1 Introduction
2.8 GERMINATION PHYSIOLOGY
2.8.12 Embryo and seedling morphology of Iridaceae
Embryo’s of the Iridaceae are straight and poorly differentiated (TILLICH, 2003). Only at the seedling stage can three different groups, defined by their cotyledon morphology, be clearly distinguished (TILLICH, 2003). The three groups are the compact cotyledon group, the tubular cotyledon group and the assimilating cotyledon group (TILLICH, 2003).
At the event of germination the cotyledonary sheath, hypocotyl and radicle are pushed through the micropyle region of the testa (TILLICH, 2003). The cotyledon then immediately bends at 90° in most cases (TILLICH, 2003). In Crocoideae the typical cotyledon is characterised by a remarkable elongation of the cotyledonary sheath and / or the development of a long coleoptile (TILLICH, 2003).
The seedling morphology of Crocus and Romulea is quite rare (TILLICH, 2003). Here an elongated tubular structure is combined with a tubular cataphyll (TILLICH, 2003).
Possible explanations for this is the pronounced hypogeous germination which is promoted by a strong contractile primary root (TILLICH, 2003). The cotyledons of these genera have also lost most of their ability to produce chlorophyll and appear white even in high light intensities (TILLICH, 2003).
62 2.9 BRIEF REVIEW OF IN VITRO CULTURE
The term ”tissue culture” is actually a misnomer inherited from the field of animal tissue culture. Plant micropropagation involves the culture of a whole individual from isolated tissues, while animal tissue culture involves the culture of isolated tissues (KYTE & KLEYN, 1996).
According to AHLOOWALIA et al. (2002), the process of micropropagation can be divided into five stages: the pre-propagation step (stage 0); the initiation of explants (stage I); the subculture of explants for multiplication or proliferation (stage II);
shooting and rooting of the explants (stage III); and hardening off the cultured individuals (stage IV). The pre-propagation stage involves preparing the explant for aseptic culture.
The explant and its response in vitro is significantly influenced by the phytosanitary and physiological conditions of the donor plant (KANE, 2004). Plant material used in clonal propagation should be taken from mother plants that have undergone the appropriate pre-treatment with fungicides and pesticides to minimize contamination in the in vitro cultures (AHLOOWALIA & PRAKASH, 2002). Such a piece of plant material is called an explant (SMITH, 2000b). A single explant can theoretically produce an infinite number of plants (KYTE & KLEYN, 1996). Explants can be obtained from meristems, shoot tips, macerated stem pieces, nodes, buds, flowers, peduncle pieces, anthers, petals, pieces of leaf or petiole, seeds, nucellus tissue, embryos, seedlings, hypocotyls, bulblets, bulb scales, cormels, radicles, stolons, rhizome tips, root pieces or protoplasts (KYTE & KLEYN, 1996). The explants should be surface decontaminated with antibiotic sprays before they are introduced into culture (AHLOOWALIA et al., 2002; KANE, 2004).
In stage I an aseptic culture is initiated by inoculating the explant onto a sterile medium (AHLOOWALIA et al., 2002). Once such an explant is established it can be multiplied a number of times (AHLOOWALIA et al., 2002). The explants are then transferred to a contaminant free in vitro environment (AHLOOWALIA et al., 2002).
63 In stage II or the propagation phase explants are cultured onto a medium that promotes the multiplication of shoots (AHLOOWALIA et al., 2002). Propagation must be achieved without excessive mutation (AHLOOWALIA et al., 2002). The culture of various organs in stage I lead to the multiplication of propagules in large numbers.
These propagules can be cultured further and used for multiplication (AHLOOWALIA et al., 2002). These cultured shoots are often placed onto different media for elongation (AHLOOWALIA et al., 2002).
The result of stage III is the production of complete plants, as the shoots derived from stage II are rooted (AHLOOWALIA et al., 2002). If shoot clumps are present, they should be separated after rooting. Many plants can be rooted on half strength Murashige and Skoog medium without any growth regulators (AHLOOWALIA et al., 2002). Successful and sufficient rooting is essential for survival of the plant during hardening and transfer to the soil (AHLOOWALIA et al., 2002).
The complete plants are weaned and hardened during stage IV (AHLOOWALIA et al., 2002). The plants should at this stage be autotrophic. Hardening consists of gradually altering the humidity, light and nutrition available to the plant. The plant is moved gradually from a high to a low humidity, from a low light intensity to a high light intensity and the agar is removed by gently washing it away with water. After sufficient hardening, plants can be transplanted to a suitable substrate and hardened further (AHLOOWALIA et al., 2002).
In vitro regeneration techniques are essential to the application of in vitro selection techniques (REMOTTI & LÖFFLER, 1995). This is not only because it enables the selected genotype to be regenerated, but also it aids commercialisation of new species and selected genotypes (DEBERGH, 1994).
Micropropagation enables the production of disease free plantlets at high rates and generally increases the efficiency of known breeding techniques (DEBERGH, 1994).
It is also essential in the breeding of plants for which no breeding methods or procedures have been established (DEBERGH, 1994). In vitro selection techniques have been used to increase genetic variability and to broaden the gene pool
64 (DEBERGH, 1994). In vitro techniques such as embryo rescue, protoplast fusion and genetic transformation enable plant breeders to accomplish wider crosses (DEBERGH, 1994).