biologia plantarum

Biologia plantarum 56:227-236, 2012 | DOI: 10.1007/s10535-012-0081-7

Nitric oxide promoted rhizome induction in Cymbidium shoot buds under magnesium deficiency

S. Guha¹, I. Usha Rao^1,2,*

¹ Department of Botany, Sri Venkateswara College, University of Delhi South Campus, New Delhi, India

² Department of Botany, University of Delhi, Delhi, India

Cymbidium shoot buds grown under Mg²⁺ deficiency without naphthalene acetic acid (NAA) showed knotted appearance. Ultrastructure of the cortical cells showed a progressive disorganization and disintegration of chloroplast membranes. The growth of shoots was resumed with the addition of 10 μM NAA. Specific NO scavenger, cPTIO induced deformation in shoot growth in 80 % of cultures. In longitudinal sections of shoots treated with cPTIO, depositions of densely stained particles in cells were observed. These inhibitory responses of cPTIO were ameliorated by 10 μM NAA. The NO donor, sodium nitroprusside (SNP), treated shoot buds displayed rapid senescence followed by necrosis of leaves. Ultrastructure of cortical cells at this stage revealed the endocytosis of mitochondria along with membrane bound cytoplasmic inclusions in the vacuole. A sharp increase in H₂O₂ generation was observed with a little change in the activity of antioxidant glutathione disulfide (GSSG), suggesting NO mediated oxidative stress. Surprisingly, after 4 weeks these necrotic shoots were converted into a globular, embryo like shoot tip with numerous structures termed here as 'neomorph' in its base. Neomorphs were different from protocorm like bodies both anatomically and morphologically. Ultrastructure of the rhizome tip exhibited numerous amyloplast and round mitochondria. At this stage, the generation of high rate of H₂O₂ was masked by GSSG, and the generation of GSSG was proportional with the concentrations of SNP, and not observed in the control (without SNP). The neomorphs were further sub-cultured to medium with different concentrations of SNP or cPTIO. After 4 weeks of culture, only the neomorphs sub-cultured on medium with SNP developed into shoots and approximately ten shoots were observed to emerge from the axils of each rhizome. Ultrastructure of cells of regenerating green neomorphs showed different shapes of mitochondria and chloroplasts and presence of active dictyosomes. The obtained shoots subjected to the acclimatization in polyhouse, expressed good growth with 85 % survival. Therefore it is reasonable to suggest that the process of de-differentiation and re-differentiation leading to rhizome formation under the condition of Mg²⁺ deficiency is NO mediated.

Keywords: NAA; neomorph; oxidative stress; rhizome; SNP; ultrastructure

Subjects: oxidative stress; rhizome; ultrastructure; magnesium; in vitro culture; naphthaleneacetic acid; anatomy - shoot buds; hydrogen peroxide; glutathione disulfite; mitochondria; chloroplasts; vacuole; starch; ex vitro acclimatization; sodium nitroprusside

Received: November 14, 2010; Accepted: May 24, 2011; Published: June 1, 2012  Show citation