Potassium requirements for growth and its related processes determined by plant analysis in wheat

Summary Potassium requirements for growth—dry matter (DM) and leaf area (LA) and related processes — relative leaf growth rate (RLGR), relative growth rate (RGR), net assimilation rate (NAR) and crop growth rate (CGR) were determined by plant analysis during the entogeny of wheat. Wheat (Triticum aestivum cv. HD 2329) plants were supplied with different amounts of K from deficient to adequate through nutrient solution. Samples were taken at specific stages for K determinations. The DM and LA were recorded at 45d, 75d and 105d. The growth related processes RGR, NAR and CGR were estimated between 30–45d, 45–75d and 75–105d. In case of RLGR the observations were carried out between 15–30d, 30–45d and 45–75d. These physiological processes and grain yield were correlated with K concentration in whole plant at 30 and 45d and top two leaves at 75 and 105d. The results indicated that k status in plants influences growth mostly through leaf area formation which inturn influences successively RLGR, RGR and CGR and finally grain yield. For vegetative growth the optimum concentration required in plants was always lower than the optimum for grain production.     

Genetic variation,heritability, divergence and biomass accumulation of rice genotypes resistant to bacterial blight revealed by quantitative traits and ISSR markers

A field experiment was carried out in order to evaluate genetic diversity of 41 rice genotypes using physiological traits and molecular markers. All the genotypes unveiled variations for crop growth rate (CGR), relative growth rate (RGR), net assimilation rate (NAR), yield per hill (Yhill^?1), total dry matter (TDM), harvest index (HI), photosynthetic rate (PR), leaf area index (LAI), chlorophyll‐a and chlorophyll‐b at maximum tillering stage. The CGR values varied from 0.23 to 0.76 gm cm^?2 day^?1. The Yhill^?1 ranged from 15.91 to 92.26 g, while TDM value was in the range of 7.49 to 20.45 g hill^?1. PR was found to vary from 9.40 to 22.34 µmol m^?2 s^?1. PR expressed positive relation with Yhill^?1. Significant positive relation was found between CGR and TDM (r = 0.61**), NAR and CGR (r = 0.62**) and between TDM and NAR (r = 0.31**). High heritability was found in RGR and Yhill^?1. Cluster analysis based on the traits grouped 41 rice genotypes into seven clusters. A total of 310 polymorphic loci were detected across the 20 inter‐simple sequence repeats (ISSR) markers. The UPGMA dendrogram grouped 41 rice genotypes into 11 clusters including several sub‐clusters. The Mantel test revealed positive correlation between quantitative traits and molecular markers (r = 0.41). On the basis of quantitative traits and molecular marker analyses parental genotypes, IRBB54 with MR84, IRBB60 with MR84, Purbachi with MR263, IRBB65 with BR29, IRBB65 with Pulut Siding and MRQ74 with Purbachi could be hybridized for future breeding program.     

Biomass accumulation and energy conversion efficiency in aromatic rice genotypes

A field experiment was conducted to evaluate photosynthetic efficiency along with different growth parameters of aromatic rice genotypes. Forty genotypes including three non-aromatic checks exhibited enormous variations for leaf area index (LAI), crop growth rate (CGR), relative growth rate (RGR), net assimilation rate (NAR), grain yield, total dry matter, harvest index and photosynthetic efficiency or energy use efficiency (Eμ) at panicle initiation and heading stages. Minimum LAI-value was 0.52 in Khazar at PI stage and maximum was 4.91 in Sakkor khora at heading stage. The CGR-value was in the range of 4.80−24.11 g m⁻² per day. The best yielder BR39 produced grain of 4.21 t ha⁻¹ and the worst yielder Khazar gave 1.42 t ha⁻¹. Total dry matter (TDM) yield varied from 4.04 to 12.26 t ha⁻¹ where genotypes proved their energy use efficiency a range between 0.58 to 1.65%. Eμ showed a significant positive relation with TDM (r = 0.80^**), CGR (r = 0.72^**) and grain yield (r = 0.66^**). A negative correlation was established between TDM and harvest index and LAI and RGR. Path analysis result showed that NAR at heading stage exerted highest positive direct effect (0.70) on Eμ.     

