Optimisation of a highly efficient shoot regeneration system using leaf explants of Chinese jujube (<i>Ziziphus jujuba</i> Mill.) by response surface methodology

<p>Chinese jujube (<i>Ziziphus jujuba</i> Mill.) is a major fruit crop in Asia. In this study, response surface methodology (RSM) was successfully employed to establish a highly efficient <i>in vitro</i> propagation and regeneration system for the ‘Teapot’ jujube via shoot organogenesis. Among the tested factors, gibberellic acid (GA₃) concentration showed the most significant positive effect. The pre-culture darkness timing and medium were also important factors for highly efficient shoot regeneration of the ‘Teapot’ jujube. The highest regeneration (> 75%) was achieved by 1 week in darkness and culture on wood plant medium (WPM) containing 0.25 mg·L⁻¹ GA₃, 0.5 mg·L⁻¹ 6-benzylaminopurine (BAP) and 0.1 mg·L⁻¹ 3-indoleacetic acid (IAA). <i>In vitro</i>-derived shoots rooted very well in the modified ¹/₂ Murashige and Skoog (MS) medium containing 0.4 mg·L⁻¹ 3-indolebutyric acid (IBA), resulting in a 100% rooting rate. These findings suggest that the RSM can be employed to optimise the protocols needed for successful <i>in vitro</i> plant regeneration of jujube cultivars, with potential applications in plant genetic transformation practices, polyploidy induction and germplasm preservation.</p

Seedling infection types (ITs) of the parents and number of F₈ recombinant inbred lines (RILs) from the Druchamp × Michigan Amber-derived recombinant inbred lines (RILs) tested in the greenhouse with races of <i>Puccinia striiformis</i> f. sp. <i>tritici</i>, theoretical segregation ratios of resistant and susceptible lines, and χ² and P values of chi-squared tests for goodness of fit of the observed numbers and expected ratios.

<p>^a Refer to references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126794#pone.0126794.ref001" target="_blank">1</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126794#pone.0126794.ref009" target="_blank">9</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126794#pone.0126794.ref015" target="_blank">15</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126794#pone.0126794.ref016" target="_blank">16</a>] for virulence of the races.</p><p>^b The IT data were recorded based on a 0–9 scale [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126794#pone.0126794.ref009" target="_blank">9</a>] with IT 0–3 as resistant, 4–6 intermediate and 7–9 susceptible.</p><p>^c Res. = resistant and Sus. = susceptible.</p><p>^d The 1:1 ratios indicate a single gene and the 1:3 ratio indicate two genes segregated in the RIL population.</p><p>^e<i>P</i> > 0.05 was used for considering the observed numbers of resistant and susceptible RILs fit the theoretical ratio.</p><p>Seedling infection types (ITs) of the parents and number of F₈ recombinant inbred lines (RILs) from the Druchamp × Michigan Amber-derived recombinant inbred lines (RILs) tested in the greenhouse with races of <i>Puccinia striiformis</i> f. sp. <i>tritici</i>, theoretical segregation ratios of resistant and susceptible lines, and χ² and P values of chi-squared tests for goodness of fit of the observed numbers and expected ratios.</p

Frequency distributions of mean relative area under the progress curve (rAUDPC) values in Druchamp × Michigan Amber derived recombinant inbred lines tested with <i>Puccinia striiformis</i> f. sp. <i>tritici</i> in various environments.

<p>rAUDPC distribution of: (<b>A</b>) Pullman, WA in 2006, 2010 and 2011; (<b>B</b>) Mt. Vernon, WA in 2005, 2010 and 2011; and (<b>C</b>) greenhouse with races PST-25 and PST-127.</p

Correlation coefficients (<i>r</i>) of mean relative area under the disease progress curve (rAUDPC) and infection type (IT) of the Druchamp × Michigan Amber-derived recombinant inbred lines tested in the eight environments.

<p>^a The field tests in Pullman (eastern Washington) and Mt. Vernon (western Washington) were conducted under natural infection of <i>Puccinia striiformis</i> f. sp. <i>tritici</i>. Greenhouse tests were conducted under high-temperature cycle (10–30^°C); plants were inoculated at the adult growth stage of booting with races PST-25 and PST-127 that were virulent on seedlings of Druchamp; and all growth stages of Michigan Amber were susceptible to the two races.</p><p>^b The <i>r</i> values based on IT data are given in the parentheses. All of the <i>r</i> values were significant at <i>P</i> < 0.001.</p><p>Correlation coefficients (<i>r</i>) of mean relative area under the disease progress curve (rAUDPC) and infection type (IT) of the Druchamp × Michigan Amber-derived recombinant inbred lines tested in the eight environments.</p

Quantitative trait loci for stripe rust resistance for the relative area under the disease progress curve (rAUDPC) and infection type (IT) in the Druchamp × Michigan Amber derived recombinant inbred line population in 2006, 2010 and 2011 at Pullman and in 2005, 2010 and 2011 at Mt. Vernon.

