Volume 6 Special Issue 1, 2012
Bulbous Ornamentals I

ISBN 978--4-907060-03-9

How to reference: de Klerk GJ (2012)Micropropagation of Bulbous Crops: Technology and Present State. In: Tuyl JM, Arens P (Eds) Focus on orchids. Floriculture and Ornamental Biotechnology 6 (Special Issue 1), 1-8

CONTENTS AND ABSTRACTS

Geert-Jan de Klerk (The Netherlands) Micropropagation of Bulbous Crops: Technology and Present State (pp 1-8)

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ABSTRACT

Invited Review: Conventional propagation of bulbous crops must be supplemented with micropropagation to satisfy the requirements of present-day horticulture with respect to fast production of disease-free, superior starting material. Adequate micropropagation protocols for bulbous crops are therefore a sine qua non. The successive steps in micropropagation of bulbous crops are reviewed: initiation, multiplication, bulb formation, dormancy breaking and planting. In the first two steps, new shoots or bulblets are generated by axillary bud outgrowth or adventitious regeneration. During initiation, endogenous contamination may be a severe problem since bulbs grow subterraneously and have often been propagated vegetatively in the field for many years. Other drawbacks are insufficient axillary branching, poor adventitious regeneration and inferior growth. The latter, inferior growth, is likely the most significant problem and is caused by poor translocation of medium ingredients to the growing regions within the explant. In micropropagation of bulbous crops, bulblets should be produced because of, among others, easy handling and acclimatization. For optimal performance after planting in soil, preparatory treatments are required in particular a dormancy breaking treatment. A phase-change from juvenile to adult and protective pretreatments are also profitable. It is concluded that when major problems like that of inferior growth have been solved, commercial micropropagation of bulbous crops will experience a second heyday.

Małgorzata Podwyszyńska (Poland) The Mechanisms of in Vitro Storage Organ Formation in Ornamental Geophytes (pp 9-23)

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Invited Review: In most ornamental geophytes, the rate of conventional vegetative propagation is low. Ten or even twenty years can pass until the commercial release of a new genotype. Therefore, numerous micropropagation methods have been developed to enhance the propagation rate, but also to obtain healthy elite stock material, speed up the breeding process, provide new genotypes on the market and restore endangered geophytes. In a number of geophyte species, formation of bulbs, corms or tubers is an essential step in the micropropagation process because only a storage organ shows high rooting ability and good field performance. In nature, abundance of photosynthesis-derived sugars together with some inducing environmental factors (low temperature, short or long day) trigger a sequence of biochemical, physiological, and finally, morphological events leading to storage organ formation. In in vitro conditions, however, due to insufficient light intensity, sugars have to be provided exogenously. In vivo, sugars exist as the multifunctional internal factors, while in vitro, they act both as internal and external factors. This can lead to certain disturbances in the course of the in vitro tuberization process. Therefore, success in storage organ formation often requires administration of the proper growth regulators whose endogenous production is insufficient (cytokinins, abscisic acid, jasmonates, auxins and polyamines), and sometimes other specific compounds such as inhibitors of gibberellin biosynthesis. This review focuses on the recent findings about the tuberization process in vitro of ornamental geophytes in relation to the newest knowledge concerning tuber formation in potato as model plant. Also some aspects of storage organ formation in vitro, the stages and the factors regulating this process on the morphological, physiological and biochemical levels are discussed in relation to storage organ formation occurring in nature (in vivo).

Avner Cohen (Israel), Frans A. Krens (The Netherlands) Genetic Transformation in the Breeding of Flower Bulbs (pp 24-34)

