Volume 6 Special Issue 2, 2012
Bulbous Ornamentals II

ISBN 978--4-907060-04-6

How to reference: de Klerk GJ (2012)Taxonomy and Phylogeny of the Genus Lilium. In: Tuyl JM, Arens P (Eds) Focus on orchids. Floriculture and Ornamental Biotechnology 6 (Special Issue 2), 1-8

CONTENTS AND ABSTRACTS

Veli-Pekka Pelkonen, Anna-Maria Pirttilä (Finland) Taxonomy and Phylogeny of the Genus Lilium (pp 1-8)

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Invited Review: Lilies have a long history as ornamental plants. Today, there is an ever increasing variety of new lily cultivars due to the significant progress in the propagation and development of new methods in breeding. The domesticated native species have retained their place along with new hybrids in commercialized horticultural industry, and they have sustained their invaluable potential for the breeding of new cultivars for garden use as well as for greenhouse culture. Systematics has always played an important role in plant breeding, giving guidelines for hybridization, although biotechnology has introduced new solutions for many problems that were evolutionary obstacles especially in inter-specific crossings before. The genus Lilium has been a subject of variable suggestions for classification systems, and the process still continues. The currently accepted concept for the phylogenetic and taxonomic system for all species is based on geographical, structural and genetic information. In our review, we give an insight into the latest progress in revealing the taxonomical relationships within the genus. According to the existing GenBank sequence data, we have constructed a phylogenetic tree consisting of the main species and sections of the genus. Provided with species photos, the tree gives a brief overview of phylogeny- and morphology-based classifications, which are not always congruent. In the tree mainly all species grouped into sections defined within the genus, but L. bulbiferum and L. dauricum grouped equally with the species in Sinomartagon and not with each other. Even though these two species share many morphological features, the phylogenetic tree questions the existence of the section Daurolirion and potentially gives a blueprint for classification in the future.

Eisuke Matsuo (Japan) Historical Survey of Easter Lily Name in Association with Lilium longiflorum (pp 9-12)

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Invited Mini-Review: Based on the English dictionaries from around 1900, the author traces back how Lilium longiflorum came to be referred to as the Easter lily. The term “Easter lily” was given to any lily-like flowering species that bloomed around the Easter day, being called Easter flowers. Among them, L. candidum became the most famous one known as “Easter lily”. The evidence suggests that when more easily forcible bulb production began in the Bermuda Islands, the superiority of L. longiflorum over L. candidum in forcing ability and price, might have led to an explosive promotion of the usage of L. longiflorum as the Easter flower instead of L. candidum. Thus, the name "Easter lily" came to be transferred from L. candidum to L. longiflorum.

Nadeem Khan (Sweden), Agnieszka Marasek-Ciolakowska (Poland/The Netherlands), Songlin Xie, Munikote S. Ramanna, Paul Arens, Jaap M. Van Tuyl (The Netherlands) A Molecular Cytogenetic Analysis of Introgression in Backcross Progenies of Intersectional Lilium Hybrids (pp 13-20)

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Invited Review: The genus Lilium is comprised of about 100 species and has been divided into seven taxonomic sections. The abundance and diversity of species within the genus Lilium offers numerous and rewarding possibilities to lily breeders. Species within the same section can be crossed by conventional hybridization and this has led to different hybrid groups of great commercial importance such as Longiflorum, Asiatic and Oriental lilies. On the other hand, the divergence of species between various taxonomic sections causes considerable difficulties for intersectional crosses. Such difficulties include crossing incompatibility barriers, embryo abortion, sterility and reduced fertility in intersectional hybrids. For these reasons, various pollination techniques followed by in vitro embryo (sac) rescue and ovary culture, chromosome doubling, and 2n gametes are used frequently to obtain progeny between parents from different sections. Being the largest genome in the plant kingdom, lily is used as a model plant for cytogenetic analysis. The genome composition of the hybrids and backcross progenies were monitored through genomic in situ hybridization (GISH). The progress in molecular cytogenetic studies has been associated with the analysis of introgression of chromosomal segments in backcross progenies of various interspecific hybrids, contribution of individual genome in the resultant progenies, and the mechanism of 2n gamete formation. Based on the cytological analysis of progenies derived from the use of both haploid and 2n gametes, cytological maps of three different genomes have been constructed and the relevance of these analyses for introgression in Lilium is demonstrated.

