This study represents the original work of the author, and no part of this work has been submitted in any form to another university. To the past and present members of the Plant Germplasm Conservation Research Group for their understanding and flexibility and most importantly for being there for me when I needed them, I am truly indebted. Thus, in addition to the size of the plant, factors such as in vitro regeneration techniques, physical injury caused upon excision and developmental status of the seed can compromise the success of cryopreservation.
This study investigated the effect of the above factors with particular attention to the developmental status of the seeds on explant in vitro development (Section 3.1), response to dehydration (Section 3.2) and cryopreservation of the desiccation-sensitive embryonic axes (Section 3.3) ) of two species: Trichilia dregeana , T. Furthermore, the sensitivity of the shoot apical meristem to desiccation was not improved with seed storage for T. Thus, the successful cryopreservation depends on the germplasm of the tested species and others that produce recalcitrant seeds. on a spectrum of species-specific factors, some of which still need to be elucidated.
Biodiversity and the need for conservation 1
Consequently, the lower yielding strains are replaced by their higher yielding counterparts, narrowing the genetic base and promoting genetic uniformity. There are many commercial and economic benefits to breeding plants with higher yields and resistance to pests and diseases. However, these are threatened if a stress occurs that the plant populations cannot adapt to due to the lack of diversity in their gene pool.
For these reasons, the Global Plant Conservation Strategy was developed (Convention on Biological Diversity, online). This strategy ultimately aims to halt the loss of plant biodiversity by: understanding and documenting plant biodiversity; the conservation of plant biodiversity; making sustainable use of plant biodiversity; The protection of 50% of the world's most important areas of plant diversity, the conservation of 70% of the genetic diversity of crops and other socio-economically valuable plant species, the conservation of 60% of the world's endangered species in situ and 60% of endangered species in accessible ex situ collections, with 10% included in recovery and restoration programs.
Germplasm conservation 2
Although agricultural practices do not necessarily use native plants, the goal is to produce crops with better and higher yields on a larger scale (Ford-Lloyd and Jackson, 1991).
Methods of germplasm conservation 3
Active collection: seed storage 4
Seed storage is the most common method of preserving plant germplasm due to its convenience and relatively low cost. The main factors determining the lifespan of seeds in storage are temperature and water content. Good quality seeds can be stored for decades or longer if stored at temperatures of -18°C or lower and a relative humidity of 5 - 7% (IBPGR). 1976; Ford-Lloyd and Jackson, 1991; Krogstrup et al., 1992; Ellis and Hong, 2006). However, this strategy cannot be applied to all plant species, as the post-harvest behavior of their seeds, which determines the most appropriate conservation method, differs.
While orthodox seeds can dry to low water content and can tolerate freezing temperatures, recalcitrant seeds are sensitive to desiccation, freezing and possibly chilling (Ford-Lloyd and Jackson, 1991; Krogstrup et al., 1992; Berjak, 19et ). .
Active collection: in vitro storage 4
To minimize this risk, it is preferable to grow organized systems such as embryos, meristems and shoot tips for in vitro storage, as these are more stable and reproducible genetic systems (Ford-Lloyd and Jackson, 1991; Krogstrup et al., 1992; Berjak et al., 1996; Engelmann, 1997; Mandal et al., 2000). Cultures can be stored in vitro by three methods: cryopreservation (which is described in more detail in section 1.3.3), actively growing cultures or slow growing cultures. Maintenance of actively growing cultures requires regular transfer of material to new media, usually at monthly intervals (Krogstrup et al., 1992; Mandal et al., 2000, Mycock et al., 2004).
This method risks losing cultures due to contamination or physiological decay; however, the remaining material can potentially be rapidly micropropagated to build reserves (Krogstrup et al., 1992; Razdan and Cocking, 1997; Mandal et al., 2000). Minimal growth storage exposes cultures to factors that limit growth; these can be either chemical or physical and include the use of growth inhibitors, reduced temperatures and reduced oxygen (Krogstrup et al., 1992; Reed and Chang, 1997; Staritsky, 1997). This method of in vitro storage can impose selection pressures and environmental stresses, producing plants with genetic modifications (Krogstrup et al., 1992; Reed and Chang, 1997; Staritsky, 1997).
