View/Open - ResearchSpace - University of KwaZulu-Natal

More documents

Recommendations

Info

Literature review misting will facilitate contamination by wetting the foliage (KANE, 2004). Placing the plant material in a less humid and dry environment a few weeks prior to taking explant material can reduce contamination of cultures (SMITH, 2000b). Plant materials growing in soil (roots, tubers, bulbs) or near the soil surface (stolons, rhizomes, orchid protocorms, etc.) are often harder to clean and disinfect than aerial plant material (SMITH, 2000b). Explants are much easier to clean if the plant has been growing in an artificial medium, such as washed sand or perlite (KYTE & KLEYN, 1996). After cutting the explant from the source plant, it should be placed in a plastic bag containing a moist paper towel and kept refrigerated until culture initiation (KYTE & KLEYN, 1996). 2.9.2 Explant preparation Explants require surface-disinfection before they can be placed in culture on the nutrient agar for in vitro culture (SMITH, 2000b). Explants are washed in sterile water and rinsed in ethanol and the surface is sterilised using chemicals with a chlorine base (AHLOOWALIA et al., 2002). There are a number of products used for surface disinfection, the most commonly used is commercial chlorine bleach (SMITH, 2000b). For soft, herbaceous material a calcium or sodium hypochlorite based solution is often used at a concentration of 1-3% (AHLOOWALIA et al., 2002). Before surface- disinfection any remaining soil or dead parts should be removed from the explant (PIERIK, 1997). An inexpensive and ready-made alternative is a 5-7% solution of Domestos® (a toilet disinfectant by Lever Bros. Ltd., UK), which contains 10.5% sodium hypochlorite, 0.3% sodium carbonate, 10.0% sodium chloride and 0.5% sodium hydroxide and a patented thickener (AHLOOWALIA et al., 2002). Explants are washed in sterile water before and after sterilization (AHLOOWALIA et al., 2002). A general procedure for preparing the explant involves washing the explant in warm, soapy water after which it is rinsed in tap water (PIERIK, 1997; SMITH, 2000b). The explant is then rinsed in a freshly made chlorine bleach solution (PIERIK, 1997). One to 2 drops of wetting agent should be added to every 100 ml of bleach solution. The explant is then rinsed in sterile water three to five times. 65
Literature review PIERIK (1997) stated that a brief alcohol rinse or swab is needed with hairy or wax coated surfaces. Epidermal hairs may trap air bubbles, in such cases these have to be evacuated under vacuum. Sterilized forceps and scalpels must be used for the transfer of explants to fresh solutions (AHLOOWALIA et al., 2002). Sterile containers must be used throughout the protocol of surface sterilization (AHLOOWALIA et al., 2002). If explants become brown or pale the strength of the sterilizing agent should be reduced (GAMBORG & PHILLIPS, 1995; PIERIK, 1997). A cut explant such as a stem or leaf that is surface sterilised often shows tissue damage from surface sterilisation (PIERIK, 1997). The damaged tissue should be removed before culture (SMITH, 2000b). GAMBORG & PHILLIPS (1995) suggest that a procedure for seed sterilization should include washing the seeds in detergent, after which they are rinsed with tap water and subsequently alcohol, a bleach solution and autoclaved demineralised water respectively. Seeds can be germinated on filter paper in Petri dishes or on an agar medium (GAMBORG & PHILLIPS, 1995). A single seed should ideally be placed in each container so that a single contaminated seed does not contaminate other seeds. Contamination resulting from improperly sterilised tissue will generally arise from the explant and be located in the medium adjacent to the explant (SMITH, 2000b). Contamination that is due to poor technique will generally appear over the entire agar surface (SMITH, 2000b). Examples of poor technique include contaminated transfer hood filters and culture cabinets and improperly sterilised media. Contamination of cultures by fungi appear as fuzzy growth whereas bacterial contamination appears as smooth pink, white or yellow colonies and contamination 66
Page 1 and 2:
Propagation of Romulea species Pier
Page 3 and 4:
Contents 2.7 GERMINATION PHYSIOLOGY
Page 5 and 6:
Contents 5.3.1 Explants from seedli
Page 7 and 8:
Abstract The suitability of various
Page 9 and 10:
Declarations I Pierre André Swart,