Michikazu Hiramatsu, Jun-ichiro Masuda, Satomi Sakazono, Hiroshi Okubo (Japan) Evolution of Early Flowering Ability in Lilium formosanum from its Progenitor L. longiflorum (pp 21-27)

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Invited Mini-Review: Attention to the evolutionary background of ‘early flowering ability’, i.e. extraordinary shorter period from seed germination to the first flowering in Lilium formosanum, could have great potential for reducing costs in commercial production of lilies. We clarified the phylogenic relationship and geographic divergence of the early flowering ability and its related life history traits using seedling populations from natural populations of L. formosanum and its progenitor species, L. longiflorum. Based on the data obtained, we propose a hypothesis for evolution of the early flowering ability in L. formosanum.

Xue Wei Wu, Li Hua Wang, Li Fang Wu, Su Ping Qu (China), Jeung Keun Suh (South Korea), Ji Hua Wang (China) Native Species of the Genus Lilium and the Closely Related Nomocharis in Yunnan, China (pp 28-38)

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Invited Review: The lily is an important ornamental flower that has been cultivated for over 3,000 years, and with the development of breeding technologies, has been widely used in the garden, as potted flower, and for cut flower, floral designing. Lily belongs to the genus Lilium of family Liliaceae, which comprises over 180 species. China is the major center of Lilium distribution in the world; and almost one-third of the species found in China are distributed in Yunnan Province, China. 34 species of the genus Lilium are reported: L. brownii, L. rownii var. viridulum, L. wenshanense, L. sulphureum, L. sargentiae, L. lophophorum, L. lophophorum var. linearifolium, L. nanum, L. souliei, L. henrici, L. henrici var. maculatum, L. bakerianum var. bakerianum, L. bakerianum var. aureum, L. bakerianum var. delavayi, L. bakerianum var. yunnanen, L. bakerianum var. rubrum, L. sempervivoideum, L. amoenum, L. pinifolium, L. nepalense, L. nepalense var. burmanicum, L. nepalense var. ochraceum, L. wardii, L. taliense, L. duchartrei, L. lijiangense, L. papilliferum, L. davidii, L. fargesii, L. stewartianum, L. habaense, L. lankongense, L. primulinum, and L. lancifolium; Seven Nomocharis: N. aperta, N. saluenensis, N. forrestii, N. basilissa, N. farreri, N. meleagrina, and N. pardanthina. Sampling quantity, environmental survey of wild lily introduction, domestication note and crossing history have been previously investigated. Present and the proposed utilization classification characteristics of 41 native species are discussed in this paper.

Keiichi Okazaki, Shotarou Nukui, Hideaki Ootuka (Japan) Application of Nitrous Oxide Gas as a Polyploidizing Agent in Tulip and Lily Breeding (pp 39-43)

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Invited Mini-Review: Nitrous oxide has been successfully applied to zygotes as a polyploidizing agent in various crops. More recently nitrous oxide treatments have been applied at male gamete formation resulting in production of diploid gametes in tulips and lilies. Additionally this treatment can be used to overcome pollen sterility of interspecific hybrids via polyploidization of archesporial cells in developing anthers. This paper provides a review of some of the literature and results of our experiments using nitrous oxide for chromosome doubling of gametes and zygotes, as well as pollen mother cells to overcome pollen sterility of the interspecific hybrids. These methods have important implications for lily and tulip breeding.

Hiroshi Okubo, Michikazu Hiramatsu, Jun-ichiro Masuda, Satomi Sakazono (Japan) New Insight into Lilium brownii var. colchesteri (pp 44-52)

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Invited Review: Lilium brownii var. colchesteri has been widely cultivated for long time by its perfect flower shape with its colour arrangement and fragrance. However, it did not receive enough attention in recent lily research programs, and information on the history and culture is lacking. An overall research project on this species including breeding, flowering control, propagation, virus-free bulb production, flower pigment and scent along with the surveys of old literature and arts to clarify the introduction history of the species into Europe and Japan, has been conducted. The major results are: 1) L. brownii var. colchesterii was probably introduced in about 1600 from Korea to Fukuoka, 2) there was confusion of the species nomenclature of this species in Europe at the time of introduction, 3) all individuals of our present collection in Japan and Korea are clones, 4) F₁ hybrids of L. formosanum ´ L. brownii var. colchesteri obtained through cut-style pollination and ovary slice culture methods showed the early flowering traits of L. formosanum, but the flower shape and colour were similar to those of the pollen parent, 5) F₂ seedlings were obtained from self-pollination of F₁ through ovary-slice culture, 6) control of flowering was successful by temperature treatments, 7) an in vitro propagation procedure was established, 8) virus-free bulblets were obtained by a combination of meristem tip culture and chemotherapy, and 9) pigments that characterize the flower colour were identified.

