Maria Dolors Serret

High-throughput phenotyping and genomic selection: The frontiers of crop breeding converge

Genomic selection (GS) and high-throughput phenotyping have recently been captivating the interest of the crop breeding community from both the public and private sectors world-wide. Both approaches promise to revolutionize the prediction of complex traits, including growth, yield and adaptation to stress. Whereas high-throughput phenotyping may help to improve understanding of crop physiology, most powerful techniques for high-throughput field phenotyping are empirical rather than analytical and comparable to genomic selection. Despite the fact that the two methodological approaches represent the extremes of what is understood as the breeding process (phenotype versus genome), they both consider the targeted traits (e.g. grain yield, growth, phenology, plant adaptation to stress) as a black box instead of dissecting them as a set of secondary traits (i.e. physiological) putatively related to the target trait. Both GS and high-throughput phenotyping have in common their empirical approach enabling breeders to use genome profile or phenotype without understanding the underlying biology. This short review discusses the main aspects of both approaches and focuses on the case of genomic selection of maize flowering traits and near-infrared spectroscopy (NIRS) and plant spectral reflectance as high-throughput field phenotyping methods for complex traits such as crop growth and yield.

Phenotyping maize for adaptation to drought

The need of a better adaptation of crops to drought is an issue of increasing urgency. However, enhancing the tolerance of maize has, therefore, proved to be somewhat elusive in terms of plant breeding. In that context, proper phenotyping remains as one of the main factors limiting breeding advance. Topics covered by this review include the conceptual framework for identifying secondary traits associated with yield response to drought and how to measure these secondary traits in practice.

Development of photoautotrophy and photoinhibition of Gardenia jasminoides plantlets during micropropagation

This paper reports on the fast fluorescence responses of Gardenia jasminoides Ellis plantlets, at two successive stages (shoot multiplication and root induction) of culture in vitro. We test whether plantlets in vitro suffer photoinhibition during culture and whether the degree of photoautotrophy of these mixotrophic plantlets has any effect on the extent of photoinhibitory impairment. In this regard the effects of different sucrose levels in the medium and PPFD during growth on the development of photoautotrophy and the extent of photoinhibition were evaluated. Plantlets were grown under low, intermediate, and high (50, 100, and 300 μmol m-2 s-1) PPFD, and at 3 different sucrose concentrations (0.5, 1.5, and 3.0%, w/v) in the medium, during shoot multiplication. During root induction the same growth conditions were assayed except for the high PPFD. The development of photoautotrophy was assessed via the difference between the stable carbon isotope composition of sucrose used as heterotrophic carbon source and that of leaflets grown in vitro. Plantlets from root induction showed more developed photoautotrophy than those from shoot multiplication. For both stages the low-sucrose medium stimulated the photoautotrophy of plantlets in vitro. In addition, intermediate PPFD induced photoautotrophy during shoot multiplication. For plantlets of both culture stages at the lowest PPFD no photoinhibition occurred irrespective of the sucrose concentration in media. However, during the shoot multiplication stage chlorophyll fluorescence measurements showed a decrease in Fv/Fm and in t1/2 as growing PPFD increased, indicating photoinhibitory damage. The decline of Fv/Fm was caused mostly by an increase in Fo, indicating the inactivation of PSII reaction centers. However plantlets growing under low sucrose showed reduced susceptibility to photoinhibition. During root induction, only plantlets cultured with high sucrose showed a decrease in Fv/Fm as PPFD increased, although t1/2 remained unchanged. In this case, the decline of Fv/Fm was mostly due to a decrease in Fm, which indicates increased photoprotection rather than occurrence of photodamage. Therefore, growth in low-sucrose media had a protective effect on the resistance of PSII to light stress. In addition, plantlets were more resistant to photoinhibition during root induction than during shoot multiplication. Results suggest that increased photoautotrophy of plantlets reduces susceptibility to photoinhibition during gardenia culture in vitro.

The effect of different closure types, light, and sucrose concentrations on carbon isotope composition and growth ofGardenia jasminoides plantlets during micropropagation and subsequent acclimationex vitro

