The aim of this study was to develop methods for cryopreservation of the germplasm of this species. Overall, the results of this study provided a better understanding of the germplasm responses of E.
CRYOPRESERVATION OF SELECTED EXPLANTS 3.1 Introduction
CHAPTER 4: OVERVIEW DISCUSSION, RECOMMENDATIONS FOR FUTURE
Mtunzini, Port Elizabeth and St Lucia 80 2.7 Effect of IBA concentration on percentage of roots and subsequent. 6 weeks, and water content of additional shoots 121 3.10 Effect of sucrose pre-culture followed by cryoprotection and flash.
GENERAL INTRODUCTION AND LITERATURE REVIEW
Plant biodiversity uses and conservation
Strategies for conservation of plant germplasm
Germplasm preserved under ex situ conditions can be maintained either as an active or a base collection (Krøgstrup et al., 1992; Ruiz et al., 1999). Active collections provide germplasm for evaluation and distribution (short- and medium-term germplasm stores) and are most often maintained as seed stocks, but also in the field (Withers and Engelmann, 1998) and in the greenhouse (Krøgstrup et al., 1992; Staritsky , 1997).
Conservation of indigenous African species: a study on Ekebergia capensis Sparrm
In this way, germplasm can be stored as collections in botanical gardens, arboreta, field gene banks, seed banks or in vitro cultures (Krøgstrup et al., 1992; Razdan and Cocking, 1997). Currently, an important additional threat is recognized that requires ex situ conservation measures: this is habitat destruction due to climate change (Hannah et al., 2002).
Seed storage characteristics
- Developmental, morphological and chemical characteristics of recalcitrant seeds
Inter- and intra-seasonal variations in the characteristics of resistant seeds are common (Berjak and Pammenter a). Another typical characteristic of resistant seeds is that in addition to being hydrated when shed, they are also metabolically active (Berjak et al., 1989; Finch-Savage, 1996).
The role of water in seeds
- Dehydration stress and tolerance mechanisms
However, dehydrin-like proteins were absent in a number of recalcitrant seeds from tropical wetland species (Farrant et al., 1996). A number of researchers have suggested that intracellular glasses can be stabilized by LEAs in the dry state (Walters et al., 1997;
Methods of seed storage .1 Storage of orthodox seeds
- Storage of non-orthodox (recalcitrant) seeds
Good quality recalcitrant seeds can be maintained in the short to medium term under conditions of hydrated storage (wet storage), where seeds are stored at the water content at which they were expelled (Berjak et al., 1989; Berjak and Pammenter, 2004a). This suggests that the deteriorating metabolic events occurring during hydrated storage of temperate and tropical recalcitrant seeds may be similar, although they may occur at a reduced rate in the former (Pammenter et al., 1994).
Cryopreservation for the long-term conservation of recalcitrant-seeded germplasm
- The theory underlying cryopreservation of plant germplasm
- Approaches to cryopreserve plant germplasm a) Controlled rate cooling
- Factors to be considered for the practical application of cryopreservation
Vitrification can be achieved by controlled rate cooling (partial vitrification) and by rapid cooling (complete vitrification) (Sakai et al., 2008). It can be manifested either somatically (where the variation is not heritable) or meiotically (where the variation is heritable and can affect subsequent generations) (Kaeppler et al., 2000).
Aims and objectives of the present study
Therefore, it is important to determine the germplasm status in terms of epigenetic changes at each stage of the cryopreservation protocol and also after acclimation. As the aim of the conservation program is to preserve the genetic and epigenetic integrity of the stored germplasm, it is essential to verify that this has been achieved. Thus, DNA was isolated from the material after each step of the cryopreservation protocol and analyzed by restriction enzyme digestion of DNA and PCR-amplification of the products, whereby the methylation status was assessed in terms of possible epigenetic changes.
Overall, the investigations of this study aimed to develop a successful cryopreservation protocol for E.
STRATEGIES FOR THE MICROPROPAGATION OF Ekebergia capensis Sparrm
INTRODUCTION
This is common practice when axillary bud explants are initiated from parent plants (Taji et al., 2002a; Wilhelm, 2003). Commonly used decontaminants include sodium and calcium hypochlorite (Niedz and Bausher, 2002), mercuric chloride (Bhat et al., 1992), ethanol (Leifert and Waites, 1994), and various fungicides (Leifert and Waites, 1990). However, it is the ratio of auxin to cytokinin that will determine the type of organogenesis (Taji et al., 2002b; Razdan, 2003); i.e.
Albizzia julibrissin Hidda hin tuqamne I Sankhla fi kkf, 1994 Albizzia julibrissin Kutaalee hundee V Sankhla fi kkf, 1996 Albizzia julibrissin Kutaalee hundee. Citrus aurantifolia Kutaa hundee D fi I Bhat fi kkf, 1992 Clitoria ternatea Kutaa hundee D fi I Shahzad fi kkf, 2007 Comptonia.
