INTRODUCTION 1
INTRODUCTION 1
In South Africa, both Pinus and Eucalyptus species contribute to approximately 90% of all commercial forests covering approximately 1.3 million ha (Department of Water Affairs and Forestry, 2005). The rest of the commercial forests consist mainly of acacia species (Department of Water Affairs and Forestry, 2005) (Figure 1.1).
EUCALYPTUS 3
EUCALYPTUS FORESTRY IN SOUTH AFRICA 3
- Introduction 3
- Breeding and improvement 4
Other desirable traits that are also included in breeding strategies are pathogen and pest resistance as well as drought and cold tolerance (Eldridge et al., 1993). Breeding strategies are generally varied, complex, multifaceted and specific to particular breeding programs (Fins et al., 1992). Different selection procedures can also be implemented in breeding strategies depending on which trait(s) the breeder selects for (Fins et al., 1992).
Molecular breeding has proven to be a valuable tool to improve the efficiency of breeding programs in agriculture (Varshney et al., 2005) as well as in forestry (Grattapaglia, 2004).
POLYPLOIDY 9
- Introduction 9
- Natural polyploidy and classification 13
Enlarged cells The cells of polyploids (including epidermal and stomatal cells) are larger than those of diploids to compensate for the increase in genetic material. Increased cell volume The increase in cell size consequently causes an increase in cell volume. Reduced growth rate The reduced number of cell divisions due to the increase in genetic material causes a decrease in the growth rate.
Increase in the size of the organ The increase in the size of the cells results in certain organs that are.
POLYPLOIDY INDUCTION 15
- Temperature induction of polyploidy 15
- Biotechnological induction of polyploidy 16
- Chemical induction of polyploidy 16
- Plant tissues utilized in the induction of polyploidy 18
- Results of inductions of polyploidy 22
Oryzalin, a dinitroaniline herbicide (Hugdahl and Morejohn, 1993) has the ability, like colchicine, to prevent microtubule formation (Morejohn et al., 1987). Another alternative to colchicine is nitric oxide (N2O), which has been successfully used to induce polyploidy in red clover (Taylor et al., 1976). Pre-soaking in colchicine before germination has been successfully used to induce polyploidy in Datura (Blakeslee and Avery, 1937) and in Acacia mearnsii (Beck et al., 2003b).
A chemical containing lanolin or agar paste has also been used to induce polyploidy in Carica papaya (Hofmeyr and van Elden, 1942) and Solanum (Chauvin et al., 2003).
POLYPLOIDY DETECTION 24
- Direct detection methods 25
- Indirect detection methods 25
In the case of the reference tetraploid (SFX 302) and the corresponding diploid (SFX 104), six visual fields per leaf of eight leaves were counted (two leaves/branch, four branches). To investigate the effects of species, colchicine concentration and exposure time on E shoot length. However, the number of putative polyploids differed between the different seed lots of the two species (Table 4.1).
The ploidy level of the putative polyploids identified using stomatal surveillance cell length measurements was confirmed by quantifying the DNA content using flow cytometry.
POLYPLOIDY IN FOREST TREE BREEDING 31
AIMS 32
MATERIALS AND METHODS 34
- INTRODUCTION 34
- MATERIALS 35
- Origin of seed 35
- Origin of axillary bud material 36
- METHODS 39
- Investigation 1: Induction of polyploidy in seed 40
- Investigation 2: Induction of polyploidy in axillary buds 43
- Investigation 3: Detection of polyploidy in the induced material 45
- STATISTICAL ANALYSIS 51
Two ramets of the corresponding diploid clone (SFX 104) were obtained for analysis from Top Crop Nursery in Pietermaritzburg and housed at the ICFR nursery. Before colchicine treatment of the seed began, the seed was first sterilized to destroy any fungal spores on the seed coat that could affect germination in the Petri dish. After placing the seeds in the appropriate dishes, 4 ml of the appropriate colchicine solution was added to the petri dish.
The treatment of the axillary buds consisted of the following steps: multiplication of the axillary buds, colchicine treatment of the axillary buds, and plant growth and maintenance. During the six-month period, lateral branches growing from untreated axillary buds were removed to ensure growth of the colchicine-treated axillary buds. The length of the guard cell of leaf operculae was measured on impressions of the abaxial side of intact leaves.
These prints were made by applying a layer of clear nail polish to the abaxial layer of the leaf. Nail polish was then dropped onto the corners of the coverslip to secure the coverslip to the slide and flatten the stomatal guard cell imprint for easier inspection and accurate measurement. This included three seedlings of each of the induction treatments (including controls) from all plots of both species.
In the preparation of samples for quantification of DNA content by flow cytometry, leaf material was treated mechanically and chemically to disrupt cells to release intact nuclei. Stomatal chloroplasts were counted in the stomata of the abaxial epidermal layer of the leaf.
RESULTS: INDUCTION OF POLYPLOIDY 52
INTRODUCTION 52
INVESTIGATION 1: INDUCTION OF POLYPLOIDY IN SEED 52
- Root and shoot length of germinated treated seed 53
- Phenotypic assessment 64
In general, an increase in colchicine concentration resulted in reduction of root and shoot growth in both 18 and 24 h exposure (Table 3.2). These analyzes were conducted to examine the effects of species, colchicine concentration, and exposure time on seedling root and shoot lengths. Exposure time (p<0.001), colchicine concentration (p<0.001), as well as the interaction between species and colchicine concentration (p<0.001) also had a significant effect on the root length of E.
