1 Introduction
4.5 SUMMARY
5.2.2 Explants from embryos
The seeds were surface sterilised as with in vitro germination experiments described in Chapter 4 and imbibed for 5 days. Decontaminated seeds were placed in a Petri dish with sterile distilled water. The seeds were left to imbibe in the laminar flow cabinet and were dissected every 24 h to test for ease of dissection. Seeds could only be dissected after 4 days, as the blade could not penetrate the periderm (fruit coat) with a clean cut with less time for imbibition. The embryos that were dissected after 7 days of imbibition did not show any growth response when placed on media.
To excise the embryo some of the surrounding endosperm had to be cut away (See Figure 5.1).
118 Figure 5.1: General embryo excision procedure for Romulea seeds. An outer view, as one would view it through a stereo microscope, as well as a view relative to the embryo is provided so that the importance of the placing of the incisions can be seen. Step 1 is viewed from the top, Step 2 is a side view, Steps 3 and 5 are bottom views 90° to the incision made in Step 2.
Step 4 is a side view.
In Step 1 the seed was gripped with a pair of fine forceps and two incisions were made. The excess tissue was scrapped to the front of the Petri dish. In Step 2 the seed was turned onto one of the flat sides created by the incision done in Step 1.
When turning on the bottom light of the dissection microscope the light radiated through the seed, making the embryo visible. Another incision was then made below the embryo. The seed was then turned onto the Petri dish as indicated in Step 3 to check for embryo visibility and to allow a better cutting angle. Small slices of tissue could then be removed as indicated in Step 4 until the embryo was clearly visible through the somewhat transparent endosperm tissue. An autoclaved dissection needle was then used to carefully flake away the sections indicated in Step 5 (Figure 5.1). The embryo was then lifted out of the endosperm tissue and placed in a Petri dish with sterile distilled water to rinse off residual pieces of endosperm.
Excised embryos were placed into 33 ml culture tubes with 10 ml nutrient media using an autoclaved Pasteur pipette with sterilised distilled water immediately after excision. Treatments with various concentrations of plant growth regulators in the growth media were prepared for each species.
119 R. diversiformis, R. flava, R. minutiflora and R. monadelpha embryos were placed on media with no plant growth regulators and media supplemented with 2.3, 4.7 and 23.2 µM kinetin. For R. diversiformis and R. minutiflora 10 embryos were used for each treatment, while 20 embryos were used per treatment for R. flava and R.
monadelpha.
Embryos of R. camerooniana and R. rosea were placed on 13 media treatments, a medium with no plant growth regulators, media with 2.3, 4.7 or 23.2 µM kinetin or mTR and media supplemented with 2.3, 4.7 or 23.2 µM kinetin or mTR in combination with 0.5 M NAA.
For R. sabulosa an experiment was designed to investigate the suitability of a set of PGR treatments for shoot culture initiation and embryogenesis. The experiment had 12 treatments and was repeated twice, first with 20 replicates and then with 10 replicates. The 12 treatments consisted of media supplemented with combinations of 0, 2.3, 4.7 and 23.2 Mkinetin, and 0, 2.2 and 4.5 M 2,4-D. An experiment with 3 treatments, media supplemented with 4.4 M BA, 22.2 M BA and 2.3 M kinetin with 5.4 M NAA was also initiated. This experiment was only repeated once with 20 replicates. This is because of the vitrification and abnormal growth of shoots cultured on media supplemented with BA.
After two months the number of shoots formed per explant, the presence or absence of roots and the morphology was recorded. The cultures that appeared embryogenic where placed in culture bottles with 30 ml of MS media supplemented with 100 mg.l-1 myo-inositol, 30 g.l-1 sucrose and 8 g.l-1 activated charcoal after 2 months.
Culture morphology was not only observed with the naked eye, but also under a light microscope. Samples of tissue suspected to have embryo initials were prepared by Epon resin embedding, sectioned using a LKB Ultrotome II microtome, stained with Ladd’s multiple stain and viewed with an Olympus AX 70 stereo microscope.
120 5.2.3 Explant comparison
R. leipoldtii seeds were used, not only because of their high germination and in vitro shooting in preliminary seedling organ experiments, but also because this species has the smallest seeds. If embryo excision is possible for a species with such a small seed it shows that seed size is not a limitation to using embryo excision in a culture initiation protocol for Romulea species.
Seeds (200) were surface sterilised and germinated at 15°C as with in vitro germination experiments described in Chapter 4. After 2 months when sufficient seeds germinated, 130 seedling hypocotyls and 130 embryos were excised simultaneously. Seedlings with stems longer than 30 mm were then removed from tubes and placed on Petri dishes and cut into 3 sections using a sterilised scalpel and blade. For this experiment only the hypocotyl was used because of its higher percentage in vitro response. The seedling hypocotyls and embryos were then placed in 33 ml culture tubes with 10 ml nutrient media with 13 medium treatments, a medium with no plant growth regulators, media with 2.3, 4.7 or 23.2 µM kinetin or mTR and media supplemented with 2.3, 4.7 or 23.2 µM kinetin or mTR in combination with 0.5 M NAA. Ten replicate explants were used per treatment and the experiment was repeated twice. After two months the number of shoots formed per explant and the presence or absence of roots was recorded.