The relation of biomass production with leaf traits varied under different land-use and precipitation conditions in an Inner Mongolia steppe

The analyses of plant leaf traits that strongly influence aboveground net primary production (ANPP) are indispensable for understanding the process of plant biomass formation. However, there are few studies that have attempted to relate patterns of ANPP under contrasting management practices to plant leaf-level traits. To assess how leaf traits affect plant biomass accumulation under different land-use practices, we examined leaf traits and biomass production in three differently managed sites in the Inner Mongolia steppe: a site fenced since 1979 (UG), a winter grazing site (WG), and a heavily grazed site (HG). Low soil water content, leaf area index, and potential growth ability of species at site HG led to low crop growth rate (CGR), net assimilation rate (NAR), and relative growth rate (RGR); resulting in lower ANPP as compared to sites WG and UG. Irrespective of land-use management, prolonged drought significantly decreases ANPP even though it systematically increases mean CGR and RGR. However, leaf N content and leaf weight ratio are the crucial components necessary to determine the RGR at site WG. This suggests that low leaf N and availability of soil N due to haymaking may be responsible for neither over-compensatory nor compensatory growth in this site. The low ANPP in dry years is not due to the low mean CGR and RGR but rather to the short effective growing days (referring to the days the vegetation actually grows), suggesting that production-adjusted grazing regimes may be the most suitable measures for precision land management and avoiding grassland degradation.     

Growth Rate, Photosynthesis and Respiration in Relation to Leaf Area Index

This work examined three possible explanations of growth rateresponses to leaf area index (LAI) in which growth rate perunit of ground area (crop growth rate, CGR) increased to a plateaurather than decreasing above an optimum LAI at which all lightwas intercepted. Single leaf photosynthetic measurements, andwhole plant 24 h photosynthesis and respiration measurementswere made for isolated plants and plants in stands using Amaranlhushybridus, Chenopodium album, and two cultivars of Glycine maxgrown at 500 and 1000 µimol m^–2 S^–1 photosyntheticphoton flux density at 25 °C. CGR, relative growth rate(RGR), and LAI were determined from 24 h carbon dioxide exchangeand leaf area and biomass measurements. Respiration increasedrelative to photosynthesis with crowding in A. hybridus andthere was an optimum LAI for CGR. In contrast, the ratio ofrespiration to photosynthesis was constant across plant arrangementin the other species and they had a plateau response of CGRto LAI. Neither increased leaf photosynthetic capacity at highLAI nor a large change in biomass compared to the change inLAI could account for the plateau responses. It was calculatedthat maintenance respiration per unit of biomass decreased withdecreasing RGR in C. album and G. max, but not A. hybridus,and accounted for the plateau response of CGR to LAI. Sincesimilar decreases in maintenance respiration per biomass atlow RGR have been reported for several other species, a constantratio of respiration to photosynthesis may occur in more speciesthan constant maintenance respiration per unit of biomass. Amaranlhus hybridus L., Chenopodium album L., Glycine max L Merr, soybean, photosynthesis, respiration, growth, leaf area index     

Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.)