Base collection: cryopreservation 5
- Cryopreservation of excised zygotic axes and whole embryos within seeds 7
- Cryoprotection 7
- Dehydration to appropriate water contents 8
- Freezing methods 8
Valladares et al., 2004), or by pre-cultivating the explants on a growth medium enriched with cryoprotectants, usually sucrose (e.g. the faster recalcitrant seeds can be dried, the lower the water content they can tolerate before viability is lost (e.g. Pammenter et al. This technique exposes the axes to moving dry air, to allow rapid drying within minutes to hours (Berjak and Pammenter, 2001; Pammenter et al., 2002).
Classical freezing is often achieved in two steps and is therefore also called "two-stage freezing" (Krogstrup et al., 1992). After treatment with cryoprotectants, the first step involves slow controlled cooling to a specific temperature before freezing, usually around -40 °C (Krogstrup et al., 1992; Engelmann, 1997), which is achieved by programming. Using high concentrations of cryoprotectants and vitrification solutions, explants can be dehydrated to a low water content, preventing the formation of ice crystals when immersed in liquid nitrogen (Krogstrup et al., 1992; Engelmann, 1997).
Post-harvest seed behaviour 10
Seed recalcitrance 11
- Seed development 11
- Desiccation 12
- Desiccation tolerance mechanisms 12
- Desiccation damage 16
- Dehydration and drying rate 17
- Seed storage potential 19
Experiments on embryonic axes of Quercus robur recalcitrant seeds (Mycock et al., 2000); In contrast, Faria et al. 2005) showed that the cytoskeleton rapidly reconstituted during imbibition of orthodox Medicago truncutula seeds. Recalcitrant seeds have also been suggested to have antioxidant and free radical scavenging mechanisms (Chaitanya et al., 2000).
In resistant seeds, LEAs have been shown to be absent in some species (eg Avicennia marina [Farrant et al., 1993]) and present in others (Kermode, 1997). However, these are a combination of more than one type of membrane (Cordova-Tellez and Burris, 2002; Walters et al., 2002a). Consequently, deleterious events associated with water stress (such as disruption of coordinated metabolism, leading to free radical damage) lead to tissue death (Dussert et al., 2006; Ratajczak and Pukacka, 2006).
Factors influencing successful cryopreservation 21
Explant size, water content and cooling and thawing rates 21
Axillary buds (Blakesley and Kiernan, 2001) and somatic embryos (Stewart et al., 2001; . Fang et al., 2004; Valladares et al., 2004) also provide explants of appropriate size for successful cryopreservation. However, the use of somatic embryos in particular does not offer the genetic diversity that seed-derived zygotic axes provide and may also be compounded by somaclonal variation during their production (Krogstrup et al., 1992). Examples of successful cryopreservation of orthodox whole seeds include those of various orchid species (Thammasiri, 2000; Nikishina et al., 2001; Popov et al., 2004), Piper nigrum (Chaudhury and Chandel, 1994) and Dendrobium candidum (Wang et al. et al., 1998).
Cryopreservation of unorthodox whole seeds has also been achieved for Wasabia japonica (Potts and Lumpkin, 1997), Warburgia salutaris (Kioko et al., 2003b) and Azardirachta indica (Berjak and Dumet, 1996). However, in several cases (Pence 1990; Vertucci et al., 1991; Kioko et al., 1998; Berjak et al., 1999b) survival was scored by callus formation, which is less than ideal since direct seedling establishment is the desired outcome. Survival after cryopreservation after slow cooling has been demonstrated for coffee (Dussert et al., 1997), immature wheat embryos (Kendall et al., butternut (Beardmore and Vong, 1998), and Landolphia kirkii (Vertucci et al., 1991).
