Development of two in vitro regeneration systems through leaf explant and callus culture and the application for genetic transformation in Alstroemeria

Alstroemeria is a popular ornamental crop cultivated for its flowers. Taxonomically, it belongs to a monocotyledonous family, the Alstroemeriaceae, and is commonly called by its genus name. An Alstroemeria plant consists of underground grown rhizomes, roots, and aerial shoots. The plant is grown perennially. Due to the good incorporation of plant breeding techniques combined with the modern greenhouse cultivation technologies of the last two decades, Alstroemeria has become a competitive greenhouse-grown cut flower in the Netherlands. Generally, the Alstroemeria plant is vegetatively propagated by rhizome division, but the multiplication rate is rather low. Therefore, the increasing demand for plantlets stimulated the development of in vitro propagation techniques. However, since the multiplication unit used in the in vitro method is limited to rhizome tips, the propagation rate is still rather low in comparison with other crops and the other plant organs seem to be of no use during subculture. In addition, a callus culture system has been developed in the last few years for plant propagation purpose. The multiplication efficiency of this system is expected to be higher than that of the rhizome culture system, but the commercial true-to-type requirement cannot be fulfilled, because the callus was initiated from zygotic embryos. Therefore, the development of an additional in vitro multiplication system based on other plant organs is considered to be desirable (Chapter 1).Plant regeneration of cultured explants has in general two pathways, either via organogenesis (the development of shoots directly on an explant) or via embryogenesis (the development of differentiated somatic embryos on an explant). A large part of this thesis research deals with the development of two plant regeneration systems, one based on organogenesis and the other based on embryogenesis. In Chapter 2, a two-step protocol for the induction of shoot formation from in vitro grown Alstroemeria leaf explants is described. Leaf explants were cut from seedlings still containing a leaf blade and a stem node. After 10 days of culture on an induction medium, the leaf explants were transferred to a shooting medium for eight weeks. New shoots were formed directly from the area adjacent to the region between leaf base and node tissue within three weeks on shooting medium. It was histologically demonstrated that these shoots were initiated from the epidermal cells at leaf axils (Chapter 3). There were no pre-existing axillary buds ever found on the aerial leaf axils, so that this kind of organogenesis suggests an adventitious nature. The leaf explants together with newly formed shoots were subcultured several times and many normal plantlets with rhizomes were formed, which then were suitable for transferring to the soil (Chapter 2, Chapter 4).The best induction was obtained on a Murashige and Skoog's (1962) medium supplemented with 10μM thidiazuron (TDZ) and 0.5μM indole butyric acid (IBA) (Chapter 2). The shooting medium contained MS medium with 2.2μM 6-benzylaminopurine (BAP). The shoot regeneration capacity of the excised leaf explants was related to the position of the leaf on the stem. The youngest explant which was located the nearest to the shoot apex, gave the highest response. A lower gradient response was found in the leaf explants derived from leaves cut off at a further distance from the apex. This was measured in percentage of shoot regeneration per leaf explant and in the number of shoots per regenerating explant (Chapter 3).A demonstration experiment was carried out in the greenhouse in order to investigate the similarity of plant growth morphology in between plants, which were obtained from either rhizome multiplication or leaf explant culture system. The plants were grown in the greenhouse to flowering, and the results indicated that plants obtained from both systems were morphologically identical (Chapter 4). This implicates that the leaf culture system seems to be a reliable in vitro propagation technique for the genotype we have investigated.Another advantage of the leaf explant culture system is that the leaf explants directly can be excised from in vivo full grown shoots, and that the disinfection of aerial shoots is easier than that of underground grown rhizomes (Chapter 4). Therefore, this technique is suitable for the initiation of in vitro propagation of existing cultivars.In the conventional micropropagation system, only the rhizome tips are multiplied and therefore, the aerial shoots are always discarded during subculture. In this thesis research, it is concluded that not only the rhizome tips can be used as propagation units, but also the discarded shoots can be used for the initiation of the other propagation system. The first three leaves excised from each shoot have an average regeneration capacity of 87.7%, and the average number of newly formed shoots per explant was 5.3 (Chapter 4). On the other hand, the rhizomes can be multiplied simultaneously. Therefore, combining the rhizome multiplication system with the leaf explant culture system, the multiplication efficiency will be enhanced.In Chapter 5, a somatic embryogenic callus regeneration system is described. A soft and sticky type of callus was induced initially