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Invited Review: Ornamental geophytes are used for the production of cut-flowers, potted flowering plants or in gardening (collectively known as flower bulbs). Most flower-bulb cultivars have been produced by cross hybridization and mutation breeding and are propagated vegetatively. Biotechnological techniques have been used to breed and propagate these plants. Plant breeders use in vitro techniques, such as cut-style in vitro fertilization, embryo rescue, ovary-slice culture and ovule culture, to overcome pre- or post fertilization compatibility barriers and generate interspecific hybrids. Recently, biotechnological tools such as molecular markers and genetic engineering have also been introduced. Genetic transformation may be defined as the utilization of isolated recombinant DNA based technology to aid the effective incorporation of a limited number of valuable traits (that are not available in the original plant genome or in closely related species) into improved cultivars lacking such traits. Transformation techniques supplement the other methods available to plant breeders and are especially valuable for clonally propagated crops, such as flower bulbs. Flower bulbs have been transformed using both Agrobacterium-mediated and microprojectile-acceleration methods. In both systems, the success of the transformation depends upon the successful assembly of several key components and the calibration of the entire system. One component is the availability of a genetic construct carrying target genes under the control of appropriate promoters. A second component is the target organ or tissue, which must be competent for genetic transformation. That is, it must be capable of accepting the foreign DNA, into the genome of its own cells, expressing the genes and maintaining the ability to regenerate into plants. Introduced genes in many agricultural crops include those that confer resistance to biotic or abiotic stresses, as well as genes that alter plant phenotype (e.g., flower color). Although transformation systems for many flower-bulb crops are available, few attempts to produce genetically engineered flower bulbs with commercially valuable traits have been successful and, to date, none have resulted in a registered cultivar. In order to be commercially viable, any genetically engineered flower bulb cultivar would contain mostly proprietary technology covered by freedom-to-operate agreements. Marker-free technology is needed to ease the risk-assessment process and to address public concerns.

William B. Miller (USA) Current Status of Growth Regulator Usage in Flower Bulb Forcing in North America (pp 35-44)

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Invited Review: North American greenhouse companies are mainly focused on potted crops, and due to lower geographical concentration, individual greenhouse firms, tend to have a very diversified product offering. As such, individual crops are often grown under less than optimum conditions. Plant growth regulators offer a set of tools that allow growers to better tailor bulb crops to containers, and the more liberal market and regulatory environment in North America continues to allow a high degree of specialization of PGR use. It is hoped the specific PGR and crop information presented herein and available online will stimulate additional research and interest in these products and crop uses worldwide.

Barbara Ruffoni, Marco Savona, Sara Barberini (Italy) Biotechnological Support for the Development of New Gladiolus Hybrids (pp 45-52)

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Invited Review: The genus Gladiolus L. (Iridaceae) includes important ornamental species and hybrids which are successfully treated on the market since the last century. Recently breeders have selected new genotypes with the aim to increase the production in winter and late spring especially in countries bordering the Mediterranean Sea. In order to speed up the volume of the new hybrids and to produce virus-free stocks of mother plants, it is possible for Gladiolus to apply biotechnological tools of in vitro propagation through liquid culture or simple bioreactors such as temporary immersion system to reduce production costs and to enhance multiplication rate and cormel quality. Gladiolus micropropagation was reported first by Ziv et al. in 1970 and subsequently by several other authors exploring the performances of different species and varieties. Bulbs and corms of several species are commercially propagated in liquid culture in semi-automatic systems as temporary immersion; in Gladiolus, Ruffoni et al. presented in 2008 data about high efficiency micropropagation using temporary immersion compared with the culture on agar-solidified medium suggesting an efficient semi automatized protocol. The present paper takes into consideration the different ways for in vitro culture initiation and the efficiency of the meristem excision for the establishment of pathogen-free cultures including data coming from direct experiences and bibliography search. Moreover it will compare the different growth strategies (solid vs liquid micropropagation) evaluating finally the performances of the temporary immersion system.

Zhanao Deng (USA) Caladium Genetics and Breeding: Recent Advances (pp 53-61)

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Invited Review: Caladiums are important ornamental aroids; they are valued for their colourful and variably-shaped leaves. Numerous advances have been made in recent decades in caladium breeding and genetic studies. Techniques have been developed to increase flower production, store pollen, and maintain seed viability. Sources of genetic resistance have been identified for important diseases and pests (such as Fusarium tuber rot, Pythium root rot, bacterial blight, and root-knot nematodes) and abiotic stress factors including chilling injury. Mode of inheritance for important foliar traits has been elucidated through analysis of trait segregation in progeny populations. Caladiums have evolved three alleles at one locus that control colour of leaf main veins (red, white or green) and two co-dominant alleles at an independent locus that determine leaf shapes (fancy, lance, or strap). Gene loci for leaf spotting and blotching are both simply inherited but tightly linked to green veins. In vitro culture and plant regeneration were successful with several types of tissues/organs through somatic embryogenesis and/or organogenesis. Shoot-tip culture has been used to eliminate viral and fungal pathogens and invigorate planting stock; protoplasts isolated from leaf callus regenerated into whole plants; foreign genes from maize or humans have been introduced into caladium through Agrobacterium co-cultivation. Molecular markers, including highly specific and informative SSRs, have been developed and applied to caladium to distinguish cultivars, assess genetic diversity, and analyze genetic relationships. The availability of these improved techniques, sources of desirable traits, and cellular or molecular tools will be very valuable for enhancing caladium breeding efficiency, achieving specific breeding objectives, and developing valuable new cultivars.