Fred Bos (The Netherlands) Lilium bulbiferum L. Subsp. croceum (Chaix) Arcang.,TheOrange Lily,A Special Plant of Lowland NW Europe (pp 53-62)

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Invited Review: The life history of the Lilium bulbiferum subsp. croceum has been described and analyzed since the orange lily was discovered around 1850 growing wild in rye fields in the northern part of the Netherlands and northwestern Germany. In this article the status of the orange lily has been discussed. The lily does not seem to be a garden escape as was thought in the 19th century, but should be treated as a native species. The orange lily belonged to the agricultural weed plant community, the Sclerantho annui-Arnoseridetum, of the so called “eternal” rye fields on the poor sandy soils in the old-morainic landscape. Due to changes in agricultural use both the orange lily and the other weeds became very rare. Recent research shows that the lily has mainly survived in gardens. In Govelin in Lower Saxony (Germany) the very last rich plant community of the Sclerantho annui-Arnoseridetum with thousands of Lilium bulbiferum subsp. croceum is still present. Because of its beauty the lily has and has had important cultural aspects. In flower symbolism, in medieval paintings and in 17th century Dutch and Flemish flower paintings it is an important symbol. For its orange colour several links to the Dutch royal family, the House of Oranje (= Orange) Nassau, were discovered.

Neil O. Anderson, Erika Berghauer, David Harris, Kari Johnson, Jenni Lonnroos, Mary Morey (USA) Discovery of Novel Traits in Seed-Propagated Lilium: Non-vernalization-requiring, Day-neutral, Reflowering, Frost-tolerant, Winter-hardy L. xformolongi.I. Characterization (pp 63-72)

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Original Research Paper: Lilium are important floricultural crops worldwide. The objectives of this research were to examine L. xformolongi hybrids and a parental species (L. longiflorum) in photoperiods (Exp. 1) and environments (greenhouses, Exp. 1; field, Exp. 2) for flowering without vernalization, post-emergence photoperiod, potted plant/field performance, frost tolerance, and winter hardiness. Seed germination (4-15%) and yield potential (3.5-12.5%) varied between genotypes. In Exp. 1, short/long days had a significant effect on visible bud date only, but not on leaf unfolding rates, plant height, leaf number, or flowering dates; cultivar differences were highly significant. Cultivar × photoperiod interactions were nonsignificant except for flowering date (P = 0.04). ‘Nellie White’ (case-cooled bulbs) flowered in 213 d, while L. xformolongi cultivars took 247 d (‘Sakigake Raizan’) to 306 d (‘Raizan No. 3’) from sowing. Both VBD (h² = 0.93) and flowering date (h² = 0.91) were highly heritable (Exp. 1) and correlated. Regardless of photoperiod and environment, L. xformolongi flowered in <1 yr from sowing without cold. Leaf number (h² = 0.81, Exp. 1) and unfolding rates (h² = 0.93) were not as tightly linked in L. xformolongi as ‘Nellie White’. No seed-propagated hybrids (98-164 cm) were as short as ‘Nellie White’ (62 cm). ‘Augusta F₁’ had the highest flower bud counts (6.9 / plant). Shoot numbers ranged from n=1 (‘Nellie White’) to n = 3.8 (‘Sakigake Raizan’). L. xformolongi reflowered continuously in the field, but varied for frost-tolerance (25-75%; Exp. 2). Winter survival ranged from 0 to 87.5% in L. xformolongi over two years (Exp. 2). Flowering and reflowering of seed-propagated L. xformolongi in <1 yr. without vernalization, frost-tolerance, day neutrality, and winter hardiness are novel trait combinations for Lilium.

