Application of in vitro selection technique towards improvement for cold stress resistance in field peas.

Abstract

Leguminous plants like pea (Pisum sativum L.) are prone to drastic environmental fluctuations and disturbances. Multiple environmental stresses can have adverse effects on plant growth and productivity. Especially, temperature extremes are known to directly impact seed quality by disrupting the biochemical pathways associated with seed quality determination. In response to environmental stresses, plants develop layers of defence mechanisms including antioxidative defence and overproduction of specific solutes. Since the economic and nutritional aspects of pea seeds are highly valued in the global market, a drop in its productivity has a strong impact worldwide. To overcome the environmental challenges, the need for pea varieties with increased tolerance to multiple environmental factors is pivotal. In vitro plant tissue culture can be a powerful tool in achieving this objective. This study was aimed at addressing the issue by implementing in vitro selection as a possible strategy in the search for abiotic stress tolerance traits in the chosen field pea varieties Ginny, Kyote, Greenwood, Aragorn, 6243 and Yellowstone supplied by Plant Research Ltd. (Christchurch, New Zealand).

In this project, an effective in vitro selection (resistance selection) approach was designed to attain novel hypdroxyproline-resistant pea cell lines. This was comprised of a two-phase selection scheme employing Hyp toxicity (0-30 mM) as selective pressure applied to pea callus cultures in generating novel Hyp-resistant cell lines. Undifferentiated cells (calli) obtained from pea seed explants were subjected to varying Hyp concentrations to identify the Hyp concentration that resulted in 50% of death of the pea callus cells during toxicity evaluation (LD50). Visual scoring was done to find

out the LD50 by evaluation of necrosis pattern of pea callus cultures. With the optimized concentration from toxicity screening, the selection for Hyp-resistance was carried out on full-strength Murashige and Skoog plant cell culture medium supplemented with 8.05 µM N6-benzylaminopurine (BA), 10.74 µM α-naphthalene acetic acid (NAA) and 0-30 mM Hyp.

The variant cells resistant to hydroxyproline (Hyp) derived from selection were characterized using different biochemical parameters, and the stability of resistance was tested by subjecting them to extreme temperature stress after the Hyp-selected cell lines were cultured in the absence of the selection pressure (Hyp). The stability of Hyp-resistance is important as the stress tolerance trait must be passed on and maintained in the next generations. Hence, the selected and unselected calli were exposed to extreme temperature stress (45 ℃ and -20 ℃ 48 h) and biochemical analyses were performed before and after stress exposure. Hyp-resistant calli in high and low temperature stress appeared to retain chlorophyll and carotenoid contents better than control calli. This was correlated positively with proline accumulation. Hyp-resistant calli from pea varieties Aragorn and Greenwood showed elevated levels of free proline compared to unselected calli. Temperature stress in both extremes had damaged the cell membranes in different aspects; thermal stress disintegrated the lipid layers, and freezing stress ruptures the cell wall, eventually leading to electrolyte leakage and membrane damage. In this case, according to spearman’s correlation matrix, electrolyte leakage and lipid peroxidation have a negative correlation with proline. This was evident in the Hyp- resistant calli showing reduced damage and electrolyte leakage compared to control calli in both varieties.

Shoot buds were regenerated from Hyp-selected pea calli on full-strength MS medium supplemented with 1.07 µM NAA, 22.19 µM BA and 43.31 µM gibberellic acid (GA3). Low frequency of root regeneration was also induced in the shoots derived from Hyp- resistant pea calli. It would take longer and beyond the present thesis to improve the root regeneration frequency and then it would be possible to characterize the mutant Hyp-resistant plants from this project.

In conclusion, the in vitro selection approach used in this study based on amino acid analogue (for example, Hyp) resistance can be applied to other varieties of pea as well as different plant species to generate the respective plants with improved abiotic stress resistance.