ananatis, P. agglomerans, and P. allii.

Assessment of genetic fidelity of Fritillaria dagana (Liliaceae) regenerated plants using ISSR markers

ISSR analysis of Fritillaria dagana, endangered ornamental geophyte from the Sayan Mountains, regenerated through direct gemmogenesis from bulb scale tissue was performed. More informative electrophoresis profile with clear and distinct bands was obtained at amplification with (CAC)3GC primer at 56 °C annealing. The genetic fidelity of F.dagana regenerants to maternal plants was confirmed.