Shugang Zhao1, Hongxia Wang2,3, Kai Liu4, Linqing Li2,3, Jinbing Yang5, Xiuhong An2,3, Pingping Li5, Linying Yun5, Zhihua Zhang6,7. 1. College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China. zshug@hebau.edu.cn. 2. Mountainous Areas Research Institute of Hebei, Baoding, 071001, China. 3. National Engineering Reseach Center for Agriculture in North Mountainours areas, Baoding, 071001, China. 4. College of Horticulture, Hebei Agricultural University, Baoding, 071001, China. 5. College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China. 6. Mountainous Areas Research Institute of Hebei, Baoding, 071001, China. zzh@hebau.edu.cn. 7. National Engineering Reseach Center for Agriculture in North Mountainours areas, Baoding, 071001, China. zzh@hebau.edu.cn.
Abstract
BACKGROUND: Tissue culture is an effective method for the rapid breeding of seedlings and improving production efficiency, but explant browning is a key limiting factor of walnut tissue culture. Specifically, the polymerization of PPO-derived quinones that cause explant browning of walnut is not well understood. This study investigated explants of 'Zanmei' walnut shoot apices cultured in agar (A) or vermiculite (V) media, and the survival percentage, changes in phenolic content, POD and PPO activity, and JrPPO expression in explants were studied to determine the role of PPO in the browning of walnut explants. RESULTS: The results showed that the V media greatly reduced the death rate of explants, and 89.9 and 38.7% of the explants cultured in V media and A media survived, respectively. Compared with that of explants at 0 h, the PPO of explants cultured in A was highly active throughout the culture, but activity in those cultured in V remained low. The phenolic level of explants cultured in A increased significantly at 72 h but subsequently declined, and the content in the explants cultured in V increased to a high level only at 144 h. The POD in explants cultured in V showed high activity that did not cause browning. Gene expression assays showed that the expression of JrPPO1 was downregulated in explants cultured in both A and V. However, the expression of JrPPO2 was upregulated in explants cultured in A throughout the culture and upregulated in V at 144 h. JrPPO expression analyses in different tissues showed that JrPPO1 was highly expressed in stems, young leaves, mature leaves, catkins, pistils, and hulls, and JrPPO2 was highly expressed in mature leaves and pistils. Moreover, browning assays showed that both explants in A and leaf tissue exhibited high JrPPO2 activity. CONCLUSION: The rapid increase in phenolic content caused the browning and death of explants. V media delayed the rapid accumulation of phenolic compounds in walnut explants in the short term, which significantly decreased explants mortality. The results suggest that JrPPO2 plays a key role in the oxidation of phenols in explants after branch injury.
BACKGROUND: Tissue culture is an effective method for the rapid breeding of seedlings and improving production efficiency, but explant browning is a key limiting factor of walnut tissue culture. Specifically, the polymerization of PPO-derived quinones that cause explant browning of walnut is not well understood. This study investigated explants of 'Zanmei' walnut shoot apices cultured in agar (A) or vermiculite (V) media, and the survival percentage, changes in phenolic content, POD and PPO activity, and JrPPO expression in explants were studied to determine the role of PPO in the browning of walnut explants. RESULTS: The results showed that the V media greatly reduced the death rate of explants, and 89.9 and 38.7% of the explants cultured in V media and A media survived, respectively. Compared with that of explants at 0 h, the PPO of explants cultured in A was highly active throughout the culture, but activity in those cultured in V remained low. The phenolic level of explants cultured in A increased significantly at 72 h but subsequently declined, and the content in the explants cultured in V increased to a high level only at 144 h. The POD in explants cultured in V showed high activity that did not cause browning. Gene expression assays showed that the expression of JrPPO1 was downregulated in explants cultured in both A and V. However, the expression of JrPPO2 was upregulated in explants cultured in A throughout the culture and upregulated in V at 144 h. JrPPO expression analyses in different tissues showed that JrPPO1 was highly expressed in stems, young leaves, mature leaves, catkins, pistils, and hulls, and JrPPO2 was highly expressed in mature leaves and pistils. Moreover, browning assays showed that both explants in A and leaf tissue exhibited high JrPPO2 activity. CONCLUSION: The rapid increase in phenolic content caused the browning and death of explants. V media delayed the rapid accumulation of phenolic compounds in walnut explants in the short term, which significantly decreased explants mortality. The results suggest that JrPPO2 plays a key role in the oxidation of phenols in explants after branch injury.
Authors: Soha Araji; Theresa A Grammer; Ross Gertzen; Stephen D Anderson; Maja Mikulic-Petkovsek; Robert Veberic; My L Phu; Anita Solar; Charles A Leslie; Abhaya M Dandekar; Matthew A Escobar Journal: Plant Physiol Date: 2014-01-21 Impact factor: 8.340
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