Neil O. Anderson (USA) Discovery of Novel Traits in Seed-Propagated Lilium: Non-vernalization-requiring, Day-neutral, Reflowering, Frost-tolerant, Winter-hardy L. xformolongi.II. Photoperiodism in Parents and Hybrids (pp 73-83)

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Original Research Paper: The discovery of seed-propagated lily hybrids which flower in <1 year from sowing in any photoperiod presents unique opportunities for transforming lilies. Recent research documented such hybrids to possess additional traits such as reflowering capabilities, frost tolerance, and winter hardiness. Objectives of this research were to examine Lilium xformolongi hybrids, backcrosses, and parental species (L. formosanum, L. longiflorum) in photoperiods (SD/LD) to clarify flowering in more stringent environments (growth chambers, greenhouses) than previously. Case cooled (CC) and non-CC bulbs (L. formosanum, L. longiflorum ‘Nellie White’); non-vernalized L. xformolongi seed-propagated cultivars (5 cultivars, 9 seed lots), L. longiflorum ‘Snow Trumpet’, and L. xformolongi backcrosses (5 BC₁F₁) were tested. Seed germination ranged from 4% to 83.3%; yield potential was similar. Flowering L. xformolongi (growth chamber) did not differ from L. longiflorum ‘Nellie White’ for VBD. Cultivar x photoperiod interactions were not significant except flowering date (P = 0.04). ‘Nellie White’ (CC) flowered in 213 d, while L. xformolongi cultivars flowered in 247 d (‘Sakigake Raizan’) to 306 d (‘Raizan No. 3’) from sowing. Non-vernalized L. formosanum, L. longiflorum bulbs never flowered in either photoperiod or environment. Both VBD and flowering date were highly heritable and correlated. Regardless of photoperiod and environment, seed-propagated L. xformolongi flowered in <1 year. One backcross was day-neutral for flowering. Two L. xformolongi BC₁F₁ and L. longiflorum ‘Snow Trumpet’ produced significantly less leaves than ‘Nellie White’. Leaf number (h² = 0.83) was not as tightly linked in L. xformolongi as ‘Nellie White’. Plant height in L. formosanum (CC bulbs, several backcross L. xformolongi hybrids) did not differ from ‘Nellie White’. In contrast, only ‘Sakigake Raizan’ was taller than ‘Nellie White’ in growth chambers.

Yoon-Jung Hwang (South Korea), Paul Arens, Jaap M. Van Tuyl (The Netherlands), Tae-Jin Yang, Ki-Byung Lim (South Korea) Genome Analysis of Lilium tigrinum by Chromosome Microdissection and Molecular Cytogenetic Techniques (pp 84-88)

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Original Research Paper: Chromosome microdissection and microcloning are powerful tools for plant genome research. Here we describe the isolation of chromosome #1 derived sequences from L. tigrinum with these techniques and their characterization. Detailed chromosome analysis was performed by FISH and then chromosome #1 was isolated from metaphase chromosomes of L. tigrinum by microbeam dissection. DOP-PCR and LA-PCR were used to amplify a DNA of chromosome #1 segments. PCR products from the microdissected chromosome were cloned into a plasmid vector to construct a chromosome #1 specific library and sequenced. BLAST-nr revealed that 28% of the sequences were matched with known genes and transposons, and the rest of 72% did not match with known sequences from NCBI database of plant taxa. The unknown sequences were putatively divided into five classes and we called them lily unique unknown repeats. FISH confirmation with some clones confirmed that the products from both methods were indeed amplified from the chromosome #1 of L. tigrinum genome. These results provide important information for not only the composition of the Lilium genome but also for detailed sequence information of huge genome sized plants.

Juozas Proscevičius, Vida Rančelienė, Violeta Kleizaitė (Lithuania) Application of Mixed Incongruous Pollen for Interspecific Crosses of Lilies (pp 89-93)

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Original Research Paper: Creating interspecific hybrids between distantly related species is the first critical step in breeding of lilies. Different methods are used to overcome pre-fertilization and post-fertilization barriers in incongruous interspecific crosses of lilies. Compatibility between female pistil and pollen play an important role on fertility of crosses. Results are presented showing that pollen incongruous to female pistil can perform fertilization if they are used in mixtures with other incompatible pollen. To overcome pre-fertilization barriers cut-style pollination and pollination of stigma by mixed incompatible pollen were performed. In some cases recalcitrant crosses were more efficient when stigma was pollinated by mixed pollen of incongruous species than when cut-style was pollination by separated pollen of one incongruous male. However, cut-style pollination was efficient in cases when long style possessing females were pollinated by short style possessing male species. Though it is known that crosses of Asiatic hybrid (Asiatic hybrids (A) with Lilium longiflorum or Oriental hybrids (O) possible perform only in one direction where Asiatic hybrid participate as male, the pollen of L. longiflorum and O stimulated fertilization of Asiatic hybrid when they were added in mixtures with pollen of L. candidum, L. henryi, L. monadelphum or Trumpet and Aurelian hybrids (T). However, any stimulation of fertilization was observed when pollen of L. longiflorum and O were added in mixtures with pollen of L. concolor or L. pumilum to pollinate native pistil of Asiatic hybrid. Pollination of Asiatic hybrid by mixed pollen of incongruous species resulted progeny among which hybrids Asiatic hybrid x L. regale and Asiatic hybrid x L. candidum were screened by inheritance of molecular markers characteristic to male species.