MATERIALS AND METHODS .1 Plant material
- Explant preparation
- Bud break
- Adventitious bud production
- Multiplication, elongation and rooting
- Decontamination of adventitious shoots
- Culture conditions
- Acclimatisation
- Photography and data analysis
Embryonic axes were excised with 2 mm3 blocks of each cotyledon attached (see Figure 2.1B), decontaminated by immersion in 1% (w/v) NaOCl for 10 min, rinsed three times in sterile distilled water, and then cultured in vitro. Two explant sizes (2 and 5 mm) from excised nodal segments of in vitro germinated seedlings were examined for bud break. Intact whole roots (15 – 25 mm long) prepared from in vitro germinated excised embryonic axes were placed in a RITA bioreactor (Rėcipient à Immersion Temporaire Automatique, CIRAD, France) for 24 or 48 h with medium containing ¼ MS- salts and vitamins, 30 g l-1 sucrose and 0, 1, 3 or 6 mg l-1 BAP.
Shoots developed by axillary bud break from nodal segments of in vitro-germinated seedlings and greenhouse-grown seedlings were placed on ½ MS salts and vitamins, 20 g l-1 sucrose, 0.33 mg l-1 IBA, 1.7 mg l-1 BAP and 8 g l-1 agar for multiplication in two cycles of 6 w each. These three protocols were tested using at least 20 root plants generated from nodal explants of in vitro germinated seedlings.
RESULTS AND DISCUSSION
- Establishment of a medium for germination of axes with cotyledonary attachments
- Shoot production from nodal segments and in vitro-germinated roots
- Rooting and acclimatisation
- Testing the micropropagation protocol to produce adventitious shoots from in vitro-germinated roots
- Decontamination of adventitious shoots for subsequent cryopreservation studies
In this regard, Denslow et al. 2005) discussed a series of gene regulation and metabolic studies that provided evidence for pyridoxine's role as an antioxidant. However, such methods are destructive and the results require careful interpretation, as these tests are predictive rather than definitive (Verleysen et al., 2004). The value of any protocol developed increases if it can be successfully applied to a range of genotypes (or germplasm from other sources) (Bhatti et al., 1997; Panis et al., 2005).
In addition, it has been reported that differences in the response of different cultivars (genotypes) to the same applied conditions in vitro may occur as a result of differences in the uptake, transport and metabolism of exogenous cytokinins (Strnad et al., 1997; van Staden). et al., 2008). In addition, the only plant growth regulator used was BAP, which has been reported to stimulate the direct production of adventitious buds and shoots from roots of Filipendula ulmaria (Yıldırım and Turker, 2009) and epicotyl explants of three citrus species (Costa et al., 2004). .
CONCLUDING COMMENTS
Additional shoots (2 – 2.5 mm) were decontaminated by immersion in 1%. w/v) NaOCl or 1% (w/v) Ca(OCl)2 for 5 or 10 min, rinsed three times with sterile distilled water and cultured on the established additional shoot regeneration medium. Thus, prior to culturing after each step in the cryopreservation protocol, additional shoots were decontaminated by immersion in 1% (w/v) calcium hypochlorite for 10 min. Additional shoots were regenerated on root explants grown for 24 h in a RITA bioreactor and then grown on semi-solid medium containing ¼ MS salts and vitamins, 30 g l-1 sucrose, 1 mg l-1 BAP and 8 g l contained -1 agar (for 6 weeks).
An essential requirement for cryopreservation is the development of appropriate in vitro regeneration protocols (Krishnapillay, 2000), which must be established in advance. In addition, established protocols avoid the callus intermediate stage, thus minimizing the risk of somaclonal variation (Skirvin et al., 1994).
CRYOPRESERVATION OF SELECTED EXPLANTS
INTRODUCTION
- Cryopreservation of recalcitrant-seeded germplasm
- Criteria governing explant selection for cryopreservation of germplasm of E. capensis
- Explant survival and onwards development after cryostorage
- Aims and objectives of the present study
As a result, the apical meristem becomes necrotic (Goveia et al., 2004; Perán et al., 2006), which prevents shoot formation. DNA methylation plays a key role in plant growth and development by regulating gene expression (Finnegan et al., 2000). In addition, a non-PCR-based method using Southern Blotting has also been described (Kumar et al., 1999).
MSAP Methylation polymorphisms present in cotton Keyte et al., 2006 MSAP Tissue culture induced methylation changes in. Seeds collected from different provenances can show different responses to cryogenic temperatures (Wen et al., 2010).
MATERIALS AND METHODS
- Explants for cryopreservation
- Explant decontamination
- Culture conditions
- Gravimetric determination of water content
- Cryopreparative procedures i) Dehydration
- Cryopreservation methods
- Recovery of explants after exposure to cryogenic temperatures a) Warming and rehydration
- Quantification of superoxide as an indication of the possible implication of ROS associated with damage
- Assessment of changes in DNA methylation status
- Photography and data analysis
The remaining explants (30) were decontaminated and plated to ascertain survival and potential for further development. In all cases where explants were only cryopreserved, such material was not previously dehydrated. iii) Sucrose preculture. Explants were sampled at 1-h intervals over a 6-h period to assess water content and potential for further development.