<p>^a QTL in bold were stable and consistently detected in multiple environments.</p><p>^b The fields tests during the 2005 and 2006 growing seasons were conducted at Mt. Vernon (western Washington) and Pullman (eastern Washington).</p><p>^c LOD = logarithm (base 10) of odds.</p><p>^d AE = additive effect. A negative value indicates that the resistance allele for rust reduction is from Druchamp.</p><p>^e ND = No data.</p><p>^f Overall mean of the six environments (two locations and three years at each location).</p><p>Quantitative trait loci for stripe rust resistance for the relative area under the disease progress curve (rAUDPC) and infection type (IT) in the Druchamp × Michigan Amber derived recombinant inbred line population in 2006, 2010 and 2011 at Pullman and in 2005, 2010 and 2011 at Mt. Vernon.</p

Frequency distributions of mean infection type (IT) values in the Druchamp × Michigan Amber derived recombinant inbred line (RIL) population tested with <i>Puccinia striiformis</i> f. sp. <i>tritici</i> in various environments.

<p>IT distribution of: (<b>A</b>) Pullman, WA in 2006, 2010 and 2011; (<b>B</b>) Mt. Vernon, WA in 2005, 2010 and 2011; and (<b>C</b>) greenhouse with races PST-25 and PST-127.</p

Effects of the number of QTL on high-temperature and adult-plant (HTAP) resistance to stripe rust illustrated by the mean infection type (IT) and relative area under the progress curve (rAUDPC) values of the recombinant inbred lines (RILs) derived from the Druchamp (Dru) × Michigan Amber (MA) in Pullman and Mt. Vernon, showing RILs with more QTL were more resistant.

<p>The data of IT are shown in (A) and those of rAUDPC in (B). MA (the susceptible parent does not have any stripe rust resistance QTL and Dru (the resistant parent) has eight QTL for HTAP resistance.</p

Linkage maps for race-specific all-stage resistance QTL <i>QYrdr</i>.<i>wgp-5BL</i>, <i>QYrdr</i>.<i>wgp-5D</i> and <i>QYrdr</i>.<i>wgp-6BL</i>.<i>1</i> constructed using the stripe rust phenotypic data obtained in the greenhouse seedling tests with different <i>Puccinia striiformis</i> f. sp. <i>tritici</i> races and simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers of the recombinant inbred lines of Druchamp × Michigan Amber.

<p>Markers with prefix <i>X</i> and SSR markers and those with prefix <i>IWA</i> are SNPs markers. The locations of the QTL are indicated by the arrows and the markers in bold.</p

Quantitative trait loci for stripe rust resistance detected in the Druchamp × Michigan Amber-derived recombinant inbred line population tested in greenhouse with races <i>Puccinia striiformis</i> f. sp. <i>tritici</i> at seedling stage and the low-temperature cycle.

<p>^a LOD = logarithm (base 10) of odds.</p><p>^b AE = additive effect. A negative value indicates that the resistance allele for rust reduction is from Druchamp.</p><p>^c An R² value measured as the percentage of the total observed variation explained indicate the effect of the QTL.</p><p>Quantitative trait loci for stripe rust resistance detected in the Druchamp × Michigan Amber-derived recombinant inbred line population tested in greenhouse with races <i>Puccinia striiformis</i> f. sp. <i>tritici</i> at seedling stage and the low-temperature cycle.</p

Analysis of variance and estimates of broad-sense heritabilities (H²) of relative area under the disease progress curve (rAUDPC) and infection type (IT) scores of the recombinant inbred lines derived ted from the Druchamp × Michigan Amber cross.

<p>^a The field tests in Pullman (eastern Washington) and Mt. Vernon (western Washington) were conducted under the natural infection of <i>Puccinia striiformis</i> f. sp. <i>tritici</i>. Greenhouse tests were conducted under high-temperature cycle (10–30^°C); plants were inoculated at the adult growth stage of booting with races PST-25 and PST-127 that were virulent on seedlings of Druchamp; and all growth stages of Michigan Amber were susceptible to the two races.</p><p>Analysis of variance and estimates of broad-sense heritabilities (H²) of relative area under the disease progress curve (rAUDPC) and infection type (IT) scores of the recombinant inbred lines derived ted from the Druchamp × Michigan Amber cross.</p