Enhancement of callus induction and its regeneration efficiency through in vitro techniques has been optimized for 2 abiotic stresses (salt and air desiccation) using 3 rice genotypes viz. BR10, BRRI dhan32 and BRRI dhan47. The highest frequency of callus induction was obtained for BRRI dhan32 (64.44%) in MS medium supplemented with 2, 4-D (2.5 mgL⁻¹) and Kin (1.0 mgL⁻¹). Different concentrations of NaCl (2.9, 5.9, 8.8 and 11.7 gL⁻¹) were used and its effect was recorded on the basis of viability of calli (VC), relative growth rate (RGR), tolerance index (TI) and relative water content (RWC). It was observed that in all cases BRRI dhan47 showed highest performance on tolerance to VC (45.33%), RGR (1.03%), TI (0.20%) and RWC (10.23%) with 11.7 gL⁻¹ NaCl. Plant regeneration capability was recorded after partial air desiccation pretreatment to calli for 15, 30, 45 and 60 h. In this case BRRI dhan32 gave maximum number of regeneration (76.19%) when 4 weeks old calli were desiccated for 45 h. It was observed that air desiccation was 2-3 folds more effective for enhancing green plantlet regeneration compared to controls. Furthermore, desiccated calli also showed the better capability to survive in NaCl induced abiotic stress; and gave 1.9 fold (88.80%) increased regeneration in 11.7 gL⁻¹ salt level for BRRI dhan47. Analysis of variance (ANOVA) showed that the genotypes, air desiccation and NaCl had significant effect on plant regeneration at P < 0.01.     

Productivity differences among loblolly pine genotypes are independent of individual-tree biomass partitioning and growth efficiency

Genetic differences in individual-tree biomass partitioning, growth efficiency, and stem relative growth rate (RGR) could confer intraspecific productivity differences and might strongly influence forest ecosystem carbon storage. We examined the relationship between genotype productivity (stem volume), whole-tree biomass partitioning, growth efficiency (stem wood production per unit leaf area), and stem RGR among nine different loblolly pine (Pinus taeda L.) genotypes from three different genetic groups of contrasting inherent genetic homogeneity: three open-pollinated (half-sib) families, three mass-control pollinated (full-sib) families, and three clonal varieties. We hypothesized that genotype productivity would be positively associated with increased partitioning to stem wood relative to other plant parts, higher stem RGR, and enhanced growth efficiency. After 3 years under plantation conditions, genotypes showed significant differences in stem volume, percent stem wood, percent branch wood, and partitioning to fine roots, yet no differences in stem RGR or growth efficiency. Furthermore, genotypic differences in stem volume were independent of genotypic differences in biomass partitioning, and overall, we found no evidence to support the hypothesized relationships. Even so, the observed variation in biomass partitioning has implications for forest C sequestration as genotypes which partition more biomass to long-lived biomass pools such as stems, may sequester more C. Moreover, the lack of a genetic relationship between stem volume and belowground partitioning suggests that highly productive genotypes may be planted without compromising belowground C storage.     

不同降湿处理后温室内番茄作物生物学性状与生理特性差异研究

研究了限量排气降湿、地下热循环冷凝除湿、膜下滴灌降湿3个降(除)湿处理后温室内番茄作物生物学性状与生理特性差异。结果表明:3个降湿处理温室内番茄群体生长率、相对生长率与净同化率均明显高于对照;番茄植株茎粗、干物重、叶面积表现出膜滴>冷凝>限排>对照规律;相接近时刻的光合速率与蒸腾速率,以壮叶>嫩叶>老叶,且日变化呈现低(上午)、高(中午)、低(傍晚)规律,并明显大于对照。     

Variation of Relative Growth Rate and Survival in Ecologically Contrasting Populations of Agrostis stolonifera

Abstract Significant differentiation in relative growth rate (RGR) was found among three ecologically contrasting populations of Agrostis stolonifera. Under low nitrogen conditions the sand dune population had the highest mean RGR. The plastic response in RGR to different levels of nitrogen supply was significantly higher in the inland meadow population than in the polder and sand dune populations. The (colonizing) polder population tended to have the highest variation for RGR. Variation in RGR within populations was mainly environmentally determined, although in the polder population significant effects of genotype and of genotype × nitrogen level were found. The repeatability for RGR in this population proved to be different from zero under both nitrogen conditions. Root/shoot ratios of the three populations were not different from each other, independent of the level of nitrogen supply. Correlations between RGR and survival of genotypes within the populations were not significant, which points at the influence of genotype × environment interactions or the possibility that genotypic differences in RGR do not necessarily indicate an adaptation to any habitat, not even the home site.     