Kathryn Kamo (USA), Bong Hee Han (South Korea) Optimized Growth and Plant Regeneration for Callus of Lilium longiflorum ‘Nellie White’ (pp 94-98)

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Original Research Paper: Rates of growth and regeneration were compared for compact callus, friable callus, and suspension cells of Lilium longiflorum ‘Nellie White’ to determine the optimal culture conditions. The highest frequencies of embryogenic callus induction (60-90%) occurred from compact callus cultured on either picloram (0.5, 1, or 2 mg/L) or dicamba (2 mg/L). Fresh weight (FW) was higher for compact callus induced from bulb scales cultured on MS medium supplemented with picloram (0.5, 1, or 2 mg/L) compared to scales cultured on MS medium supplemented with dicamba (2, 4, or 8 mg/L). Compact callus cultured on picloram (0.5, 1, or 2 mg/L) or dicamba (2, 4, or 8 mg/L) grew slowly with a 1.2X increase in FW/month compared with suspension cells grown in 0.5 mg/L picloram that increased 1.7X in FW/month. Regeneration rates were similar (23-35 plantlets/g FW callus) for compact callus cultured on either dicamba (2 or 4 mg/L) or picloram (0.5 or 1 mg/L), but 3% of the plantlets regenerated from dicamba were phenotypically abnormal while none were abnormal with picloram. Suspension cells showed a lower regeneration rate than compact callus with a maximum of only 12 plantlets regenerated from one g fresh weight suspensions cells grown in 0.5 mg/L picloram. A fast-growing, friable callus was induced and selected from compact callus cultured on MS medium with 2 mg/L dicamba and 9% sucrose but not from 3, 6, or 12% sucrose. Friable callus grew 5X faster than compact callus and formed numerous somatic embryo-like structures when cultured on MS medium with 1% activated charcoal, but only a few embryo-like structures germinated to form plants with roots.

Chad T. Miller, Benham Lockhart, Margery Daughtrey, William B. Miller (USA) Iron Deficiency May Result in Interveinal Chlorosis of Shamrock Plant (Oxalis regnellii) (pp 99-103)

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Original Research Paper: Oxalis regnellii is a geophytic ornamental pot plant grown primarily for its clover-like leaves. During greenhouse production, the leaves often become chlorotic for unknown reasons, possibly including virus infection, iron (Fe) and/or manganese (Mn) deficiencies, and improper greenhouse forcing temperatures. We conducted a series of experiments to address these hypotheses. Shamrock chlorotic ringspot virus (SCRV) has been reported before in Oxalis regnellii. Oxalis plants exhibiting virus-like symptoms were analyzed and a potyvirus was detected, although this virus was not further confirmed to be SCRV. To confidently test other hypotheses, any suspected viral infected material was discarded. Plants grown at 13°C exhibited slowed growth and development; however, the incidence of leaf chlorosis did not increase compared with plants grown at warmer temperatures of 21/16°C (day/night); 22°C constant; or 22 to 16°C (plants were moved to 16°C when 50% of the plants were in first flower). To assess the ability to correct an iron (Fe) deficiency, a media drench of ferric ethylenediaminedi (o-hydroxyphenylacetic) acid (Fe-EDDHA) was applied to chlorotic, Fe-deficient oxalis plants and plants successfully re-greened within 5 days.

