Conclusiones

In the developmental process to flowering, the initiation and development of buds was seen as being the starting point of the process. In order to determine the factors influencing this initial stage, as described below, it was considered necessary to describe the types of buds present in gentians.

1.5.1.1 Apical, axillary and adventitious buds

Plants can be viewed as consisting of banks of different types of buds, that can give rise to vegetative regeneration (Vesk and Westoby, 2004). These banks of buds can be utilized following termination of dormancy, to supplement developing shoots during the growth cycle or, to replace growing shoots lost (Stafstrom, 1995). Types of meristems/buds can be categorised based on their location of origin on plants and their initiation, as being apical, axillary, or adventitious (Evert, 2006; Kerstetter and Hake, 1997). The apical bud is located at the apex of the shoot, is derived from the apical meristem, and lays the foundation for the subsequent development of the whole shoot via cell division. Axillary buds typically develop from the axils of the leaves, and adventitious buds arise de novo from any plant tissue. Hence adventitious buds may appear on leaves, stems or roots.

With gentians possessing a determinate shoot growth habit and sympodial branching (Ho and Liu, 2001), the involvement of the apical meristem in the first flowering of a

gentian plant from seed is known. In subsequent growth cycles however, in gentians, the types of buds that develop into floral shoots (continuation buds) have not previously been described, i.e. whether they are axillary or adventitious in origin. If both axillary and adventitious buds are present in gentians, they could both potentially give rise to floral shoots. In contrast to axillary buds, leaf expansion and shoot development rates are typically slower in adventitious buds, as noted in other species (Del Tredici, 2001; Vesk and Westoby, 2004). If, therefore, different types of buds are present in gentians, their growth and development may differ, giving rise to different times at which shoots reach anthesis in any given season (refer Section 1.3.1). It was therefore necessary to identify which types of buds exist, and the morphological and physiological changes during growth and development of shoots originating from the differing bud-types present (Chapters 3, 4 & 5).

Morphology, anatomy and ontogeny of buds could be used in determining whether these buds in gentians are axillary or adventitious. Axillary meristems, which give rise to axillary buds, are typically associated with a leaf primordium. This axillary meristem could be derived from the primary shoot apical meristem (SAM), which arises during embryogenesis (Evert, 2006; Kerstetter and Hake, 1997) or from groups of meristematic cells which originate directly from a detached part of the primary SAM in the axils of leaf primordia (Shimizu-Sato et al., 2009). In contrast, adventitious buds develop from endogenous (i.e. tissues deep within the parental axis) or exogenous tissue (i.e. tissue positioned at a relatively superficial depth) of any plant organ (Evert, 2006). In order to distinguish between axillary and adventitious buds present in gentians, characteristic features relative to the origin, associated tissues, and vascular connections from the bud to the tissue of origin could be utilised as a research tool when investigated both macroscopically and microscopically (Chapter 5).

In herbaceous perennials, vegetative propagules (including stems, rhizomes, tubers, bulbs, stolons etc) contain axillary and adventitious buds capable of producing new shoots under permissive environments (Anderson et al., 2001). Most of the cultivars of gentians used in the current research programme have been developed from G. scabra and G. triflora, as dominant parental lines. As mentioned previously, the published literature (Ohkawa, 1983), and growers, refer to buds present in gentians as “crown buds”, “winter buds”, “dormant buds” or “buds arising from an underground stem”. The origin of these particular buds or their function however, has not previously been

described. As it was considered important for the current study to develop a more appropriate descriptive terminology for crown tissue and crown buds, anatomical studies were conducted in order to identify the types, ontogeny and role of the buds in the gentians of interest (Chapter 3, 4 and 5). Further to that, it was considered necessary to understand the developmental sequence of any bud type from initiation till anthesis, in order to investigate what if any correlations might exist between developmental stages and, therefore, what potential might exist for manipulation of initiation, development, emergence of buds and, development of shoots.

1.5.2 Methods for morphological and anatomical investigations

As detailed in Section 1.5.1.1, in order to identify what if any association existed between types of buds and their developmental process, as explored below, both macroscopic and microscopic techniques were used during the current thesis.

