Studies on in vitro manipulation of male and female reproductive systems of flowering plants
The overall aim of this research project is to investigate the possibility of using pollen as a vector for transporting extracellular substances to the site of gamete fusion in the embryo sac. Manipulation of plant male and female gametophytes included studies on pollen culture in vitro, pollen viability and developmental state, loading of fluorescent probes by plasmolysis/endocytosis and via vascular system, clearance of embryo sacs, ovule culture and the in vitro fertilisation and production of genetically uniform lines.Pollen from Impatiens glanduiifera cultured under a range of nutrients (sucrose, H(_3)B(_O4), Ca. K and Mg), temperature and humidity conditions revealed that 5% sucrose, lOOppm H(_3)BO(_4) with 100 ppm potassium nitrate, gave longer pollen tubes (463.20 pm after 1 h). Pollen tubes were longer at room temperature; however, they also grew under temperatures down to 4 C. The effect of humidity levels was also significant, and pollen tube length increased with the increase of relative humidity (RH) over the range 0.0 to 92.0%. Plasmolysis followed by deplasmolysis of pollen gave a non-significant effect on tube growth compared to the control treatment.Assessment of pollen viability using fluorescein diacetate (FDA) and Calcofluor White M2R (CFW) highlighted some drawbacks on the most widely used technique for assessing pollen viability, the fluorochromatic reaction. Pollen developmental state in I. glandulifera was assessed using the Feulgen staining technique. The use of DNA-specific 4,6-diamidino-2- phenyl indole (DAPl) has clearly shown the vegetative and generative nuclei. Pollen tubes were monitored using aniline blue.When pollen were plasmolysed and deplasmolysed in the presence of 5 mg ml (^-1) Lucifer Yellow CH (LY-CH), the fluorescent probe was taken into pollen and the most likely mechanism by which it was taken up was through plasmolysis/endocytosis. The loading up into pollen of FDA by enzymic cleavage and fluorescein by endocytosis is also discussed. Fluorescein, LY-CH and Calcofluor White M2R were loaded via the vascular system of Nicotiana tabacum, I. glandulifera and Brassica napus. Using fluorescence microscopy, the path of these probes was followed from the pedicel cells up to the ovules.The demonstration of loading of fluorescent probes into embryo sacs, whether via germinating pollen or via the vascular system, required manipulation of ovaries and clearance of embryo sacs. The fixing and clearing technique revealed, to some extent, embryo elements in I. glandulifera and N. tabacum. The enzymic maceration technique, however, resulted in the isolation of I. glandulifera embryo sacs. The embedding in London Resin White (LR White) technique was used to reveal additional information.In vitro stigmatal pollination of I. glandulifera ovaries resulted in pollen tubes penetrating into the ovules. When this was conducted in the presence of Lucifer Yellow CH, B. napus pollen tubes were seen carrying the probe and penetrating into the ovule. Fully grown N. tabacum plants were obtained from ovaries cultured and pollinated in vitro.Micro-propagation of I. glandulifera, N. tabacum and B. napus in Murishige and Skoog, and Nitsch and Nitsch -based media resulted in plantlets from B. napus and Nicotiana tabacum. Acclimatisation of the latter, under humid conditions, resulted in fully grown plants.