Resistance of wheat genotypes to boron toxicity is expressed at the cellular level

The effects of toxic boron (B) concentrations on the growth of wheat genotypes at organ level and cellular level were investigated using excised root culture techniques. At the organ level, wheat genotypes differed for root elongation and lateral root development in response to toxic B concentrations. Genotypes classified as resistant from field studies had longer root axes and more lateral roots than sensitive or moderately sensitive genotypes, but there was no difference in axis elongation between sensitive and moderately sensitive genotypes. At the cellular level, callus production of root explants among genotypes still different at toxic B concentrations: resistant genotypes could produce more callus than sensitive or moderately sensitive genotypes. These results suggest that differences among genotypes in resistance to toxic B concentrations may be related to cell membrane permeability to B since they were also expressed in undifferentiated callus cells. The distinct and consistent differences among genotypes in response to B toxicity both at the organ level and at the cellular level could serve as a basis for selection in a breeding program. The methods were precise and efficient.     

Analysis of Growth, Photosynthesis and Light Interception for Single Plants and Stands

The two traditionally distinct treatments of growth analysis,using either relative growth rate (RGR) or crop growth rate(CGR), can be integrated in a single treatment in which RGRis a component of CGR. CGR can alternatively be analysed asthe product of incident light receipt, efficiency of light interception(as determined by leaf area index and extinction coefficient)and efficiency of use of intercepted light in dry-matter productionor in canopy net photosynthesis. Further, the net assimilationrate or net photosynthetic rate can be resolved into two componentswhich quantify the dependence of light interception on leafarea and of CO₂ assimilation on intercepted light. These relationsprovide increased flexibility in the analysis of assimilationand growth in terms of light interception and the structureof plants and stands. The usefulness of growth analysis in elucidatingphysiological mechanisms is discussed in relation to the useof more complex mechanistic models. Crop growth rate, light interception, growth analysis, leaf area index     

Effect of genotype on callus induction,shoot regeneration,and phenotypic stability of regenerated plants in the greenhouse of Primula ssp.

Genotypic differences between six genotypes of Primula vulgaris could be observed in callus induction rate, type of callus, root formation during the callus phase, and shoot regeneration rate. The shoot regeneration rate ranged from zero to 11.6 shoots per explant. There was no correlation between callus induction rate and shoot regeneration rate. Callus consistency and colour were an indicator of the organogenetic capacity of callus. An experiment with different periods of treatment with 4.0 mg l 2,4-dichlorophenoxyacetic acid and 2.0 mg l21 thidiazuron revealed that the shoot regeneration rate varied tremendously between genotypes. In two genotypes a period of 8 weeks on medium with plant growth regulators was sufficient to induce shoot regeneration. In three other genotypes a longer induction period was not able to overcome low regeneration capacity. However an increase in shoot regeneration rate was observed after 16iV32 weeks of induction. Phenotypic stability was also strongly dependent on genotype. In three genotypes the majority of regenerated plants looked normal and were diploid. Aberrations like abnormal growth habit, crinkly leaves, deviation of flower colour or lack of pollen formation occurred in only one genotype at a very low frequency (1.5 genotypes between 12.5 and 18.1 regenerants was tetraploid.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

The Effects of Vernalization on the Growth of the Wheat Shoot Apex

he effect of vernalization on the growth of the wheat shootapex was examined by comparing three genetic lines of ChineseSpring (CS) wheat having strong [CS (Hope 5D)], medium (CS Euploid),or no [CS (Hope 5A)] vernalization requirement. The mean volumeof the apical dome increased gradually in all lines, and thenthe apical dome enlarged rapidly as its relative growth rate(RGR) increased prior to double ridge formation. Phytomer volumeat initiation remained constant, so that the ratio of phytomerto apical dome at primordium initiation decreased in successiveplastochrons. In CS Euploid and in unvernalized CS (Hope 5D),the RGR of the apical dome tended to decrease at least untilinitiation of the collar primordium. The rate of primordiuminitiation at double ridge formation increased in proportionto the RGR of the apex at that time; i.e. it increased greatlyin CS (Hope 5A) and vernalized CS (Hope 5D), less so in CS Euploid,but no increase was observed in unvernalized CS (Hope 5D). Thetime of formation of double ridges seemed to be independentof the growth rate or size of the apical dome. The number oftillers present at ear emergence was inversely proportionalto vernalization requirement and was reduced by vernalization.Vernalization resulted in a decrease in the RGR of the newly-initiatedleaf primordia in relation to the RGR of the apical dome andthe axial part of the phytomer. Transfer of plants from longto short days at various times during growth showed that vernalizationincreased the number of labile primordia which could developinto either leaf, collar or spikelet. Vernalization thereforeseems to alter the ability of the apex to respond to subsequentphotoperiod rather than to affect its growth directly. Triticum aeslivum, wheat, chromosome substitution lines, shoot apex growth, vernalization     