Emmy Dhooghe, Dirk Reheul, Marie-Christine Van Labeke (Belgium) Cytological and Molecular Characterization of Intertribal Hybrids Between the Geophytes Anemone coronaria L. and Ranunculus asiaticus L. (Ranunculaceae) (pp 104-107)

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Original Research Paper: Anemone coronaria L. and Ranunculus asiaticus L. both belong to the Ranunculaceae, a large plant family with many ornamentals of horticultural importance. There are considerable differences between these species leaves, flower morphology and flower colour therefore intergeneric crosses between the species might result in new interesting hybrids. Crosses between Anemone coronaria L. and Ranunculus asiaticus L. were performed and the F1 progeny was examined. In this study, the F1 hybrid generation was investigated at morphological, molecular and cytogenetic levels. More than 85% of the F1 plants had very similar flowers to the maternal plants and although seeming to have a limited paternal contribution, AFLP analyses confirmed the partial hybrid character of the F1 plants. GISH experiments revealed that the F1 plants were mixoploids (plants composed of cells with different chromosome numbers) or showed many chromosome rearrangements.

Marian Saniewski (Poland), Hiroshi Okubo, Kensuke Miyamoto, Junichi Ueda (Japan) Evidence for a Role of Auxin in the Stem Elongation of Dark-grown Tulips (pp 108-113)

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Original Research Paper: Shoot growth of fully cooled tulip bulbs cvs. ‘Gudoshnik’ and ‘Apeldoorn’ grown in continuous dark conditions was investigated in relation to the role of exogenously applied auxin. Continuous darkness caused much more stem elongation than natural light conditions in the greenhouse. In both cultivars, all internodes were longer in the dark than those in the light. Auxin (indole-3-acetic acid, IAA, applied in the place of a removed flower bud on a stem with no leaves) greatly stimulated the growth of all internodes in the dark in comparison to that in the light whereas almost no growth in all internodes was observed in the absence of exogenously applied auxin both in the dark and in the light. These results confirm that auxin is a major factor responsible for growth of all internodes in etiolated tulip stems. The hormonal control and its metabolic significance during the etiolation of tulips are discussed.

Dariusz Sochacki, Małgorzata Podwyszyńska (Poland) Virus Eradication in Narcissus and Tulip by Chemotherapy (pp 114-121)

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Original Research Paper: The aim of the research was to obtain virus-free stock plant material of several cultivars and breeding clones of narcissus (Narcissus L.) and tulip (Tulipa L.) by in vitro culture using chemotherapy with ribavirin. Virus indexing by ELISA was done several times to detect the most important viruses infecting tulips and narcissus. Genotypes of both crops, totally or heavy infected by viruses, were subjected to chemotherapy in a few experiments. The results of virus eradication showed that none of the three concentrations of ribavirin (12.5, 25 and 50 mg l^-1) did result in death. However, shoot regeneration and growth on the medium with the highest concentration of ribavirin was significantly retarded, both for tulips and narcissus. Chemotherapy joined with further consecutive virus indexing and roguing the virus-suspected plantlets of tulip S2 and S3 enabled selection of virus-negative plants. The results of chemotherapy obtained for the new tulip cultivars (S2, S6 and S7) in the next experiment were very promising. The ribavirin treatment resulted in virus eradication from the newly forming shoots. In turn, in the old cultivars (E and F) and one breeding line (P7), whose shoots were totally infected, chemotherapy appeared to be ineffective. Virus eradication was unsuccessful for all treated plantlets of narcissus ‘Lajkonik’ infected by Narcissus mosaic virus and potyviruses in two experiments. Virus eradication for breeding clone 0.985T infected by Narcissus latent virus and potyviruses was successful in 27 among the 80 plantlets which started the experiment. The results leads to conclusion that the effect of ribavirin depends on the concentration of this antiviral agent, kind of virus and their concentration in plant tissue and on the genotype of plant.

Regina Juodkaitė, Angelė Meilutė Baliūnienė, Zenonas Jančys (Lithuania) Assessment of the Vegetative Reproduction Potential of Tulips (Tulipa L.) (pp 122-132)

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Original Research Paper: The principal aim of this research was the assessment of the vegetative reproduction potential of different size tulip bulbs. Bulbs were arranged by size into 7 fractions. Vegetative reproduction capacity of different size tulip bulbs of 299 cultivars was calculated using a number of specific reproduction coefficients: total reproduction coefficient (TRC), generative bulb reproduction coefficient (GRC) and forcible bulb reproduction coefficient (FRC). Reproduction coefficients were calculated individually for each different bulb size class of the investigated tulip cultivars. TRC is a quantitative indicator specifying the mean number of all daughter bulbs per clone. GRC is a qualitative indicator specifying the mean number of bulbs per clone that is capable to blossom next year. FRC is a qualitative indicator specifying the mean number of forcible tulip bulbs per clone. By modulating the data on TRC, GRC and FRC of all cultivars of different size bulbs, indexed reproduction coefficient (IRC) was deduced. IRC indicates a comparative reproduction value. Empirical tulip cultivar dispersion analysis demonstrated that this coefficient most objectively reflects reproduction capacity of all bulbs of the studied tulip cultivars. Based on IRC, the investigated tulip cultivars were grouped into 5 classes of reproduction capacity. Most tulip cultivars were ascribed to 2nd–4th classes (correspondingly 24, 30 and 30%), whereas a small number of the studied cultivars were attributed to one of the outer classes 1st and 5^th (8%).