A Holm-Sidak test was performed to determine which species and colchicine concentration interactions were significantly different from each other with respect to root length of E. The REML analysis showed that colchicine concentration (p<0.001), exposure time (p<0.001), the interaction between species and colchicine concentration (p<0.001) and the interaction between species and exposure time (p<0.05) had a significant effect on shoot length of E. A Holm-Sidak test was performed as a post hoc test to determine which interactions between species and colchicine concentration were significantly different from each other with respect to seedling shoot length (Table 3.10).
A generalized linear mixed model (GLMM) analysis was performed to examine the effects of species, colchicine concentration and exposure time and their interactions on survival of E. Colchicine concentration (p<0.001) and the interaction between species, colchicine concentration and exposure time ( p<0 .05) had a significant effect on the survival of E. A Holm-Sidak test was performed to determine which species, colchicine concentrations, and exposure time interactions were significantly different from each other with respect to E.
In general, the higher the colchicine concentration and exposure time, the higher the number of atypical seedlings found. Only species (p<0.05) and colchicine concentration (p<0.001) had a significant effect on the number of atypical E observed.
INVESTIGATION 2: INDUCTION OF POLYPLOIDY IN AXILLARY
- Growth of axillary buds 76
- Growth of bud sports 78
The comparison of the number of emerging bud spores between treatments and within genotypes revealed no clear pattern (Table 3.22). It would be expected that the colchicine would have a toxic effect on the meristematic cells to some extent, leading to the lower number of emerging bud spores treated with the higher. However, only SGR 1266, SGR 1238 and GxU 082 had a lower number of emerging bud spores from the 1.50% colchicine treatment than the number from the control treatment, indicating a possible sensitivity to colchicine in only three of the six genotypes.
Comparing the number of new buds between treatments and for all genotypes (Table 3.22), shows that genotypes treated with 0.50% colchicine had the lowest average number of bud spores (12.17). A generalized linear mixed model (GLMM) analysis was performed to examine the effect of genotype on the number of new buds from the treated axillary buds (Table 3.23). Genotype had no significant effect (p>0.05) on the number of bud spores emerging from the treated axillary buds.
A generalized linear mixed model (GLMM) analysis was performed to investigate the effect of colchicine concentration on the number of buds formed from treated axillary buds (Table 3.24). Colchicine concentration had no significant effect (p>0.05) on the number of buds that emerged from treated axillary buds. The results of average bud length from emerging buds showed a small trend similar to the number of emerging buds (Table 3.25).
Additionally, some genotypes (SGR 1266, SGR 1238 and GxN 075) had longer bud sports grown from colchicine-treated axillary buds than control-treated axillary buds. A Restricted Maximum Likelihood (REML) analysis was performed to investigate the effect of genotype and colchicine concentration on the length of bud spurs that emerged from treated axillary buds (Table 3.26).
RESULTS: DETECTION OF POLYPLOIDY 80
INTRODUCTION 80
INVESTIGATION 3: DETECTION OF POLYPLOIDY IN SEEDLINGS 80
- Pre-screening of seedlings for polyploidy 81
- Confirmation of polyploidy in seedlings and reference material 87
- The application of additional detection methods 92
- Comparison of phenotypic characteristics of pure tetraploid and
The true ploidy of the selected seedlings was then confirmed by quantifying the DNA content using flow cytometry. In both species, the average stomatal guard cell length per seed group of diploid controls did not match the values found for putative polyploids. When the average stomatal guard cell length of the reference material was compared to that of the two species, it was found that the reference diploid had significantly shorter guard cells than the average stomatal guard cell length for both species controls.
On the other hand, the average stomatal guard cell length of the reference tetraploid was in the lower order of the average stomatal guard cell frequency of the putative polyploids measured for both species. The average stomatal guard cell length for the reference tetraploid was found to be approx. 35% longer than the reference diploid. This indicates that the DNA content of the tetraploid was approximately double that of the reference diploid.
The guard cell chloroplast frequency of four tetraploids identified by flow cytometry and a reference hybrid tetraploid was compared with that of their corresponding diploids. In general, putative polyploids displayed regions of bud leaves that differed significantly in stomatal size. In mixoploids, this can be explained by the different rates of cell division of diploid and tetraploid leaf tissue (Otto, 2007).
The difference between stomatal guard cell size, frequency and stomatal chloroplast frequency of the reference material and the species could be attributed to the genomic constitution of the hybrid. Mutations in the periphery of the meristematic tissue result in sectional and mericlinal mutations (Lineberger, 2008). Furthermore, due to the lower success rate of polyploidy induction in axillary buds compared to seeds (Table 5.1), it is recommended to use seeds in future induction experiments to maximize results.
Demonstration of the three germ layers in the shoot apex of Datura by induced polyploidy in periclinal chimeras.
INVESTIGATION 3: DETECTION OF POLYPLOIDY IN BUD SPORTS 97