Thawing 23
In vitro regeneration 23
- Culture conditions 23
- Excision of explants 24
However, explants excised in this way, even before dehydration and/or cryopreservation, often fail to develop shoots with the shoot pole becoming necrotic or, at best, forming a callus (Kioko et al., 1998; . Perán et al. , 2006). Callus formation at the shoot pole can form in response to injury as shown by Grunweld et al. This response has also been reported in lichens (Beckett and Minibayeva, 2003) and in macroalgae (Ross et al., 2006).
Orthodox seeds have well-developed antioxidant mechanisms (Abdallah et al., 1997; Chandru et al., 2003; Edreva, 2005) that scavenge free radicals, whereas antioxidant mechanisms are thought to be either insufficient, impaired, or absent in recalcitrant seeds (reviewed by Berjak and Pammenter, 2007). If free radicals are not effectively scavenged, their build-up will have harmful consequences (Hendry, 1993; . Bailly, 2004; Bailly et al., 2004). It has previously been suggested that axis development continues after germination in recalcitrant seeds that can be stored for weeks to months under hydrated conditions before signs of germination become macroscopically visible (Berjak et al., 1989).
The present study 26
It is likely that the developmental state of axes when removed will affect continued development either negatively or positively, and that this critical factor must be resolved before any further cryopreservation manipulation is attempted. Despite the many biochemical and procedural complications underlying successful cryopreservation, there is currently no alternative for long-term ex situ storage of genetic resources of resistant seed-producing species.
Species studied 27
Trichilia emetica Vahl. 28
There are also differences between the flowering and fruiting seasons, which are September to November and January to April respectively for T. The fruits have a distinct neck (absent in T. dregeana) and are slightly smaller than those of T. The fruits are eaten by baboons, monkeys and antelopes, and are also used to make a milky soup similar to that of T.
Preparations of bark, roots, leaves and seed oils are used for medicinal purposes against stomach, intestinal and kidney disorders, indigestion, fever, parasites and eczema (Hutchings et al., 1996; van Wyk et al., 1997). Resin and tannin are found in the bark (Hutchings et al., 1996) and limonoids such as trichilin A have also been isolated (van Wyk et al., 1997) and may contribute to the medicinal properties of this species. Few studies have been published on the behavior of the seeds of this species, but they have been classified as recalcitrant (Maghembe and Msanga, 1988; Kioko et al., 2006).
Strychnos gerrardii N.E. Br. 29
MATERIALS AND METHODS
Seed processing 32
- Surface sterilization 32
- Seed storage 32
Cleaned seeds were surface sterilized using a 1% NaOCl solution containing a few drops of Tween 20/80®, a wetting agent, for 20 min (T. dregeana and S. gerrardii) or 10 min (T. emetica ) contained. These fungicides have been shown to limit fungal contamination in storage (Calistru et al., 2000; Berjak et al., 2004). Seeds were then placed on paper towel on a laboratory bench, and dried back to the original batch fresh weight, and either used immediately or prepared for storage.
Seeds of all three species were dusted with Benomyl 500 WP (active ingredient, benzimidizole; Villa Protection, S. Africa) and stored at 16°C as a layer on a sterilized plastic mesh suspended 200 mm over paper towels saturated with water in a closed towel. 5-l bucket.
Germination 33
- Whole seeds 33
- Gravimetric determination of water content 33
- Embryonic axes 33
Cryoprotection has no significant effect on shoot production by non-dehydrated explants excised with basal cotyledon segments from stored seeds (Figure 3.46, Chi-square, p > 0.05). Survival after cryopreservation was not affected (Chi-square, p > 0.05) by the cooling technique used (Figure 3.53). Nevertheless, 10% of explants from seeds of newly harvested, yellow fruit produced shoots after cryopreservation (Figure 3.56).