Faouzi Haouala, Emna Chaïeb (Tunisia) Effects of Explant Position and Polarity on Callus Induction and Shoot Regeneration of Gladiolus (Gladiolus hybridus Hort.) (pp 133-139)

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Original Research Paper: Different types of explants were used for callus induction in Gladiolus tissue culture of cultivars ‘ChaCha’ and ‘Priscilla’. Different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA) were tested. Explant polarity was studied for leaves by placing fragments of these organs horizontally and vertically on the culture medium. Also, explants were taken from the apical, middle and basal parts of leaves and petals. Apical buds, leaves and flower stalks showed excellent callus formation (100%). However, petals were characterized by a low callus formation ability (10%) while floral stems, bracts and floral spikes showed no callus formation. The rate of callus development was significantly higher for horizontally cultured leaves and decreased from basal explants to middle and apical ones. The highest rate of callus formation was obtained on media containing either 2,4-D (1 or 3 mg l^-1) or NAA (2 or 5 mg l^-1). Callus budding was significantly higher on medium supplemented with 1 mg l^-1 BA. The budding rate of callus obtained from apical buds was 93.3 and 100%, respectively, for cultivars ‘Priscilla’ and ‘ChaCha’. For callus derived from leaf fragments, the rate of budding was 100% for both cultivars.

Rosalia Paladines, Diandra Jurado (Ecuador), Tjitske Riksen-Bruinsma (The Netherlands), Ana Maria Quiñones (Ecuador) Prospects of Isolated Microspore Culture for Haploid Production in Anemone coronaria L. (pp 140-145)

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Original Research Paper: The aim of this study was to establish a procedure to obtain haploid plants from microspore cultures of Anemone coronaria L., an important ornamental crop known worldwide due to its commercial value in the cut flower industry. Microspores were isolated from two genotypes of A. coronaria: ‘Blue’ (plants obtained through one cycle of selfing) and ‘Lilac’. The effect of different treatments to interrupt the gametophytic development of microspores and promote sporophytic development was evaluated. High temperature, culture media composition and developmental stage of microspores at the moment of isolation were the assessed factors. Achieved microspore-derived embryo formation was 0.53% for ‘Blue’ and 0.06% for ‘Lilac’. Different treatments were tested for microspore-derived embryo germination. Organic supplements had a positive effect on triggering germination, while growth regulators were needed to complete the development of the plantlets. Germination percentage was 2.13 and 2.41 for ‘Blue’ and ‘Lilac’, respectively. The ploidy analysis revealed the existence of haploid and doubled haploid plants of both genotypes. We identified 18 haploid plants and 9 doubled haploid plants of ‘Blue’, and 4 haploid plants and 3 doubled haploid plants of ‘Lilac’.

Yukiko Kashihara, Koichi Shinoda, Hajime Araki, Yoichiro Hoshino (Japan) Towards Intergeneric Hybridization between Alstroemeria L. and Bomarea Mirb. (pp 146-149)

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Original Research Paper: There are many interspecific hybrids of Alstroemeria. In this study, the possibility of intergeneric hybridization between Alstroemeria and Bomarea Mirb. was examined through the development of pollination procedures and ovule culture based on the histological observation of embryo and endosperm development after intergeneric pollination. Three methods of pollination (stigmatic, cut-style, and non-style) were combined with four different pollen types (fresh, frozen, non-germinated, and pre-germinated). We observed that the pollen tubes of Bomarea coccinea (Ruiz & Pav.) Baker could reach to the ovules of Alstroemeria aurea Graham 48 hours after stigmatic pollination with frozen pollen. Histological observations revealed that a primary embryo was formed, but subsequently aborted during development. This study demonstrates the possibility of intergeneric hybridization between Alstroemeria and Bomarea, but showed that there are post-fertilization barriersbetween A. pelegrina and B. coccinea. Further study is needed to investigate the optimum conditions for obtaining hybrid progeny.

