Identification and Characterization of Reduced Epicuticular Wax Mutants in Rice

doi:10.1016/S1672-6308(14)60295-X

Abstract

Abstract:

Epicuticular wax forms the outermost protective barrier of the aerial surfaces of land plants, working in concert with other components of the plant cuticle to prevent uncontrolled loss of water and to provide protection against an array of external environmental stress. In this study, chemically- mutagenized populations of rice (Oryza sativa L.) derived from approximately 4 750 M₂ families were screened for adhesion of water droplets resulting in a wet leaf/glossy (wlg) phenotype. Mutants were identified in 11 independently-derived M₂ families. Scanning electron microscopy analysis confirmed the association of the wlg phenotype with changes in the epicuticular wax crystals of these plants. The phenotypes of five mutants (7-17A, 26.1, 524.2, 680.2, and 843.1) were confirmed to be the result of single recessive gene mutation. Evaluation of mutants from 3 (6-1A, 7-17A, and 11-39A) of 11 M₂ families revealed significant reductions (> 50%) in surface wax content and increases in cuticle membrane permeability.

Key words: chemical mutagenesis, cuticle wax, forward genetics, membrane permeability, rice, scanning electron microscopy

H. Tai Thomas. Identification and Characterization of Reduced Epicuticular Wax Mutants in Rice[J]. Rice Science, 2015, 22(4): 171-179.

Figures/Tables 6

References 27

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27	(Managing Editor: Wang Caihong)

M₁ line	M₂ line	Seed source	Mutagenesis	Phenotype of M₃	Single gene recessive mutation	Mutant designation
6-1	6-1A	2009	1 mmol/L NaN₃/ 16 h presoak	Wet leaf/glossy segregating	Nd	wsl3
6-1	6-1B	2009	1 mmol/L NaN₃/ 16 h presoak	Wet leaf/glossy	Nd
7-17	7-17A	2009	1 mmol/L NaN₃/ 16 h presoak	Wet leaf/glossy segregating	Yes	wsl4
11-39	11-39A	2009	1 mmol/L NaN₃/ 20 h presoak	Wet leaf/glossy	Nd	wsl5
26	26.1	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl6
264	264.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Nd	wsl7
524	524.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl8
680	680.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl9
843	843.1	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl10
1064	1064.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy	Nd	wsl11
1086	1086.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy	Nd	wsl12
1558	1558.1	2011	2 mmol/L NaN₃/ 16-20 h presoak	Intermediate	Nd
1558	1558.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Intermediate	Nd	wsl13
Phenotypic ratios of segregating M₃ populations were evaluated using Pearson’s Chi-square test for goodness-of-fit to the 3:1 (wild-type:mutant) ratio expected for single gene recessive mutations. Nd, Not determined; wsl, Wax crystal-sparse leaf.

M₁ line	M₂ line	Seed source	Mutagenesis	Phenotype of M₃	Single gene recessive mutation	Mutant designation
6-1	6-1A	2009	1 mmol/L NaN₃/ 16 h presoak	Wet leaf/glossy segregating	Nd	wsl3
6-1	6-1B	2009	1 mmol/L NaN₃/ 16 h presoak	Wet leaf/glossy	Nd
7-17	7-17A	2009	1 mmol/L NaN₃/ 16 h presoak	Wet leaf/glossy segregating	Yes	wsl4
11-39	11-39A	2009	1 mmol/L NaN₃/ 20 h presoak	Wet leaf/glossy	Nd	wsl5
26	26.1	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl6
264	264.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Nd	wsl7
524	524.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl8
680	680.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl9
843	843.1	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy segregating	Yes	wsl10
1064	1064.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy	Nd	wsl11
1086	1086.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Wet leaf/glossy	Nd	wsl12
1558	1558.1	2011	2 mmol/L NaN₃/ 16-20 h presoak	Intermediate	Nd
1558	1558.2	2011	2 mmol/L NaN₃/ 16-20 h presoak	Intermediate	Nd	wsl13
Phenotypic ratios of segregating M₃ populations were evaluated using Pearson’s Chi-square test for goodness-of-fit to the 3:1 (wild-type:mutant) ratio expected for single gene recessive mutations. Nd, Not determined; wsl, Wax crystal-sparse leaf.

Population	M₃ seeds planted ^a	M₃ plants phenotyped ^a	Segregation ratio (wild-type:mutant)	Pearson’s Chi-square test (3:1)
Population	M₃ seeds planted ^a	M₃ plants phenotyped ^a	Segregation ratio (wild-type:mutant)	χ²	P value
26.1	77	57	40:17	0.708		0.400
680.2	77	61	43:18	0.661		0.416
843.1	77	73	53:20	0.224		0.636
524.2	95	68	53:15	0.314		0.575
^a includes initial screening of M₃ plants.

Population	M₃ seeds planted ^a	M₃ plants phenotyped ^a	Segregation ratio (wild-type:mutant)	Pearson’s Chi-square test (3:1)
Population	M₃ seeds planted ^a	M₃ plants phenotyped ^a	Segregation ratio (wild-type:mutant)	χ²	P value
26.1	77	57	40:17	0.708		0.400
680.2	77	61	43:18	0.661		0.416
843.1	77	73	53:20	0.224		0.636
524.2	95	68	53:15	0.314		0.575
^a includes initial screening of M₃ plants.

Line	Wax content (mg/g)	Reduction in wax content (%)
Sabine (wild-type)	3.01 ± 0.49
6-1A/B ^a	0.91 ± 0.31 **	69.77
7-17A	0.68 ± 0.42 **	77.41
11-39A	1.38 ± 0.10 **	54.15
Values are given as mean ± SD with four replicates except where noted. ^a, Due to insufficient tissue from 6-1A M₃ mutants, additional samples were taken from 6-1B M₃ mutants and content was determined using a total of five replicates (three from 6-1A plants, two from 6-1B plants included). ** means significant difference between mean values at P < 0.01 by t-test between the wild-type and each of the mutant line.