Rina Kamenetsky (Israel), John Dole (USA) Herbaceous Peony (Paeonia): Genetics, Physiology and Cut Flower Production 62-77)

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Invited Review: Peony (Paeonia spp.) is one of the most popular garden plants in temperate regions. They were introduced into cultivation in China hundreds of years ago, and have since been spread widely to many countries. According to morphological traits and life form, the genus is divided into tree and herbaceous peonies. Numerous cultivars of herbaceous peonies have been developed to satisfy demand for colors, fragrance, flowering time, and disease resistance. In the last two decades, the popularity of peonies as cut flowers has resurged, and has resulted in additional requirement for new research, production methods and postharvest technology. Today, more than 25 countries produce cut peony flowers, with the primary markets being in Europe and the USA. Despite the popularity of herbaceous peonies, their production and use are restricted due to a lack of reliable systems for mass propagation, a long juvenile period, complicated flowering physiology and ineffective postharvest handling procedures. In this review, highlights of the recent scientific research in herbaceous peony are presented, along with up-to-date information on peony propagation, postharvest handling and cut flower marketing.

Marija Petrić, Angelina Subotić, Milana Trifunović, Slađana Jevremović (Serbia) Morphogenesis in Vitro of Fritillaria spp. (pp 78-89)

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Invited Review: The genus Fritillaria includes 100 species of bulbous plants and is found throughout the temperate region of the Northern Hemisphere. Fritillaria species are often used as ornamental plants, but various species have also been used in traditional Chinese, Japanese and Turkish medicine. Many species from the genus Fritillaria are endangered, rarely found in the wild and protected by law. Micropropagation techniques have great importance for germplasm conservation and commercial multiplication of fritillaries. Successful propagation methods have been developed for the following Fritillaria species: F. anhuiensis, F. alburyana, F. camtschatcensis, F. cirrhosa, F. hupehensis, F. imperialis, F. meleagris, F. pallidiflora, F. przewalskii, F. roylei Hook, F. sinica, F. sichuanica, F. thunbergii, F. taipaiensis, F. unibracteata, F. ussuriensis and F. whitallii. This paper summarises the various techniques of in vitro morphogenesis induction and rapid propagation of fritillaries, as well as successful acclimatisation. The most potent explant types for the induction of morphogenesis in vitro are bulbs, bulb scales, inflorescence parts and immature or mature zygotic embryos. Whole plant regeneration of fritillaries has been achieved by bulblet production, as well as by direct or indirect somatic embryogenesis. The influence of different media compositions, hormone concentrations and temperature requirements for the induction of morphogenesis and overcoming of dormancy are discussed. This review also describes major secondary metabolites in Fritillaria (alkaloids and non-alkaloid constituents), their nature and perspective for production by methods of in vitro culture which can be used in the pharmaceutical industry.

Agnieszka Marasek-Ciolakowska (The Netherlands/Poland), M.S. Ramanna, Paul Arens, Jaap M. Van Tuyl (The Netherlands) Breeding and Cytogenetics in the Genus Tulipa (pp 90-97)

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Invited Review: Tulip (Tulipa) is one of the most important ornamental bulbous plants, which has been cultivated for cut flower, potted plant, garden plant and for landscaping. Species from the different sections display complementary agronomic characteristics and breeding techniques are used to combine desired features. The main goals of modern tulip breeding are the introgression of resistance against Tulip Breaking Virus (TBV), Botrytis tulipae and Fusarium oxysporum (bulb-rot), and also characteristics such as a short forcing period, good flower longevity and new flower colours and flower shapes into the commercial assortment of T. gesneriana. T. gesneriana has been crossed successfully with only 12 out of the approximately 55 tulip species by using conventional breeding methods. Many successful crosses have been made between T. gesneriana cultivars and TBV resistant T. fosteriana cultivars resulting in highly resistant Darwin hybrids tulips. The majority of tulip cultivars are diploid (2n = 2x = 24) however, there have been many attempts to obtain polyploid tulips. The production of tetraploids was described in the late sixties when young ovaries were treated, under pressure, with laughing gas (N₂O). In breeding of polyploid tulip laughing gas has also been used to induce 2n gametes. Several new tetraploids were also obtained by making crosses between tetraploid lines. Polyploids have been derived from interploidy crosses between diploid, triploid, and tetraploid cultivars. Several other polyploids have resulted from 2n gametes, spontaneously produced by diploid F1 hybrids. Molecular cytogenetic tools such as FISH and GISH permitted detailed studies of genome composition and chromosome recombination in the progenies of interspecific hybrids. In this context, tulip breeding and the use of cytogenetic techniques for genome analysis of hybrids are discussed.