Differences in developmental plasticity and growth rate among drought-resistant and susceptible cultivars of durum wheat (Triticum turgidum L. var. durum)

Understanding how growth and development of durum wheat cultivars respond to drought could provide a basis to develop crop improvement programmes in drought-affected tropical and subtropical countries. A greenhouse experiment was conducted to study the responses of five durum wheat cultivars to moisture stress at different developmental phases. Phenology, total dry matter (TDM), relative growth rate (RGR), leaf area ratio (LAR), net assimilation rate (NAR), leaf weight ratio (LWR), specific leaf area (SLA) and shoot:root ratio were compared. Pre-anthesis moisture stress delayed phenological development, whereas post-anthesis moisture stress accelerated it. TDM accumulation rate was different between drought-resistant and susceptible cultivars. RGR and its components changed with age and moisture availability. Drought-resistant cultivars had a high RGR in favourable periods of the growing season and a low RGR during moisture stress. In contrast, the drought-susceptible cultivar (Po) showed an opposite trend. LAR explained the differences in RGR (r=0.788) best, whereas the relationship between NAR and RGR was not significant. Even though both LWR and SLA were important factors determining the potential growth rate, LWR was of major importance to describe cultivar differences in LAR, and consequently in RGR. The drought-resistant cultivars Omrabi-5 and Boohai showed vigorous root development and/or a low shoot:root ratio. It is concluded that biomass allocation is the major factor explaining variation in RGR among the investigated durum wheat cultivars.     

In vitro callus induction from adult tissues of peach (Prunus persica L. Batsch)

We describe an efficient protocol for callus induction from adult tissues of Prunus persica (L.) Batsch. Three different commercial peach genotypes, Early May®, Zise May®, and UFO-3®, plus three other genotypes from hybrid crosses performed in February 2006, PS108, PS208, and PS708, were used in the study. Thirteen explant treatments were tested using nine different plant parts. Murashige and Skoog and woody plant medium salts were assayed with several concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KN), and thidiazuron, and two different photoperiods were tested, a 16-h photoperiod or continuous darkness. In terms of the quantitative response, two parameters were assessed: the number of d to callus induction and relative callus growth recorded after 30 d. Woody plant medium supplemented with 2,4-D and KN significantly increased the rates of callus induction in the majority of treatments. And no significant differences among the P. persica genotypes were found. The explants derived from stem and calyx produced up to 85 and 96% callus induction, respectively. The protocol described here could be used for efficient callus induction in a range of Prunus spp.     

