Plant tissue culture investigations on Capsicum annuum L. : Somatic embryogenesis, in vitro flowering, fruiting and seed formation
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Four cultivars of Capsicum annuum L., namely Sweet Banana, California Wonder, Yolo Wonder and Ace were used in different experiments initially to examine more closely some of the critical factors influencing somatic embryogenesis in zygotic embryo explants. Using the protocol of Buyukalaca and Mavituna (1996) only callus formation without embryogenesis was observed in the mature zygotic embryo explants of the four cultivars. With some modifications to the protocols of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), improved somatic embryogenesis in immature zygotic embryo explants of the cultivars used in the present study was achieved. When the immature zygotic embryo explants were placed on induction medium longer than 2 weeks, the percentages of plantlet conversion were significantly reduced from 75% and 65% to 40% and 28% in cvs. Sweet Banana and California Wonder, respectively. Somatic embryogenesis of cv. Yolo Wonder was not observed if sucrose was not added to the induction medium or if it was supplemented either with 10% glucose or fructose. Maltose could replace sucrose as far as somatic embryo induction and eventual plantlet conversion in the cv. Yolo Wonder is concerned. For all 4 cultivars, somatic embryos were initiated from immature zygotic explants on medium with or without coconut water, in the light or dark condition. Somatic embryo structures of C. annuum L. cv. Sweet Banana were easily observed under a stereo-microscope after 14 days of culture on the induction medium. These structures turned green 5-7 days after the explants were transferred to the germination or conversion medium, and then they germinated into plantlets about 3 days later. The origin and developmental sequence of the somatic embryos were investigated using the resin infiltration and embedding technique. Periclinal cell division firstly began underneath the protoderm of the cotyledons of the explant after 3 days of culture on somatic embryo induction medium. The number of cells obviously increased after 5-7 days of culture as a result of both periclinal and anticlinal cell divisions. After 14 days, the globular structures were clearly visible on the surface of the cotyledons of the immature zygotic embryo explant. Upon further culture, other well known somatic embryo developmental stages, including heart-shaped, torpedo and cotyledonary stages were observed. The germinated somatic embryos of C. annuum L. cv. Sweet Banana grew well on the agar-gelled plantlet development medium comprising the basal salts and vitamin mixture of Murashige and Skoog (1962) supplemented with 1 mg/l NAA and no sucrose. Upon further culture, the somatic embryo-derived plantlets were found to be capable of flowering in vitro in small tissue culture containers. The in vitro flowers were compared with those produced by the mature plants grown in the glasshouse. It was found that there were no obvious differences in the outer parts (calyx colour, corolla number, corolla colour and corolla length) of flowers from both sources. The inner parts of both in vitro and in vivo flowers were slightly different as far as stamen number, anther length, filament length, carpel length and style length are concerned. However, anther colour, filament colour, carpel number, carpel colour, style colour, and stigma colour were the same. Pollen from both in vitro and in vivo flowers also appeared to be the same when observed under a scanning electron microscope. In contrast, pollen physiology of in vitro and in vivo flowers in relation to both viability and germination tests were slightly different (ANOVA, P<0.05). The percentages of viability and germination of pollen from in vitro flowers were about 94% and 25%, respectively, while those of pollen from in vivo flowers were about 96% and 34%, respectively. In vitro floral buds were first formed on plantlets that grew on the plantlet development medium in a growth room at 22°C under continuous illumination provided by white fluorescent lamps. However, the floral buds rarely developed further into mature flowers as they abscised soon after their formation. This problem was overcome using the vented autoclavable plant tissue culture containers (Phytacon TM, Sigma Chemical Co.). In vitro fruit formation and ripening was observed when liquid half strength MS basal medium supplemented with 5 μg/ml Ag2S20 3, 1 mg/l NAA and 3% sucrose was added to the surface of the plantlet development medium. Further improvement to this in vitro fruiting system was achieved with the aid of hand pollination. Clonal propagation starting with both nodal and excised shoot tip explants from the plantlets of C. annuum L. cv. Sweet Banana regenerated via somatic embryogenesis was investigated. It was found that sucrose in MS basal medium free of plant growth regulators is an important factor for plantlet regeneration from the explants. Furthermore, the plantlets were capable of flowering, fruit setting, fruit ripening and seed setting in vitro. The harvested seeds germinated and developed into normal-looking seedlings. This is the first report on the completion under in vitro conditions of the whole developmental cycle starting from somatic embryogenesis and resulting in the formation of germinable seeds of C. annuum L.