Page 11 and 12:
Declarations DETAILS OF CONTRIBUTIO
Page 13 and 14:
Publications from this thesis ASCOU
Page 15 and 16:
List of Figures Figure 2.13: Suther
Page 17 and 18:
was significantly different from th
Page 19 and 20:
List of Tables Table 2.1: Names of
Page 21 and 22:
List of Abbreviations 2,4-D 2,4-Dic
Page 23 and 24:
Die vlakhaas spits sy oor hy het di
Page 25 and 26:
As die mens dan ook so sy dankbaarh
Page 27 and 28:
Introduction where a great number o
Page 29 and 30:
Introduction The subgenera Romulea
Page 31 and 32:
2 Literature review Leef van daad e
Page 33 and 34:
Literature review Figure 2.2: Life
Page 35 and 36:
Literature review Figure 2.4: Life
Page 37 and 38:
Literature review The plants are sh
Page 39 and 40: Literature review flowers (DE VOS,
Page 41 and 42: Literature review often found on cl
Page 43 and 44: Literature review flowers of R. lei
Page 45 and 46: Literature review flattened upper s
Page 47 and 48: 2.2.16 Romulea tabularis Literature
Page 49 and 50: Literature review extinct, R. papyr
Page 51 and 52: Literature review R. leipoldtii occ
Page 53 and 54: Literature review Figure 2.8: Calvi
Page 55 and 56: Literature review Figure 2.14: Fras
Page 57 and 58: Figure 2.20: Nieuwoudtville weather
Page 59 and 60: Literature review Figure 2.26: Grah
Page 61 and 62: Literature review Figure 2.29: Diag
Page 63 and 64: Literature review Figure 2.30: A te
Page 65 and 66: Literature review Macronutrients ca
Page 67 and 68: Literature review The soils of the
Page 69 and 70: Literature review The embryo is the
Page 71 and 72: Literature review Phase 2 is seen a
Page 73 and 74: Literature review endogenous gibber
Page 75 and 76: Literature review tolerable level (
Page 77 and 78: Literature review these channels pe
Page 79 and 80: Literature review 2.8.7 Seed dorman
Page 81 and 82: Literature review the embryo (COPEL
Page 83 and 84: Literature review germinated under
Page 85 and 86: Literature review (COPELAND, 1976).
Page 87 and 88: 2.9 BRIEF REVIEW OF IN VITRO CULTUR
Page 89: Literature review (DEBERGH, 1994).
Page 93 and 94: Literature review The distinguishin
Page 95 and 96: Literature review The essential fun
Page 97 and 98: 2.9.3.4 Abscisic acid Literature re
Page 99 and 100: Table 2.6: Example of a matrix to e
Page 101 and 102: Literature review Young embryos req
Page 103 and 104: Literature review and the addition
Page 105 and 106: Literature review organ is then pro
Page 107 and 108: Literature review environmental str
Page 109 and 110: 2.11 IN VITRO FLOWERING Literature
Page 111 and 112: Literature review higher regenerati
Page 113 and 114: Subfamily Ixioideae (continued) Tri
Page 115 and 116: Literature review Table 2.8: Corm i
Page 117 and 118: 3 Investigation into the habitat of
Page 119 and 120: Soil sampling and analysis The firs
Page 121 and 122: Soil sampling and analysis Table 3.
Page 123 and 124: 4 Germination physiology 4.1 INTROD
Page 125 and 126: 4.2.2 Water content and imbibition
Page 127 and 128: Germination physiology (-N), phosph
Page 129 and 130: Germination physiology rosea seeds
Page 131 and 132: Germination physiology Figure 4.4:
Page 133 and 134: Germination (%) 50 40 30 20 10 0 77
Page 135 and 136: Germination (%) 100 80 60 40 20 0 c
Page 137 and 138: Germination physiology The relative
Page 139 and 140: Germination physiology seeds of R.
Page 141 and 142:
5.2 MATERIALS AND METHODS In vitro
Page 143 and 144:
In vitro culture initiation and mul
Page 145 and 146:
5.2.3 Explant comparison In vitro c
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
Page 159 and 160:
5.4 DISCUSSION In vitro culture ini
Page 161 and 162:
Page 163 and 164:
6 In vitro corm formation and flowe
Page 165 and 166:
In vitro corm formation and floweri
Page 167 and 168:
6.3 RESULTS 6.3.1 Corm formation In
Page 169 and 170:
Page 171 and 172:
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
Commercialization potential of Romu
Page 179 and 180:
Commercialization potential of Romu
Page 181 and 182:
BASKIN, C. C., and BASKIN, J. M. (1
Page 183 and 184:
Literature cited DOWLING, P. M., CL
Page 185 and 186:
Literature cited HILTON-TAYLOR, C.
Page 187 and 188:
Literature cited KUMAR, A., SOOD, A
Page 189 and 190:
Literature cited PIERIK, R. L. M. (
Page 191 and 192:
Literature cited STEINITZ, B., COHE
show all

View/Open - ResearchSpace - University of KwaZulu-Natal

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?