The growth ofGardenia jasminoides Ellis plantlets and the development of photoautotrophy during two successive culture stages (shoot multiplication and root induction)in vitro was analyzed. We examined the effects of changes in growth conditions (type of tube closure, light, and sugar levels) on the development of photoautotrophy and growth during micropropagation and sought to establish whether they affected later acclimation to conditionsex vitro. During the two stagesin vitro, plantlets were grown in tubes under two different PPFD (50 and 110 µmol m−2 s−1), in media with three different sucrose concentrations (0, 1.5, and 3.0%, w/v) and with two different CO2 levels inside the tubes (controlled by either tightly closed caps or loosely sealed caps, and with an external CO2 concentration of 750 µmol mol−1). The development of photoautotrophy was assessed by determining the difference between the stable carbon isotope composition (δ13C) of sugar cane sucrose used as a heterotrophic carbon source and that of leaflets grownin vitro. Plantlets from the root-induction stage showed a more highly developed photoautotrophy than those from the shoot- multiplication stage. At both stages, utilization of closed caps was the treatment which most stimulated development of photoautotrophy in plantlets. Also, lowering PPFD or sucrose concentration induced a greater degree of photoautotrophic development, the strongest effect being observed in plantlets cultured inside loosely sealed tubes. During acclimationex vitro, plantlets taken from loosely sealed tubesin vitro performed better than those cultured inside tightly sealed tubes. The former, as well as recording a larger increase in fresh weight during this stage, also showed more negative δ13C in the newly developed leaves, which would seem to indicate a better water status during acclimation. Present results validate the usefulness of δ13C analysis of leaflets as a simple technique in assessing the development of photoautotrophy during culturein vitro. In addition, δ13C analysis can be extended to evaluate growth conditions during acclimation toex vitro conditions.

Contribution of the ear and the flag leaf to grain filling in durum wheat inferred from the carbon isotope signature: Genotypic and growing conditions effects

The ear, together with the flag leaf, is believed to play a major role as a source of assimilates during grain filling in C3 cereals. However, the intrusive nature of most of the available methodologies prevents reaching conclusive results in this regard. This study compares the carbon isotope composition (δ(13)C) in its natural abundance in the water-soluble fractions of the flag leaf blade and the ear with the δ(13)C of mature kernels to assess the relative contribution of both organs to grain filling in durum wheat (Triticum turgidum L. var. durum). The relative contribution of the ear was higher in landraces compared to modern cultivars, as well as in response to nitrogen fertilization and water stress. Such genotypic and environmentally driven differences were associated with changes in harvest index (HI), with the relative contribution of the ear being negatively associated with HI. In the case of the genotypic differences, the lower relative contribution of the ear in modern cultivars compared with landraces is probably associated with the appearance in the former of a certain amount of source limitation driven by a higher HI. In fact, the relative contribution of the ear was far more responsive to changes in HI in modern cultivars compared with landraces.

Physiological traits contributed to the recent increase in yield potential of winter wheat from Henan Province, China

This experiment aims to test the traits responsible for the increase in yield potential of winter wheat released in Henan Province, China. Seven established cultivars released in the last 20 years and three advanced lines were assayed. The results showed that grain yield was positively correlated with harvest index (HI), kernel number per square meter, and aboveground biomass. In addition, the HI and aboveground biomass showed an increasing trend with the year of release. Therefore, we can conclude that bread wheat breeding advances during recent decades in Henan Province, China, have been achieved through an increase in HI, kernel number per square meter, and aboveground biomass. A higher δ(13)C seems also to be involved in these advances, which suggests a progressive improvement in constitutive water use efficiency not associated with a trend towards lower stomatal conductance in the most recent genotypes. However, genetic advance does not appear related to changes in photosynthesis rates on area basis when measured in the flag leaf or the spike, but only to a higher, whole-spike photosynthesis. Results also indirectly support the concept that under potential yield conditions, the spike contributed more than the flag leaf to kernel formation.

Low-cost assessment of wheat resistance to yellow rust through conventional RGB images

RGB imagery is a low-cost approach for field phenotyping of rust tolerance in wheat.It performs better than chlorophyll content and gas exchange of individual leaves.Hue, Green Fraction, Greener Fraction, a and u are the most effective color traits. Establishing low-cost methods for stripe (yellow) rust (Puccinia striiformis f. sp. tritici) phenotyping is paramount to maintain the breeding pipeline in wheat. Twelve winter wheat genotypes were grown to test rust resistance and yield performance. Physiological traits, including leaf chlorophyll content (Chl), net photosynthesis rate (Pn), stomatal conductance (gs), transpiration rate (E) and canopy temperature depression (CTD), together with diverse color components derived from Red, Green and Blue (RGB) images, were measured at different crop stages. Grain yield (GY) and grain yield loss index (GYLI) were assessed through comparison with the previous normal planting year. Genotypes exhibited a wide range of resistance to yellow rust, with GYLI values ranging from about -3% for the more resistant (Zhoumai 22) to 89% for the most susceptible (Lankao 298) genotypes. Moreover yellow rust reduced Chl and to a lesser extent, Pn, while traits related to water status were lower (gs) or not affected (E and CTD). The color parameters Green Fraction, Greener Fraction, Hue, a and u measured during grain filling were much better correlated with GY and GYLI (r2 ranging between 74% and 81%) than the set of photosynthetic and transpirative traits (Chl, Pn, gs, E, CTD) measurements in the same stage. Conventional digital imaging appears to be a potentially affordable approach for high-throughput phenotyping of yellow rust resistance.