Takejiro Takamura, Hideki Yamashita (Japan) Specific Differences in Nuclear DNA Content in the Genus Cyclamen (pp 150-153)

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Original Research Paper: Relative nuclear DNA contents in Cyclamen species were estimated by flow cytometry with 4’, 6-diamidino-2-phenylindole (DAPI) staining and propidium iodide (PI) staining. The relative fluorescence intensity (RFI) values in each species ranged from 1.521 to 8.071 and from 1.485 to 7.941 in flow cytometry with DAPI and PI staining, respectively. The ratio of RFI with PI staining to that with DAPI staining ranged from 0.93 to 1.15. Species belonged to the same subgenus indicated almost the same ratio of RFI with PI staining to that with DAPI staining. Subgeneric difference of the ratio was also observed. Flow cytometry seemed to be useful for the identification for interspecific hybrids in Cyclamen species, because the RFIs of interspecific hybrids were intermediate between that of their maternal and paternal species. The results of the present study should indicate that flow cytometry might be one of the effective tools for classification and identification of interspecific hybrids in the genus Cyclamen.

Peter J.M. Knippels (The Netherlands) Advanced in Vivo Propagation Techniques for Specialty Bulbs (pp 154-157)

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Original Research Paper: Bulbs can be propagated by natural propagation techniques like by seeds and off setts, as well as by techniques like scoring, chipping, scaling and leaf cuttings. These techniques are mostly used for in vivo propagation, less for in vitro propagation. This article concerns the first case. Specialty bulbs can successfully be propagated by scoring and chipping, either starting at the beginning of the dormant period or at the end of this period. Of various species of the genera Eucomis, Lachenalia and Ornithogalum, it is known that they can successfully be propagated by leaf cuttings. In the presented experiments the techniques scoring, chipping and leaf cuttings were applied on Eucomis autumnalis and Hymenocallis festalis. Scoring and chipping resulted in new newly formed bulblets with E. autumnalis. Propagation by leaf cuttings resulted only with E. autumnalis to the formation of adventitious bulblets. Crucial is the timing of propagation, which will count for all genera and species. In previous research with Lachenalia species it has been proved that the regeneration potential is highest when leaf cuttings are taken at visible bud stage.. A preliminary conclusion is that with Eucomis the regeneration potential is the highest before flowering. It is not possible to draw conclusions for Hymenocallis, because this genus belongs to another family than Eucomis and Lachenalia and the experiment with leaf cuttings failed with this genus. Due to the low number of used bulbs a statistical evaluation was not possible.

Antra Balode (Latvia) Effect of Microbiological Products on Bulblet Development of Lilium spp. in Scale Culture (pp 158-160)

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Short Communication: The aim of the present study was to investigate the effects of two locally produced microbiological products Trihodermins B-J and Vitmins on scale promotion and bulblet development in Lilium. Lily scales were given three treatments: untreated controls; Vitmins solution (10 ml L^-1) and Vitmins solution (10 ml L^-1) + Trihodermins B-J (dry powder form, at 10 g kg^-1 in the substrate). After treatment, scales were placed in a polyethylene bag in peat moss. Bulbs of three cultivars from different groups were used: Asiatic hybrid ‘Gardenja’, Longiflorum × Asiatic (LA) hybrid ‘Sonora’ and Trumpet hybrid ‘Elegija’. The scales were incubated in a plastic greenhouse at 15°C for 12 weeks; at 5°C for 10 weeks; and 18°C for 4 weeks. The effectiveness of microbiological products was evaluated by the number of bulblets per scale, the diameter of bulblets, shoot height, root length, and the percentages of dead plants. Significant difference (P < 0.05) between shoots and roots among the three cultivars and variants were found. The mean values obtained for shoots by treatment with Vitmins + Trihodermins B-J were: ‘Gardenja’ - 138.5 ± 9.8 mm, ‘Sonora’ – 102.0 ± 4.7 mm and ‘Elegija’ – 7.0 ± 3.0 mm. The coefficient of variation for number of bulblets per scale was recorded in the range from 19.7 to 39.2%. Treatment with Vitmins + Trihodermins B-J resulted in a significantly higher number of bulblets per scale and greater shoot height (P < 0.05 vs. control).