from the stem segments of one month old seedlings of two tetraploid Alstroemeria genotypes. The soft calli turned into compact type after subculture on a medium (MS with 30 g/l sucrose) containing 6-benzylaminopurine. Subsequently, two other different morphotypes of callus, friable and granular, were obtained by subculturing the compact callus on different culture media. The friable callus can be maintained on a single medium (PCA) containing 10 mg/l picloram for a long period without loosing its friability. Subculturing the friable callus on plant growth regulator free media or on 6-benzylaminopurine containing media stimulated the granular callus formation, and the subsequent somatic embryogenesis. The somatic embryos were able to develop into complete plants.The granular callus proved to be an intermediate between friable callus, somatic embryo, and compact callus. The friable callus could also be induced from granular callus, and vice versa. Therefore, a cyclic reproduction system was established in this research. This system provides two types of callus with a high embryogenic capability, which were initially derived from the stem segments. Thus, this system is considered to be applicable for the in vitro propagation of Alstroemeria .In addition to the purpose of plant propagation, the development of a plant regeneration system is also considered to have the potential for genetic modification in Alstroemeria . Some characteristics, for example virus resistances, are very important in the continuously greenhouse-grown cultivars. However, virus resistance genes are not generally present in the Alstroemeria gene pool yet, so that the traditional breeding techniques are not sufficient for this purpose. Genetic transformation of Alstroemeria is considered to be useful for breeding in the future (Chapter 1). For genetic transformation, four important factors should be taken into account: a) an efficient DNA delivery system, b) the appropriate target cells competent for both transformation and regeneration, c) an adequate promoter, and d) a good selection system (Chapter 1).In this research, the particle bombardment delivery system was chosen for the monocot A lstroemeria , because of its expectedly higher transformation efficiency than the Agrobacterium vector system (Chapter 1). The leaf explant regeneration system was tested for gene transformation by using the particle bombardment. Although the gene expression could be detected after particle bombardment, the gene activities were only transiently expressed on leaf tissues, and they disappeared within two months (Chapter 7). On the other hand, the somatic embryogenic callus regeneration system was successfully used for particle bombardment mediated gene transformation. Two tetraploid Alstroemeria genotypes were transformed, and many transgenic plants were obtained (Chapter 6).Both granular and friable calli were used as bombardment targets, and the subsequent somatic embryogenesis resulted in the formation of complete transgenic plantlets. Two plasmids containing different selection and reporter genes were used. Firstly, a plasmid containing a firefly luciferase reporter gene, driven by the maize ubiquitin promoter ( Ubi1 ), was bombarded into both granular and friable calli. The luciferase activity was measured by a luminometer after spraying the bombarded plant material with a luciferin solution. Visual selection of the luciferase positive calli, assisted by the luminometer, was effective. This kind of selection has a nondestructive nature, without injuring the plant material, and the luciferase activity can be assayed periodically over the whole developmental process from callus to embryo and plantlet. It was shown that the granular callus is more suitable for particle bombardment mediated transformation using luciferase activity as selection marker than the friable callus (Chapter 6).Secondly, another plasmid containing the selectable Basta (herbicide) resistance gene ( bar ) encoding phosphinotricin acetyltransferase (PAT) together with an uid A reporter gene encoding ß-glucuronidase (GUS) was used. Both genes were driven by the Ubi1 promoter. The granular calli were bombarded in this experiment. Selection of the phosphinotricin (PPT) resistant calli was accomplished by culturing the bombarded calli on a medium containing 5 mg/l PPT. The PPT resistant calli were the friable type of calli which were already regenerated from the granular calli, and they developed into somatic embryos, and subsequently into the plantlets. Stable expression of the GUS gene was confirmed by histochemical staining. The blue color was detectable in all tissues of the transgenic plants tested by the GUS assay. The PPT selection proved to be a more efficient and labor-saving method compared to the luciferase selection (Chapter 6).The results described in this thesis are beneficial for both the in vitro propagation and the genetic modification of Alstroemeria . The use of leaf explants as in vitro propagation units is rather unique in Alstroemeria , which opens an alternative way for enhancing the plant propagation efficiency. The embryogenic callus regeneration system described in this thesis is not only applicable for plant propagation, but also for genetic transformation. The establishment of particle bombardment mediated transformation techniques will push the molecular breeding in Alstroemeria forward into a luminous future.