1.5.2.1 Macroscopy

Tracking of changes occurring within the crown of geophytes, such as Asparagus officinalis L., at regular intervals, has been successfully used to identify the types of buds as well as the timing of their emergence and subsequent fate (Danningsih, 2004). It was envisaged during the current thesis that by using a similar procedure the emergence of buds and subsequent shoot development could be traced over time, to identify and quantify the different bud types and the influence of timing of their initiation, emergence and development, on the timing of harvest maturity (Chapter 3). Macroscopic examinations could also be useful in determining the origin of buds relative to the associated tissue, and their progressive development (Chapters 4 and 5). Further to this, dissections (Chapter 3, 4 & 5) could be used to reveal the progressive arrangement of structures such as buds, and leaf or floral primordia (Inouye, 1986; Sabatier and Barthélémy, 2001).

1.5.2.2 Microscopy

Different microscopic techniques such as light, confocal, fluorescence or electron microscopy could be usefully applied in identifying anatomical features of plant tissues. Histological studies have been undertaken successfully using light microscopy to identify the origin and development of buds on seedlings of herbaceous perennials like Euphorbia esula L. (Myers et al., 1964), Cardopatum corymbosum L. (Chiatante et al.,

2008), as well as many woody perennials like Quercus species (Pascual et al., 2002), Myrtaceae species (Burrows, 2000) and Araucariaceae species (Tomlinson and Huggett, 2011). Light microscopy was therefore considered as a possible technique to use in the histological studies presented in this thesis (Chapter 4 & 5). Laser confocal microscopy has been used as a method in studying anatomical features of plant tissues (Pawley, 2006), including SAMs (Lemon and Posluszny, 1998), young shoots (Kitin et al., 2005), and xylem vessels (Kitin et al., 2004). This technique offers the advantage of enabling observation of tissues, regardless of thickness of the section. Given the wide divergence of tissue types/sizes that could be evident in gentians, it was considered that in the current study laser confocal microscopy might also offer a possible alternative, if light microscopic techniques were found to be limiting.

Proper sample preparation is a key requirement for any microscopic technique. During the current study it would be necessary to achieve progressive sections of the samples in order to identify the vascular connections to the associated tissues. Sample preparation could be conducted using protocols such as wax (Eaton et al., 2010; Spence, 2001) or resin embedding (Busse and Evert, 1999), as this facilitates sequential sectioning of tissues using a microtome. Based on the facilities available at Massey University, wax embedding protocols were considered to be economical and feasible for the current study. The success of sectioning following wax embedding however depends upon the softness of tissues. In contrast to light microscopy, confocal microscopic techniques do not require having thin sections, therefore could be used to handle even fibrous tissues (Chapter 5).

1.5.2.3 Staining of the tissues for microscopy

1.5.2.3.1 Light microscopy

Various stains reacting to components of different cell types can be used to examine a particular tissue during light microscopy. Toluidine Blue has been used in numerous studies of buds and their associated tissues (Burrows, 2000; Burrows et al., 2008; Burrows, 2002; Foster et al., 2007; Mibus and Sedgley, 2000; Waters et al., 2010). Toluidine blue stains lignified walls of xylem vessels in a blue-green colour (O'brien et al., 1964), therefore could be used as a stain that would enable identification of vascular connection of a bud to the associated tissue. By identifying the vascular connection of

the buds to the tissue of origin, and the morphological identity of this tissue (i.e. stem or root), an understanding could be achieved of the types of buds present.

1.5.2.3.2 Confocal microscopy

In confocal microscopy various fluorescent dyes can be used to identify the tissue or, in some cases, cells naturally give an autofluorescence without a stain. Autofluorescence of lignin in secondarily thickened cell walls, has been used to detect the presence of vascular bundles (Pawley, 2006). Stains such as Acridine Orange and Ethidium Bromide have been used in combination, to distinguish between lignified and non- lignified cells (Yang et al., 2007). Safranin and Acridine Orange have been used in identifying structures such as the cambium (Kitin et al., 2000). During the preliminary stages of the current study, autofluorescence of the tissues and the potential use of several stains, individually or in combination, were therefore investigated in order to identify the suitable protocol for buds and associated tissues in gentians (refer Chapter 5).

1.6 Influence of plant and environment related factors during the