Riana Kleynhans, Paula Spies, Johan J. Spies (South Africa) Cytogenetic and Phylogenetic Review of the Genus Lachenalia (pp 98-115)

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Invited Review: The genus Lachenalia (family Asparagaceae), endemic to southern Africa, is a horticultural diverse genus, with many species featuring in the red data list of southern Africa. The extensive morphological variation within some species complicates species delimitation and has led to taxonomic confusion. The genus is utilised in a breeding programme where cytogenetic and phylogenetic information is important for the development of breeding strategies. Chromosome numbers of 89 species have been recorded in literature, with 2n = 10 to 56 and n = 5 to 28. B-chromosomes have been described in some species. Basic chromosome numbers include x = 5, 6, 7, 8, 9, (probably 10), 11, (probably 12), 13 and (probably 15). Polyploidy was reported in 19 taxa (23%), and is most common in the x = 7 group. Molecular cytogenetic studies using 5S rDNA, 18S rDNA probes and DAPI staining, as well as molecular systematic studies using trnL-F and ITS1-2 were used to assess the phylogeny of the genus. All these studies indicated that species with the same basic chromosome number are closely related. The one deviation is that it appears as if there are two separate groups within the x = 7 group. The cytogenetic and molecular studies are further supported by breeding studies, where improved results are generally obtained from crosses within a phylogenetic group or between closely related groups. This review of the literature reveals how different studies obtain similar results regarding the phylogenetic relationships within the genus and how these results can be utilized to improve breeding strategies. It also accentuates that further multidisciplinary studies are needed to solve the evolutionary history of the complex genus Lachenalia.

M. S. Ramanna (The Netherlands), Agnieszka Marasek-Ciolakowska (Poland), Songlin Xie (The Netherlands), Nadeem Khan (Sweden), Paul Arens, Jaap M. Van Tuyl (The Netherlands) The Significance of Polyploidy for Bulbous Ornamentals: A Molecular Cytogenetic Assessment (pp 116-121)

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Invited Mini-Review: Most of the bulbous crops, viz., Crocus, Narcissus, Tulipa, Alstroemeria and Lilium that are commercially important, share certain common characteristics. The present day cultivars are all derived from hybrids between distantly related species, and in almost all cases spontaneous polyploidization has played a prominent role and there is a tendency to replace diploids by polyploid cultivars. Molecular cytogenetic techniques such as genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH), along with other techniques, have greatly facilitated our understanding of the modes of origins of polyploids. Because the bulbous crops generally have large chromosomes, the parental genomes, individual chromosomes, as well as intergenomic recombinant chromosomes, can be accurately identified in the interspecific hybrids and their backcross progenies. This enables an assessment of the potential genetic variation that might occur in the progenies as well as the extent of introgression. Although the superiority of polyploids as compared to their diploid parents is beyond doubt, the actual explanation for their superiority is still elusive. Of the several explanations, chromosome dosage, optimal amounts of 4C DNA values of the complements, heterozygosity and favourable gene interactions transmitted by the 2n gametes to polyploid progenies are some of the factors that might be considered at present. Undoubtedly, more studies on the bulbous ornamental crops using molecular techniques might be rewarding.

