Project Number
P04000060
Project Title
Evaluation of induced mutation methods to increase the genetic variability of Pinotage
Project Leader
Burger, P
Team Members
Burger, P
Viviers, M A
Burger, J T
Husselmann, J
Van Schalkwyk, D
Daniels, E A
Harris, I
Stander, C
Korkie, M
Objectives and Rationale
The aim was to conduct a pilot study to optimise protocols to induce possible mutations in the cultivar Pinotage towards obtaining potentially new and novel clones of this flagship South African wine cultivar. The approach was to test sub-lethal doses of irradiance on different source materials (hardwood or in vitro buds) to identify the strongest irradiance dose that still allowed sufficient survival rates and regeneration of plantlets/embryos of the irradiated buds.
Methods
i) Hardwood shoots from three clones (7, 45 and 48) were collected from nuclear plants at Vititec, irradiated at multiple irradiance levels (20, 30 and 40 Gy) and one bud cuttings rooted in a mist bed. ii) In vitro plantlets (clones 7, 45 and 48) were established and micropropagated from nuclear plants at Vititec and three populations of plantlets were irradiated on three separate occasions at multiple irradiance levels (September 2016, March and October 2017). Afterwards, a new stock of plantlets was established from the irradiated plantlets, using single-bud mini cuttings as explants. The nodal outgrowth was evaluated for growth and development over two to three months. iii) One set of plants, originating from irradiated in vitro nodes, were subjected to a second round of irradiance and used as explants for a stock of plants that experienced two cycles of irradiance. (iv) Some irradiated in vitro plants were used to initiate somatic embryogenesis from the irradiated in vitro nodes and evaluated for their ability to produce calli and regenerate into plants and subsequent somaclonal variation analyses. (v) Plants were also raised from seeds after self-pollinating
clones 45 and 48 of Pinotage to screen the range of variability within the cultivar.
Key Results
After the first rounds of irradiation, it was clear that the 30 and 40 Gy doses were too high to allow sufficient survival of both the hardwood and in vitro buds. The doses for in vitro plants of the three clones were thus adjusted to 10, 15 and 20 Gy (irradiated in March 2017) and again after rating survival and regeneration rates to 15 and 20 Gy doses for the October 2017 irradiations. Plants were either micropropagated (single bud mini cuttings) afterwards, with some undergoing a second round of irradiance, or some buds were subjected to callus and somatic embryo initiation (clones 45 and 48 in March 2017. The ability to form callus was slightly different between the two clones and the irradiation caused a slight increase in callus-forming potential in clone 45 at the lower doses, but none of the irradiated materials successfully completed somatic embryogenesis. Instead, these cultures became necrotic on the embryo-induction media compared to the controls. Two Pinotage clones were also allowed to self-pollinate to successfully establish a population of seedlings to evaluate potential variation obtained this way.
Conclusion and Discussion/ Recommendation
The results provided three main outcomes: (i) Information was obtained to establish a protocol of the irradiance conditions that are sub-lethal to the materials (irradiation doses for in vitro plants should not exceed 20 Gy as higher doses are too severe, while 10 Gy may not induce sufficient variation); (ii) embryogenic callus lines could not be regenerated into plantlets to evaluate the impact of irradiance and somatic embryogenesis in the likelihood to induce mutations; (iii) a large collection of irradiated nodal cuttings were obtained for three clones of Pinotage that can now be tested further for possible mutations generated through the irradiation.