The Effect of In Vitro Culture Conditions on the Pattern of Photoinhibition during Acclimation of Gardenia Plantlets to Ex Vitro Conditions

We tested the effect of growing conditions during micropropagation on the fast kinetics of chlorophyll (Chl) fluorescence of Gardenia jasminoides Ellis plantlets during a 4-week acclimation to ex vitro. We studied whether photoautotrophic growing in vitro produced plantlets with less photoinhibition impairment during acclimation. Of the growing conditions stimulating photoautotrophy in vitro, only loose tube caps had a positive effect, whereas low sucrose or sucrose-free content in the medium and high PPFD showed a negative effect. Thus, plantlets cultured with 3 % (m/v) of sucrose were subsequently less photoinhibited throughout acclimation than those cultured with low sucrose (0.5 %) or sucrose-free media. Moreover, at the end of acclimation the former plantlets showed Fv/Fm and Fv/F0 ratios typical of unstressed ex vitro plants as well as a higher Chl content and ratio of Chls to carotenoids. Plantlets cultured at a photosynthetic photon fluence density (PPFD) of 50 µmol m−2 s−1 also showed a better performance at the end of acclimation than those cultured at a higher (110 µmol m−2 s−1) PPFD. Thus except in the case of loose-tube closure, gardenia plantlets cultured in vitro under conventional sucrose concentration and PPFD are the least photoinhibited during acclimation. Nevertheless, significant interactions between the in vitro growing factors were observed at the end of acclimation.

Comparative effects of growth irradiance on photosynthesis and leaf anatomy of Flaveria brownii (C4-like), Flaveria linearis (C3–C4) and their F1 hybrid

Photosynthetic rates and related anatomical characteristics of leaves developed at three levels of irradiance (1200, 300 and 80 umol · m−2 · s−1) were determined in the C4-like species Flaveria brownii A.M. Powell, the C3–C4-intermediate species F. linearis Lag., and the F1 hybrid between them (F. brownii × F. linearis). In the C3–C4 and F1 plants, increases in photosynthetic capacity per unit leaf area were strongly correlated with changes in mesophyll area per unit leaf area. The C4-like plant F. brownii, however, showed a much lower correlation between photosynthetic capacity and mesophyll area per unit leaf area. Plants of F. brownii developed at high irradiance showed photosynthetic rates per unit of mesophyll cell area 50% higher than those plants developed at medium irradiance. These results along with an increase in water-use efficiency are consistent with an increase of C4 photosynthesis in high-irradiance-grown F. brownii plants, whereas in the other two genotypes such plasticity seems to be absent. Photosynthetic discrimination against 13C in the three genotypes was less at high than at low irradiance, with the greatest change occurring in F. brownii. Discrimination against 13C expressed as δ13C was linearly correlated (r2 = 0.81; P<0.001) with the ratio of bundle-sheath volume to mesophyll cell area when all samples from the three genotypes were combined. This tissue ratio increased for F. brownii and the F1 hybrid as growth irradiance increased, indicating a greater tendency towards Kranz anatomy. The results indicated that F. brownii had plasticity in its C4-related anatomical and physiological characteristics as a function of growth irradiance, whereas plasticity was less evident in the F1 hybrid and absent in F. linearis.

Nitrogen assimilation and transpiration: key processes conditioning responsiveness of wheat to elevated [CO2] and temperature

Although climate scenarios have predicted an increase in [CO(2)] and temperature conditions, to date few experiments have focused on the interaction of [CO(2)] and temperature effects in wheat development. Recent evidence suggests that photosynthetic acclimation is linked to the photorespiration and N assimilation inhibition of plants exposed to elevated CO(2). The main goal of this study was to analyze the effect of interacting [CO(2)] and temperature on leaf photorespiration, C/N metabolism and N transport in wheat plants exposed to elevated [CO(2)] and temperature conditions. For this purpose, wheat plants were exposed to elevated [CO(2)] (400 vs 700u2009µmolu2009mol(-1)) and temperature (ambient vs ambientu2009+u20094°C) in CO(2) gradient greenhouses during the entire life cycle. Although at the agronomic level, elevated temperature had no effect on plant biomass, physiological analyses revealed that combined elevated [CO(2)] and temperature negatively affected photosynthetic performance. The limited energy levels resulting from the reduced respiratory and photorespiration rates of such plants were apparently inadequate to sustain nitrate reductase activity. Inhibited N assimilation was associated with a strong reduction in amino acid content, conditioned leaf soluble protein content and constrained leaf N status. Therefore, the plant response to elevated [CO(2)] and elevated temperature resulted in photosynthetic acclimation. The reduction in transpiration rates induced limitations in nutrient transport in leaves of plants exposed to elevated [CO(2)] and temperature, led to mineral depletion and therefore contributed to the inhibition of photosynthetic activity.