Rodrigo Barba-Gonzalez, José Manuel Rodríguez-Domínguez, Ma. Claudia Castañeda-Saucedo, Aaron Rodríguez (Mexico), Jaap M. Van Tuyl (The Netherlands), Ernesto Tapia-Campos (Mexico) Mexican Geophytes I. The Genus Polianthes (pp 122-128)

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Invited Mini-Review: Members of the genus Polianthes L. are bulbous ornamentals in the Agavaceae, includes 15 species three varieties and a two cultivars native to Mexico. Polianthes tuberosa L.(Tuberose) is the only species cultivated as an ornamental cut flower in tropical and subtropical areas. The cultivation of tuberose occupies a prime position in the floriculture industry in countries such as Mexico, China, India, New Zealand and Taiwan. The Flower colour of all known cultivars of P. tuberosa is white; however, attempts have been made to introduce colors from related species. Besides its use as an ornamental, it is cultivated for use in manufacturing: as a source of fragrant essences in perfumery, to extract polysaccharides and glycosides; in addition P. geminiflora (Llave & Lexarza) Rose is utilized as a source of saponins for soap. The main diseases in this crop are caused by virus and it is affected by a coleoptera (Scyphophorus acupunctatus) whose larva feeds on the bulbs. In this review we will cover the uses, distribution, species, of the genus and the current state of tuberose breeding as a further reference for tuberose breeding programmes.

Ernesto Tapia-Campos, Jose Manuel Rodriguez-Dominguez, María de los Milagros Revuelta-Arreola (Mexico), Jaap M. Van Tuyl (The Netherlands), Rodrigo Barba-Gonzalez (Mexico) Mexican Geophytes II. The Genera Hymenocallis, Sprekelia and Zephyranthes (pp 129-139)

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Invited Review: Among hundreds of bulbous ornamental plants native to tropical and subtropical America, different genera from the Amaryllidaceae family such as Hymenocallis, Sprekelia and Zephyranthes present an enormous potential as ornamental crops. The genus Hymenocallis comprises over 60 species distributed from the north of Brazil to the south east of the United States; many of them are endemic to Mexico. The flowers are star shaped and white. The different species grow in a wide and contrasting diversity of habitats, near rivers and streams, on occasion completely submerged under water and sometimes in dry areas. The genus presents a complicated phylogeny, where in some cases it is difficult to distinguish species from hybrids. The genus Sprekelia is a monotypic genus native to Mexico; Sprekelia formosissima is cultivated as an ornamental pot plant in many countries. It presents solitary red flowers, their stems reaches up to 80 to 90 cm. The genus Zephyranthes comprises over 70 species distributed in tropical and subtropical America; different species are cultivated all over the world as an ornamental crop. The different species have beautiful flowers from white to yellow with various tints from lemon to sulphur and pink. In this review we will cover taxonomical, chromosomal and phenological aspects of these genera, with the aim of providing a reference of useful traits for breeding programs.

Guadalupe Palomino, Javier Martínez, Rodrigo Barba-González, Ignacio Méndez, Benjamín Rodríguez-Garay (Mexico) Mexican Geophytes III. Cytotypes and Meiotic Behavior in Mexican Populations of Species of Echeandia (Anthericaceae) (pp 140-152)

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Original Research Paper: Echeandia Ortega includes about 85 perennial herbaceous species. The subgenus Echeandia is distributed from USA, to Argentina and Chile. Mexico is considered to be the genus center of origin and diversity. Echeandia is considered as a monobasic genus with x = 8. Diploid plants (2n = 16, n = 8, x = 8) have been reported for 35 species of Echeandia. Chromosome numbers for 22 polyploid species for the genus have been the reported (4x, 5x, 6x, 8x, 10x and 11x-4). These reports detail karyotype, meiotic chromosome behavior and, pollen fertility of 23 populations of eight species: Echeandia echeandioides, E. hintonii, E. mexicana, E. montalbanensis, E. nana, E. pubescens, E. reflexa and E. tenuis. All species of Echeandia were diploid (2n = 16, n = 8, x = 8). Each species had a distinctive karyotype that varied among populations of the same species. Spontaneous heterozygotic exchanges in species and cytotypes of Echeandia have a common behavior pattern in karyotype variation. The exchanges were observed in heteromorphic pairs of chromosomes with satellites, and, in metacentric, submetacentric and subtelocentric chromosomes. The origin of these rearrangements was evident in heteromorphic bivalents (IIs) and quadrivalents (IVs) observed in MI. Additional evidence for translocations and chromatid exchange comes from the low level of meiotic irregularities observed in anaphase I (AI), including U-type bridges, side arm bridges and lagging chromosomes. Populations of E. nana, display only two cytotypes. Based on these results, the translocations and chromatid exchange follow a behavior pattern common to species and cytotypes of Echeandia, and these chromosome aberrations have played a major role in evolution of the genus, providing a larger potential of colonization and distribution in new habitats.