Callus induction and regeneration in sugarcane under drought stress

Tissue culture methods are useful in assessing the tolerance of various stresses due to the ease of controlling stress under in vitro conditions. This study aimed to investigate the response of sugarcane genotyps to drought stress using calli as a model system. For inducing sugarcane callus, the medium of Murashige and Skoog (MS) was used with different mannitol concentrations (100, 200, and 300 mM) to measure their effects on callus frequency, the day of callus initiation, embryogenic potential, relative growth rate (RGR), water and proline contents, K⁺ and Na⁺ contents, as well as the formation of shoot and roots for three sugarcane genotypes (e.g., GT 54-9, G 84-47, and pH 8013). The RAPD-PCR analysis was carried out using five oligonucleotide primers to identify the genetic variation among sugarcane genotypes. The results indicated that the degree of callus proliferation varied from 70 − 86%. The highest value of callus proliferation, PGR, shoot formation was recorded for the genotype GT 54-9 compared to the other two genotypes (G 84-47 and pH 8013). Calli treated with 100 mM mannitol showed the highest RGR, proline and waer contents for the genotype GT 54-9, while, those treated with 300 mM recorded the lowest values of these parameters for the genotype pH 8013. The genotype G 84-47 collected highest Na⁺ content, while the genotype pH 8013 collected highest K⁺ content. The results of this study recommend preference for GT 54-9 genotype, which is considered the most promising genotype, showing more tolerance to drought stress based on all studied traits.     

Genotypic difference of potato in carbon budgeting as a mechanism of phosphorus utilization efficiency

Observed genotypic difference in P utilization efficiency in soil grown potatoes led to the present study to investigate possible mechanisms of P utilization efficiency in potato genotypes grown in nutrient solution under three P regimes (low, medium and high). For all genotypes relative growth rate (RGR), leaf P content, net assimilation rate (NAR) and leaf area ratio (LAR) increased while P utilization efficiency and leaf starch content decreased at the two higher P regimes compared to the low P regime. The P-efficient genotypes CGN 17903 and CIP 384321.3 had higher RGR compared to the P-inefficient genotypes CGN 22367 and CGN 18233, which resulted from enhanced NAR rather than from LAR. Net photosynthetic rate was similar for all genotypes. However, for P-inefficient genotype CGN 22367, the lower NAR could be explained by increased leaf dark respiration. For P-inefficient genotype CGN 18233 we speculate that increased carbon cost of root respiration or exudation or both, caused low NAR, since leaf dark respiration of this genotype was similar to that of P-efficient genotypes.     

影响小麦成熟胚培养及植株再生因素的研究

对3个不同栽培品种小麦的成熟胚进行离体培养,研究影响小麦愈伤组织诱导和植株再生的一些因素。结果表明,东农7742的苗分化率明显高于龙麦9814和龙麦26;高浓度的玉米素可明显提高芽的分化率;附加低浓度NAA的1/2 MS培养基可有效促进生根。可见,基因型对小麦愈伤组织的分化有很大的影响,附加一定的外源激素有利于提高植株的再生频率。     

A Study of <Emphasis Type="Italic">in vitro</Emphasis> Regeneration in Relation to Doses of Growth Regulators in Hybrids of Upland Cotton

The frequency of in vitro callus induction and plant regeneration is influenced by several factors, including composition of culture medium, explant source, and the genotype. Crosses between regenerable and non-regenerable upland cotton cultivars were evaluated for hybrid vigour towards regeneration responses, which is consequential in recalcitrant crop species like cotton where regeneration is limited only to a few cultivars. The results indicated that regenerable and non-regenerable parental cultivars had similar potential of producing callus, but differed in producing callus weight and embryogenic calli. Mean performance of crosses, regarding callus induction, callus weight, callus growth rate, percent embryo induction, and percentage of germinating embryos, deviated considerably from the performance of their parents, signifying the presence of hybrid vigour for the expression of these traits. Magnitude of hybrid vigour varied across hormonal levels. Genetic component was evident for all the traits although of lower magnitude. The results indicated that genetic component in the phenotypic expression of callus growth, percentages of embryo induction and germinating embryos was higher than that of callus induction, callus weight and percentage of embryogenic calli. Hormonal concentration in the media had affect on the degree of gene expression responsible for regeneration in upland cotton. Over, partial- and additive-dominance types of gene effects were apparent in the expression of these traits. Genotype × growth regulator level interaction caused considerable variation in the expression of regeneration responses, suggesting that determination of specific level of growth regulator concentration in the medium was necessary for a particular genotype to obtain optimum response. Genotype × explant source interaction was, however, relatively less important. Differences among genotypes for percent embryo induction were clearly evident.