For the slowest rate, explants were subjected to the two-step cooling method, placing them in 2 ml polypropylene cryovials (5 explants per cryovial with 6 cryovials per treatment) and cooling at 1°C min-1 down to -40°C in a -70°C freezer using the Mr Frosty® apparatus (Nalgene, Thermo Scientific, USA) with 250 ml of isopropyl alcohol in the outer reservoir, after which the cryobales were immersed in liquid nitrogen. Explants were then removed from cryovials and immersed in a solution containing 0.5 µM calcium chloride and 0.5 mM magnesium chloride (CaMg [Mycock, 1999]) for 30 minutes in the dark at 23oC.
RESULTS
- Explant type 1: in vitro ‘broken’ buds
- Explant type 2: adventitious shoots generated from intact roots derived from in vitro-germinated material
- Explant type 3: embryonic axes with attached cotyledonary segments
- Assessment of potential ROS-mediated damage caused by cryopreparative stages and exposure to cryogenic temperatures
- Probing the germplasm for possible epigenetic changes following cryopreparative stages and cryopreservation
Since the water content of explants was sufficiently reduced with acceptable levels of survival (Table 3.5), all cryopreserved and flash-dried explants were subjected to cryopreservation trials. Accordingly, cryoprotected additional shoots were flash-dried (Table 3.8) in an attempt to reduce the water content of explants to levels suitable for non-damaging cooling and subsequent heating. The only anomaly observed (which is currently unexplained) was the 67% of axes that produced roots after cryoprotection with glycerol and then flash-drying (Table 3.13).
The highest percentage of ash producing shoots (10%) was obtained when explants were cryoprotected with glycerol, rapidly dried and then cooled in cryovials (Table 3.14). Explants were sampled at 10 minute intervals during flash drying for up to 60 minutes (see Table 3.11).
DISCUSSION
- Cryopreparative procedures a) Choice of explants and dehydration
- Exposure of explants to cryogenic temperatures a) Survival of explant types 1 – 3 after cooling
- Assessment of potential ROS-mediated damage caused by the cryopreparative stages and exposure to cryogenic temperatures
- Probing the germplasm for possible epigenetic changes following cryopreparative stages and cryopreservation
During rapid cooling (>100°C min-1), the cytoplasm can become supercooled, promoting the formation of small, potentially non-lethal (intracellular and extracellular) ice crystals (Muldrew et al., 2004). Explants in cryovials cool much more slowly than explants directly exposed to nitrogen slurry (Sershen et al., 2007). It has been reported that seeds from different origins do not necessarily exhibit similar responses to cryopreservation (Wen et al., 2010).
In the present study, some of the adventitious shoots were produced from non-cryopreserved axes using the protocol of Perán et al. 2006) and were observed to develop from calluses produced by the axes. Damage resulting from successive (repeated) oxidative bursts will therefore be cumulative (Whitaker et al., 2010).
CONCLUDING COMMENTS
However, it should be noted that a disadvantage of the method used was that it only evaluated cytosines that were present within the restriction enzyme recognition sites (Portis et al., 2004). Thus, it has been suggested that results from molecular biological analyzes should be complemented with phenotypic assessments to determine whether the observed polymorphisms will appear as heritable or stable phenotypic traits (Watt et al., 2009; Snyman et al., 2011). It is also important to ascertain whether any detected epigenetic changes are transient or whether they persist and may possibly be expressed as altered phenotypes (Peredo et al., 2009).
Future work could focus on using seedling meristems as vegetative explants as they may be more resistant to the cumulative effects prior to, including and after cryostorage (Panis et al., 2005; Varghese et. al., 2009) . ). For adventitious shoots, the application of alternative pre-cultivation methods with various additives such as ABA (Reed, 1993), proline (Burritt, 2008), antioxidants and anti-stress compounds such as vitamins C and E, lipoic acid, glutathione and glycine betaine (Uchendu et al., 2010a and b) ) may be considered when attempting to promote survival after cryostorage.
CHAPTER 4: OVERVIEW DISCUSSION, RECOMMENDATIONS FOR FUTURE RESEARCH AND CONCLUDING REMARKS
The first aspect of this study focused on the development of micropropagation protocols for selected explants of E. Micropropagation protocols (for each species under investigation) must be determined in advance (Krishnapillay, 2000) before cryopreservation can be attempted. A novel aspect of this work was the development of a protocol for the production of adventitious shoots from intact root explants (Table 2.4; Hajari et al., 2009).
Examination of the available literature has revealed that there are some reports on the use of intact roots as a source of explants for micropropagation (see Table 2.1), in most cases excised root segments are used for this purpose. This was considered essential because the aim of the current work was to maintain the intrinsic genetic diversity of stored germplasm without artificially inducing variation.
