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Plant material and nutrient media
Immature zygotic embryos (IZEs) ranging from 0.8-1.2 mm in length were used as explants. These were derived from sorghum seeds harvested 12-15 days post anthesis. The immature seeds were surface-sterilized in 70% (v/v) ethanol for 3 min, and for 15 min in 2.5% sodium hypochlorite solution containing 0.1% Tween-20 before a thorough rinse with sterile distilled water. Tissue culture experiments were performed under aseptic conditions. Callus cultures were transferred to fresh callus induction medium (CIM) every two weeks until the onset of somatic embryogenesis. The IZEs were placed on CIM with the scutellum cells facing upwards and the embryogenic axis in contact with the CIM for somatic embryo formation. After somatic embryo formation, the calli were transferred to callus maintenance medium (maturation medium) before transfer to regeneration medium for plantlet production. In total, 100 to a 150 IZEs (10 embryos per petri dish) were cultured per genotype per tissue culture medium (Table 2.4). The IZE explants consisted of three biological replicates, i.e. independent panicles and harvest dates, with 15-30 explants per replication. Tables 2.1 to 2.3 list a summary of the nutrient media contents and the culturing conditions for each solid medium used in this study. For tissue culturing on medium J (O’Kennedy et al., 2004), the CIM contains L3 based salts and vitamins (see Annexure), 2.5 mg l-1 of the auxin 2,4- dichlorophenoxyacetic acid (2,4-D), the carbon source maltose, 4 g/l gelrite as the gelling agent and 20mM L-proline. In the root and regeneration medium (RRM), 2,4-D and L-proline were not included. A 4-week culture on CIM was followed by a two-week period on callus maturation medium, prior to regeneration and rooting regimes/phases. The maturation medium contained double the amount of carbohydrate, which in this case was maltose (Table 2.1).
For tissue culturing on Tadesse’s medium, somatic embryogenic calli formed within 4 weeks of culture on CIM were transferred to a modified CIM with reduction in 2,4-D (2.5 – 2.0mg/l) and increment in kinetin (0.2 – 0.5 mg/l) until somatic embryos were ready to germinate (Table 2.2).
The somatic embryos were then transferred to shoot induction medium (SIM) until shoots developed (Tadesse et al., 2003) and subsequently to root induction medium (RIM).
The CAPD medium was described by Casas et al. (1993). This medium was used with the following modifications: 1 g/l asparagine and 2 mg/l 2,4-D was added in the CIM. After 14 days on CAPD2 and 7 days on CAPD1 to initiate somatic embryoids, the cultures were transferred to callus maintenance medium (CCM) for 4-7 days, followed by subsequent culture for 2-6 weeks on regeneration medium. The callus regenerating medium (CMR) was responsible for shoot formation within 14-28 days. The callus shoot elongation (CSE) medium cultures take 10-14 days before shoots are cultured on Casas Rooting Medium (CROOT) for 14 days. Finally, transfer and culturing of rooted shoots on Casas Root Elongation (CRE) for 7-14 days was performed (Table 2.3).
Plasmids
Two plasmids were used for transforming sorghum. Plasmid pAHC25 (Christenson and Quail, 1996) contains the bialaphos resistance gene bar (Figure 3.2 A). The plasmid pNOV3604-ubi, obtained from Syngenta, USA, carries the manA gene (O’Kennedy et al., 2004) which confers resistance to mannose selection (Figure 3.1 A). Both genes are driven by the maize ubiquitin promoter and the nopaline synthase terminator (Nos-ter). All plasmid DNA preparations were carried out using the Qiagen Maxiprep Kit (Southern Cross Biotechnologies, South Africa) according to the manufacturer’s recommendation. The plasmid pABS encoding the RNAi co suppression cassette is shown in Figure 3.1 A, B and C. This was generously supplied by Drs Rudolf Jung and Kimberly Glassman from Pioneer Du Pont, Iowa, USA (members of the ABS consortium). The seed endosperm specific promoter from maize 19GZ was used to drive the co-suppression of the LKR, δ-kaf-2, γ-kaf-1 and -2 genes. The rice ADH1 intron was used as a hairpin part of this double stranded DNA transgene. The target genes were isolated from a cDNA library of a developing seed. cDNA clones from a developing seed were analyzed and sequenced to generate expressed sequence tags (EST). These ESTs were classified on the basis of sequence homology to known protein sequences. Domains for silencing targeted genes were selected from the EST sequences (Jung, 2007) and the 5’-3’ sense strand sequences are displayed in Figure 3.1 C. Selected domains of the LKR and kafirin genes were cloned in tandem and designed into a hairpin construct in which the tandemly cloned domains and the inverted versions are separated by the ADH-1 intron to form a loop and cloned into a binary vector through Gateway cloning (Invitrogen, USA). The γ-kaf-1 gene sequence translates into a 186 amino acid long protein that contains only one lysine residue and a total of 12 sulphur containing cysteine residues (see NCBI protein sequence database, Accession number AAS73290. The γ-kaf-2 protein is a 211 amino acid long preprotein that comprises one lysine and 13 cysteine residues (Accession number CAA44347). The δ-kaf-2 protein is 187 amino acid long containing 2 lysine and 14 cysteine residues. This δ-kaf-2 protein sequence has not been identified and reported into any database to date. These three target proteins are implicated as major contributors to the seed lysine content deficiency problem and the low seed protein digestibility due to their high content of cysteine, thus, increasing the disulphide bond formation potential with other proteins. To create plasmid pABS (Figure 3.1 B) minimal transgene cassettes (MTCs), the full plasmid was prepared by endonuclease digestion with EcoRI to remove the backbone carrying the kanamycin resistant gene. For the pmi MTC, a double digest of Asp 718 and Hind III released the backbone carrying the ampicillin resistance gene. These kafirins were identified and chosen on the basis of their inherently poor lysine content and contribution to poor protein digestibility as a result of cross-linkages with other polymers in grain endosperm.
CHAPTER 1 INTRODUCTION
1.1 The sorghum plant
1.2 Grain utilization
1.3 Genetic engineering of plants
1.4 Genetic engineering of sorghum
1.5 Nutritional quality of the grain
1.6 Strategies for improving plant nutritional value: advantages of RNAi
1.7 Rationale of the study
1.8 References
CHAPTER 2 SCREENING OF SORGHUM GENOTYPES FOR TISSUE CULTURE AMENABILITY
2.1 Abstract
2.2 Introduction
2.3 Materials and methods
2.4 Results
2.5 Discussion
2.6 References
CHAPTER 3 TRANSFORMATION OF SORGHUM FOR SUPPRESSION OF SELECTED SEED PROTEINS
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.4 Results
3.5 Discussion
3.6 References
CHAPTER 4 CHARACTERIZATION OF TRANSGENIC PLANTS FOR SUPPRESSION OF TARGET KAFIRINS AND LKR
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.5 Discussion
4.6 References
CHAPTER 5 DISCUSSION
5.1 The need for establishing protocols for genetic engineering of sorghum
5.2 Optimization of parameters for transformation
5.3 The RNAi approach to enhance lysine through reduction of storage proteins
5.4 Limitations and product drawbacks
5.5 Future research
5.6 References
Annexure
